8. Kirst M, Cordeiro C, Rezende G, Grattapaglia D: Power of microsatellite markers for fingerprinting and parentage analysis in Eucalyptus grandis breeding populations. *J Heredity* 2005, 96(2):161.

9. Wagner H, Sefc K: Identity 1.0. Centre for Applied Genetics. University of Agricultural Sciences, Vienna 1999, Available at http://www.boku.ac.at/zag/forsch/identity.htm.

10. Faria D, Mamani E, Pappas G, Grattapaglia D: Genotyping systems for Eucalyptus based on tetra-, penta- and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests. *Tree Genetics & Genomes* 2011, 7:63-77.

P59

Selection of SSR markers for population studies in *Eucalyptus globulus*
seed orchards

Diego Torres-Dini¹*, Zohra Bennadji², Laura Lima-Aliano³, Natalia Nikichuk ¹,
Fernando Resquin², Gustavo Balmelli²

¹Forest Biotechnology Laboratory, INIA Tacaurembo, Uruguay; ²Fosrestry Breeding Program, INIA Tacaurembo, Uruguay; ³Uruguay.University of the Republic, Montevideo Uruguay

E-mail: dtorres@tb.inia.org.uy

BMC Proceedings 2011, 5(Suppl 7):P59

**Background:** *Eucalyptus globulus* is a widely planted species in temperate regions of the world for pulpwood production. Its good characteristics for kraft pulping in addition to a broad adaptability to different site conditions have led this species to be employed in commercial plantations and to be included in breeding programs [1]. Uruguay has approximately 300.000 hectares forested with *E. globulus* being the most cultivated species in the country and representing 45% of the total forested area. The main objectives of many Uruguayan breeding programs for *E. globulus* are the increase of volume per hectare, basic density and pulp yield. The strategies for genetic improvement used in many breeding programs in Uruguay require seed multiplication in seed orchards to obtain genetic gain [2]. In long-term breeding programs, the strict selection of reproductive populations restricts the number of genotypes involved in the final orchard, thus reducing genetic diversity and increasing the risk of depression through inbreeding in the following generations. The use of SSRs (simple sequence repeat) markers as selecting strategies of seed orchards in other species such as *Eucalyptus dunnii*, has proved to be an excellent tool to reduce inbreeding [3,4]. The transferability of SSR markers across Eucalyptus species has been widely studied [5-8]. The aim of this work was to select highly polymorphic SSR markers of *E. globulus* to assist breeding programs.

**Methodology:** Eighteen *E. globulus* samples were collected from the breeding program of the National Agricultural Research Institute of Uruguay .DNA was extracted with CTAB 2X buffer [9]. Eight SSR markers, previously reported for *E. grandis* and *E. urophylla* were used: EMBRA 8, EMBRA 11, EMBRA 18, EMBRA 32, EMBRA 47, EMBRA 51, EMBRA 58 and EMBRA 155 [6,7] The annealing temperature was specifically adjusted for *E. globulus* using a temperature gradient of 52°C – 56°C. The other reaction parameters did not differ from those previously reported. The results were visualized in polyacrilamide gels 8% under denaturing conditions at 80 W constant power over 3h 30min and were stained with silver nitrate. The estimation of the molecular weight of the alleles was made by comparison with a ladder. The matrix information was analyzed with the software Identity v 1.0 [10] and the following parameters were calculated: alleles number per loci, expected heterozygosity (He), observed heterozygosity (Ho), identity probability (Pi) and paternity exclusion probability (Pe).

**Results and conclusion:** From the eight tested markers, EMBRA 8, EMBRA 18 and EMBRA 13 did not amplify at any of the tested temperatures. EMBRA 11 and EMBRA 47 showed good amplification at 52°C of annealing. The rest of the primers worked optimally in the conditions previously described for *E. grandis* and *E. urophylla*[7]. The five SSRs markers amplified a total of 42 alleles. With a maximum of 14 alleles for EMBRA 11, 9 alleles for the markers EMBRA 47 and EMBRA 58 and 5 alleles for the markers EMBRA 51 and EMBRA 155. The range of heterozygosity expected for all the loci ranged from 0.64 to 0.88. However, the observed heterozygosity showed values of 0.1 to 0.9. With this information the probability of identity (IP) was 17 x 10⁻⁵ and the paternity exclusion probability was 0.99. The considerable number of SSR markers currently published [6-8] confirm how necessary the availability of these tools is to carry out precise population analyses. The five identified markers turned out to be

promising candidates to be used in *E. globulus* seed orchards population studies.

References

1. Freeman J, Whittock S, Potts B, Vaillancourt R: QTL influencing growth and wood properties in Eucalyptus globulus. *Tree Genetics & Genomes* 2009, 5:713-722.

2. Balmelli G, Kubota M: Estimación de parámetros genéticos en la segunda generación de Eucalyptus grandis y su utilización para el manejo de la población multipropósito. *Serie Aftercare Forestal INIA-JICA* 2002, 41-11.

3. Marcucci-Poltri S, Zelener N, Traverso J, Gelid P, Hopp E: Selection of a seed orchard of Eucalyptus dunnii based on genetic diversity criteria calculated using molecular markers. *Tree physiology* 2003, 23:625.

4. Zelener N, Marcucci-Poltri S, Bartoloni N, López C, Hopp E: Selection strategy for a seedling seed orchard design based on trait selection index and genomic analysis by molecular markers: a case study for Eucalyptus dunnii. *Tree Physiology* 2005, 25:1457-1467.

5. Sansaloni C: Desenvolvimento, caracterização e mapeamento de microsatelites tetra y pentanucleotidos em Eucalyptus ssp. Brazilia: Universidade de Brazilia 2008.

6. Brondani RPV, Brondani C, Tarchini R, Grattapaglia D: Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla. *Theor Appl Genet* 1998, 5:816-827.

7. Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. BMC Plant Biology 2006, 6:20-36.

8. Faria D, Mamani E, Pappas G, Grattapaglia D: Genotyping systems for Eucalyptus based on tetra-, penta- and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests. *Tree Genetics & Genomes* 2011, 7:63-77.

9. Ferreira M, Grattapaglia D: Introdução ao uso de marcadores RAPD e RFLP em análise genética. EMBRAPA.CENARAGEN 1995, 1-220.

10. Wagner H, Sefc K: Identity 1.0. Centre for Applied Genetics. University of Agricultural Sciences, Vienna 1999, Availableathttp://www.boku.ac.at/zag/forsch/identity.htm.

P60

Genomic selection using a realized genomic relationship matrix in a
Pinus taeda L. cloned population

Jaime Zapata-Valenzuela¹, Fikret Isik¹, Christian Maltecca², Jill Wegryzn³,
David Neale³, Steven McKeand¹, Ross Whetten¹

¹Cooperative Tree Improvement Program, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA;

²Department of Animal Science, North Carolina State University Raleigh, NC, USA; ³Department of Plant Science, University of California-Davis, CA, USA E-mail: ross_whetten@ncsu.edu

BMC Proceedings 2011, 5(Suppl 7):P60

Genetic merit can be considered the finite sum of thousands of allelic
effects, each physically located at some place on the genome, whose
transmission can be traced through molecular markers. Traditionally, best
linear unbiased prediction (BLUP) of breeding values relies on average
additive genetic covariances (the numerator relationship matrix **A**) derived
from pedigrees to utilize information from relatives. For example, all pairs
of full-sib offspring of a cross are assumed to share 50% of alleles in
common. Such assumptions ignore variation in Mendelian segregation of
alleles among progeny within family. With advances in marker genotyping
technology and reduction in genotyping cost, it is now feasible to estimate
genetic covariances from markers.

Linear mixed models that utilize realized genomic relationship matrices could predict genomic estimated breeding values (GEBV) more accurately than those that use expected average genetic covariances derived from pedigrees. Dense markers can be used to trace identity by descent probabilities at each locus, and those probabilities used to construct an incidence matrix. The incidence matrix is used to estimate the genomic relationship matrix (G), which is used in place of the A matrix in solving the mixed model equations. This may allow more accurate estimation of individual breeding values than the traditional model based on average genetic covariances.

We estimated realized genetic covariances between cloned progeny of a
*P. taeda* population. There were 165 cloned progeny derived from nine
full-sib families. The realized genomic relationships were based on a set of 3,461 biallelic SNP markers. We used the following linear mixed model **y** = **Xb** + **Zu** + **e** to estimate GEBV. In the model **X** and **Z** are incidence matrices, **b** is the vector of fixed mean, **u** is the vector of additive genetic effects that correspond to allele substitution effects for each marker with Var (**u**) = Iσ²_m; where σ²_m is the marker variance and **I** is the identity matrix. The term **e** is the vector of residuals. The dimension of **Z** is the number of individuals (n) by the number of loci (m). The regression method used to construct our **G** matrix did not require allele frequencies; instead, the inverse of the **G** matrix was generated by regressing **ZZ**' as a dependent variable on the **A** matrix as the independent variable. Therefore, the expected value of **G** is **A** plus a constant matrix.

Different cross-validation methods were used to test performance of the **G** matrix. Clones were divided into a training group with both marker and phenotypic information and a validation group for which only marker genotypes were used. In one scenario ~90% of the clones (148) were sampled for training, either randomly selecting within each of the nine families or at random without family consideration. The remaining ~10% were used for validation (17 clones). In the second scenario, ~50% of clones (84) were sampled either within family or randomly from the whole population for training, and the remaining ~50% were used for validation (81 clones). The model parameters estimated in the training set were used to predict GEBV in the validation set. For each scenario, six independent samplings were carried out. The mean correlation between the GEBV based on **G**-BLUP and breeding values based on **A**-BLUP were determined for each scenario, along with the accuracy of the BLUP predictions for both **G** and **A** based models. The mean correlation varied from 0.37 to 0.61 across the four validation methods. The accuracies of the predictions for any validation scenario were always higher for **G**-BLUP (range of 0.65 to 0.75) than **A**-BLUP (0.60 to 0.62), which is related to the smaller standard error of the predicted **G**-BLUP for the validation clones (17 or 81) under the different scenarios.

Estimating realized genetic covariances based on the genotypes of biallelic markers and incorporating those estimates into G-BLUP helps to more accurately characterize Mendelian segregation of alleles, and could allow more accurate selection within family. Such a method would increase genetic gains in forest tree breeding. The major impact would be on reducing the need for expensive field testing, but it may also be possible to shorten the breeding cycle and thus increase genetic gain per unit time and cost. The impact of genomic selection applications in forest tree breeding may be greater than for other crop or animal species, because of the biology of trees and their much longer breeding cycles.

P61
Imputing missing genotypes: effects of methods and patterns of missing data

Funda Ogut¹, Fikret Isik², Steven McKeand², Ross Whetten2*

¹Cooperative Tree Improvement Program Department of Forestry and Environmental Resources, North Carolina State University Raleigh, NC, USA;

²Cooperative Tree Improvement Program, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA

E-mail: ross_whetten@ncsu.edu

BMC Proceedings 2011, 5(Suppl 7):P61

Costs of high-throughput genotyping have decreased to the point where it appears economically feasible to use molecular genetic marker information in applied breeding programs. Some practical questions remain to be addressed about how best to deal with missing data in the resulting genotype datasets, to minimize the impact of the missing data on the accuracy of breeding value prediction. Data can be missing for two reasons – first, genotyping assay failure is likely for at least some loci in some samples; and second, it may prove economically desirable to invest more resources for high-density genotyping of a few individuals and fewer resources for lower-density genotyping of many individuals [1]. The proportion of missing genotypes may range from less than one percent due to genotyping assay failure, to over 80% if a selective genotyping strategy is used. Many methods for predicting genetic merit of trees using marker genotype data require complete genotype information for mathematical reasons. It is therefore important to use efficient statistical methods to accurately impute missing genotypes. In species with complete reference genome sequences available, the map

order of markers and linkage disequilibrium (LD) information can be used to guide imputation of missing genotypes. Completely sequenced reference genomes are available for only two forest tree species, so these methods are not suitable for most forest trees.

Gengler et al. [2] described a method to impute missing genotypes using mixed linear models and BLUP. We determined the effect on accuracy of BLUP estimated breeding values of imputation with different levels (10%, 20%, 40%, 60% and 80%) of missing genotypes. Analyses were conducted both with empirical data (3461 SNP markers in a cloned loblolly pine population of 165 genotypes) and simulated data, using missing data created by random sampling (some loci missing in all individuals) or by structured sampling (all loci missing in some individuals). Simulations were used to examine the effect of family and progeny size, mating design, proportion of missing genotypes, genotyping strategy and the method for imputation on the accuracy of breeding values. Imputed genotypes were obtained using the numerator relationship matrix (the **A** matrix) and solving the mixed model equations of **y** = **Xb** + **Mu** + **e**, where **y** is the vector of gene content predictions, **X** is the design matrix (vector of 1s) for the mean, **M** is the design matrix connecting trees to the gene content vector **y**, **u** is the individual tree effect and **e** is the error variance. The solutions of mixed model equations produce predicted SNP genotypes for trees with missing genotypes. The solutions would be continuous, centered on 1 because the gene content values are 0, 1 or 2.

Imputation of missing genotypes in empirical data from an unbalanced mating design with family sizes ranging from 1 to 35 was more powerful for data with structured missing genotypes at all levels of missing data than for data with random missing genotypes with same proportions of missing data. The accuracy of imputation for 10% and 80% missing genotypes ranged between 0.96 to 0.23 and 0.96 to 0.16 for structured and random missing genotypes in the data, respectively. As the proportion of missing genotypes increased in the data, the power of imputation decreased. With simulation, we found that the imputation was less affected by the distribution of missing genotypes in a balanced mating design with families of equal size. The accuracy of imputation ranged between 0.97 to 0.75 for the 10% and 80% missing genotypes in the data, respectively.

References

1. Habier D, Fernando RL, Dekkers JCM: Genomic selection using low-density marker panels. *Genetics* 2009, 182:343-353.

2. Gengler N, Mayeres P, Szydlowski M: A simple method to approximate gene content in large pedigree populations: application to the myostatin gene in dual-purpose Belgian Blue cattle. *Animal* 2007, 1(1):21-28.

S4. REPRODUCTION, GROWTH AND DEVELOPMENT

P62
Variation in gene expression profile with aging of *Pinus radiata* D. Don

Carolina Alvarez¹*, Luis Valledor², Rodrigo Hasbún³, Manuel Sanchez-Olate¹,
Darcy Ríos¹

¹Plant Tissue Culture Laboratory, University of Concepción, Chile; ²Molecular Systems Biology, University of Viena, Austria; ³Genómica Forestal S.A. Biotechnologyy center, University of Concepción, Chile

E-mail: caalvarez@udec.cl

BMC Proceedings 2011, 5(Suppl 7):P62

Phase change in higher plants, from juvenile to mature, affects the reproductive competence, morphology and growth rate, as well as the regenerative potential of tissue explants. However, in conifers these changes can be achieved before the onset of flowering and at the beginning of the seed production. Early maturational changes in conifers are very obvious, severe and overall irreversible [1], and are characterized by lost of morphogenetic capacity. This may affect clonal multiplication programs and genetic manipulation, due mainly to the decline in adventitious rooting capacity which is the most marked maturation and aging effect. Despite the serious effects of these processes, little is known about their basic regulation in forest clonal propagation programs. The latest researches regarding this subject are related to auxins changes during maturation [2], epigenetic variations during phase-change, like DNA methylation [3], and proteomic changes during needle maturation [4].
This work has been performed to gain deeper insight into the genetic mechanisms that regulates the loss of morphogenetic capacity with the aging process in *P. radiata*.

*P. radiata* ortets of different age were brought from La Posada nursery at BioBio region, Chile. Needles were collected in August, frozen in liquid nitrogen and stored at -80°C until its RNA extraction. RNA was isolated from 100 mg of frozen tissue according to Chang [5], the RNA extracts were purified with RNeasy Clean up and treated with RNase-free DNase (both from QIAGEN). Gene expression analysis was done by a reverse dot blot hybridization technique by comparing the transcription levels of 174 genes in 1, 3 and 5 years old ortets using the methodology described by Valledor *et al.*[4]. The 174 genes analyzed were classified into categories being the most important: Chloroplast and photosynthesis, protein translation folding, modification and degradation and proteins involved in transcription and DNA replication. Gene expression was quantified as signal intensity using the Gel Pro-analyzer 3.1.The values were normalized dividing every spot intensity by the average of the intensity of each membrane.

From the 174 studied sequences only 56 were differentially expressed among ortets of different ages. From the 56 genes differentially expressed, 27 showed a clear variation tendency with age, from which 21 were down-regulated and 6 were up-regulated, reflecting a higher number of active pathways in the younger ortets. GBBS1, PPI-phosphofructokinase and α-L-fucosidase (Figure 1) genes are related with carbohydrates and carbon metabolism that were affected by the ortet age; all were down-regulated with the increasing age, showing a significantly lower level of transcription in ortets of 5 years old. Both PPI-phosphofructokinase and α-L-fucosidase have been correlated to a higher level of expression in younger and metabolically active tissues. Specifically the *Arabidopsis thaliana* fucosidase (AtFXG1) has shown a higher expression level in younger leaves than in the older ones, and it has been proposed that it has a role as a growth regulator. The previous results are consistent with the higher expression level in RUBISCO

activase (figure 1) in the younger ortets (1 and 3 year old), which in turn agree with different studies, indicating that photosynthesis is reduced with increasing age of shrubs and trees [7] and higher in the younger ones.

Transcription regulated protein family were mainly over expressed in 1 year old ortets. It is the case of the chromosome condensation gene regulator (RCC1) (figure 1), which inhibit the chromosome condensation until G2 phase of the cell cycle and is required for the formation of the mitotic spindle [7].This result indicates that 1 year old ortets are more transcriptionally active. Likewise, proteins like Putative valyl-tRNA synthase and T-complex polypeptide protein like (TCP-1) (figure 1), involved in processes of protein modification and translation, were up-regulated in 1 year old ortets, indicating that juvenile plants are more active at protein synthesis and modification level. In contrast 5 year old ortets presented a down-regulation in these genes expression. Additionally, in 5 year old radiata pines genes like histidine-rich glycoproteins (figure 1) were up-regulated, this type of protein have been found to be a component of the cell wall, like histidine-rich extensin in *Zea mays* [9], this is consistent with the increase in cell wall synthesis in older plants.

Thus, this work has provided an initial insight of the pathways and mechanisms that may be involved in the loss of morphogenetic capacity caused by aging in *P. radiata* ortets. Juvenile ortets showed higher expression in proteins related with transcription activators, photosynthesis and carbon and carbohydrate metabolism, while older *P. radiata* ortets showed a down-regulation in the same genes and a increase in genes related to cell wall synthesis, indicating that these might be candidates genes to be markers of the aging and maturation process, results that will be complemented and validated with qRT-PCR and proteome studies.

References

1. Diaz-Sala C, Hutchinson K, Greenwood M: Maturation-related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity. *Physiol Plant* 1996, 97:481-490.

<figure><img src="image_34.png" /><figcaption>**Figure 1 (abstract P62)** Normalized gene expression of: regulator of chromosome condesation (RCC1), granule-bond starch synthase (GBBS1), putative valyl-tRNA synthase, T-complex polypeptide protein (TCP-1), RUBISCO activase, histidine-rich glycoprotein and α-L-fuocosidase *Pinus radiata* genes. Values are means ±SE.</figcaption></figure>
2. Greenwood M, Cui X, Xu F: Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (*Pinus taeda*) stem cuttings. *Physiol Plant* 2001, 111:373-380.

3. Fraga M, Cañal M, Rodríguez R: Phase-change related epigenetic and physiological changes in *Pinus radiata* D. Don. *Planta* 2002, 215:672-678.

4. Valledor L, Jorrín J, Rodríguez J, Meijón M, Cañal M: Combined proteomic and transcriptomic analysis identifies differentially expressed pathways associated to *Pinus radiata* needle maturation. *J Proteome Res* 2010, 9:3954-3979.

5. Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees. *Plant Mol Biol Rep* 1993, 11:113-116.

6. Grulke N, Miller P: Changes in gas exchange characteristics during the life span of giant sequoia: implications for response to current and future concentrations of atmospheric ozone. *Tree Physiol* 1994, 14:659-668.

7. Fredericksen T, Steiner K, Skelly J, Kolb T, Kouterick K: Diel and seasonal patterns of leaf gas exchange and xylem water potentials of different-sized prunus serotina trees. *Forest Sci* 1996, 42:359-365.

8. Hao Y, Macara I: Regulation of chromatin binding by conformational switch in the tail of the Ran exchange factor RCC1. *J Cell Biol* 2008, 182:827-836.

## P63
### Competing MYB networks as switches in primary and secondary metabolism in spruce

Claude Bomal¹*, Isabelle Giguère¹, Sébastien Caron¹, Brian Boyle¹, Élise Fortin¹, Isabelle Duval², Armand Séguin², John MacKay¹

¹Centre d'Étude de la Forêt, Université Laval, Québec (QC), G1V A06, Canada;

²Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec (QC), G1V A06, Canada

E-mail: Claude.Bomal@sfb.ulaval.ca

BMC Proceedings 2011, 5(Suppl 7):P63

**Background:** R2R3-MYB transcription factors are regulatory genes that have been linked to key aspects of plant development and secondary metabolism. They have been implicated in the transcriptional control of phenylpropanoid, and flavonoid metabolic pathways in model plant systems. However, in trees, knowledge is still limited about the role of R2R3-MYBs in such processes. Microarray transcript profiles (9K custom cDNA array) obtained from comparison between wild type and transgenic spruces that constitutively overexpressed *Pinus taeda* MYB1, MYB8 [1], and MYB14 [2] identified putative targets in flavonoid and phenylpropanoid metabolism. Cross-comparison of these data sets identified 70 sequences that were common to the three transgenic backgrounds: interestingly, 66 of these 70 sequences were co-expressed between PtMYB1 and PtMYB8 transgenics but produced opposite profiles with PtMYB14 transgenics. Pairwise comparisons between these transgenic data sets identified 121 additional sequences showing opposite profiles. Predicted annotations and KEGG classification showed that many of the sequences were linked to the metabolism of amino acids and carbohydrates as well as flavonoids, phenylpropanoids, and terpenoids. It was hypothesized that MYB1/MYB8 and MYB14 may be part of competing transcriptional networks for the regulation of primary metabolism and secondary metabolism. The opposite transcriptional responses might also support structural or defensive oriented response, respectively.

**Material and methods:** A qPCR based experimental approach was used to test these hypotheses by evaluating transcript profile of the negatively co-expressed sequences together with MYB1, MYB8 and MYB14. Transcript levels were monitored during a diurnal cycle in three year-old wild type spruces, based on the fact that some genes associated to phenylpropanoid metabolism [3] and stress [4] were reported to follow a diurnal transcript variation. Our analysis also included a set of 10 additional MYBs putatively linked to secondary cell wall deposition [1] and stress oriented response [2]. A Spearman correlation rank test was used to estimate significant correlation (adjusted *P* val ≤ 0.01, FDR 1%) between MYB transcript profiles and selected co-expressed sequences. A functional assay system developed in embryogenic spruce cells was used to screen for possible interactions between transcription factors and promoters of putative target genes.

**Results:** Among the co-expressed sequences tested, five sequences coding for 4CL, DHS2, OMT1, SMT4, and a Lipase thioesterase displayed transcript profile that positively correlated with those of PgMYB1, PgMYB8

but negatively with those of PgMYB14 and PgMYB15, the closest homologue of PgMYB14 [2]. Interestingly, these oppositions were observed in secondary xylem differentiating tissues but not in bark/ phloem. Significant correlations were also observed between the co-expressed sequences tested and the others MYBs. A separate analysis of large scale expression profiles for spruce transcription factors gave some additional evidences that these putative competing MYB networks may be tissue-preferential. Transient expression results obtained with our functional assay system further suggested interactions between these MYBs and some of their proposed targets. More extensive testing is in working progress.

**Conclusions:** The results of the present study strongly suggest that putative MYB networks may compete to allow rapid metabolic switch within wood forming tissue in order to direct metabolic flux towards specific aspects of primary and secondary metabolism for structural, or defensive-oriented response.

## References

1. Bomal C, Bedon F, Caron S, Mansfield SD, Levasseur C, Cooke JEK, Blais S, Tremblay L, Morency MJ, Pavy N, Grima-Pettenati J, Séguin A, MacKay J: Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. *J Exp Bot* 2008, 59:3925-3939.

2. Bedon F, Bomal C, Caron S, Levasseur C, Boyle B, Mansfield SD, Schmidt A, Gershenzon J, Grima-Pettenati J, Séguin A, MacKay J: Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses. *J Exp Bot* 2010, 61:3847-3864.

3. Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, Zhu T, Wang X, Kreps JA, Kay SA: Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. *Science* 2000, 290:2110-2113.

4. Walley JW, Coughlan S, Hudson ME, Covington MF, Kaspi R, Banu G, Harmer SL, Dehesh K: Mechanical stress induces biotic and abiotic stress responses via a novel cis-element. *PLoS Genet* 2007, 3:e172.

## P64
### Expression of auxin carrier genes during adventitious rooting in *Eucalyptus globulus*

Arthur Fett-Neto*, Marcia De Almeida, Carolina Ruedell Gabriela Pereira Center for Biotechnology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

E-mail: fettneto@cbiot.ufrgs.br

BMC Proceedings 2011, 5(Suppl 7):P64

**Background:** *Eucalyptus globulus* and its hybrids are important for the cellulose and paper industry, mainly due to their relatively low lignin content. However, rooting of cuttings of this species is often recalcitrant and exogenous auxin application is necessary for adventitious root development. Auxin plays a central role in rooting capacity, which is particularly affected by its endogenous content and transport rate. The shoot apex is a major source of endogenous auxin, which is mainly transported by both influx (*AUX1*) and efflux (*PIN*) carriers in a specific basipetal active transport through the vascular parenchyma in stems. As part of a larger study to investigate the causes of low rooting in *E. globulus* microcuttings without exogenous auxin, we evaluated the expression profiles of *AUX1* and *PIN1* during the process of adventitious rooting using qPCR.

**Material and methods:** *E. globulus* in vitro tip microcuttings obtained from 14 week-old seedlings were submitted to a culture system consisting of a two-step protocol: an initial step of induction, which lasted 96 h (induction medium composition: 0.3x MS salt concentration, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ inositol, zero (control) or 10 mg l⁻¹ indolyl-butyric acid (IBA – root promoting auxin), equivalent to 49.3 µM, 30 g l⁻¹ sucrose and 6 g l⁻¹ agar, followed by a formation step (same composition of induction medium, but devoid of auxin and supplemented with 1 g l⁻¹ activated charcoal). The expression analysis of the selected genes was monitored along the rooting process and the harvest of microcuttings for RNA extraction was done at 6, 12, 24, 48 and 96 h of exposure to induction medium and 24 and 48 h after transfer to formation medium (formation step), for both treatments (with and without auxin in the first step). For the formation step harvest, the microcuttings remained for 96 h in the induction medium before transfer to formation medium. Total RNA was extracted, and the first strand cDNA synthesis was performed for all of the samples starting from about 500 ng total RNA, using oligo-dT primers and
reverse transcriptase M-MLV (Invitrogen) in a final volume of 20 µl. The final cDNA products were diluted 50-fold in RNase-free distilled water prior to use in qPCR. The analysis was carried out using specific primers for *Arabidopsis thaliana* orthologue genes in eucalypt and both *Histone H2B* (*H2B*) and *Alpha-Tubulin* (*TUA*) genes were used as references [1]. The data were analysed with the comparative Ct method [2].

**Results and conclusion:** The gene encoding the auxin influx carrier (*AUX1*) did not show differences in expression profile between treatments (with and without exogenous auxin), suggesting that *AUX1* is not critical to the process of adventitious rooting promoted by exogenous auxins in microcuttings. This would be in line with the fact that the rate of endogenous auxin transport is probably not limiting under exogenous auxin supply. The auxin efflux carrier gene encoding *PIN1* showed an expression increase during the first 24 hours of the induction step in microcuttings exposed to exogenous auxin when compared with the control treatment (without exogenous auxin supply). This result seems to indicate a requirement of *PIN1* to redistribute and perhaps concentrate auxin, possibly IBA-derived IAA (indolyl-3-acetic acid), in specific areas of the base of microcuttings, in order to allow root development. Although IBA is a natural auxin recognized as an IAA precursor, its use as exogenous auxin instead of IAA, a common practice in clonal propagation by cuttings, may have also involved the expression of other sets of recently described IBA-specific transporters [3]. In conclusion, despite the unusual exogenous auxin entrance pathway into the microcutting compared to the endogenous auxin fluxes, *PIN1* likely takes part in the process of auxin concentration required to program founder cells involved in the establishment of new root meristems.

**Acknowledgements:** Funding for this investigation was provided by the Brazilian agencies National Council for Scientific and Technological Development (CNPq) and Rio Grande do Sul State Foundation for Research Support (Fapergs).

**References**

1. De Almeida MR, Ruedell CM, Ricachenevsky FK, Sperotto RA, Pasquali G, Fett-Neto AG: Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in *Eucalyptus globulus* Labill. *BMC Molecular Biology* 2010, 11:73.

2. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. *Methods* 2001, 25:402-408.

3. Strader LC, Bartel B: Transport and metabolism of the endogenous auxin precursor Indole-3-Butyric Acid. *Molecular Plant* 2011, 1-10.

P65

**Genome characterization of a *Eucalyptus* natural mutant**

Maria CP Fuchs¹*, Juliana C Lourenço¹, Evandro V Tambarussi²,
Tânia M Bortoloto¹, Shinitiro Oda³, Fábio TS Nogueira¹, Celso L Marino¹
¹Departamento de Genética, Instituto de Biociências, Universidade Estadual
Paulista (UNESP), Distrito de Rubião Jr s/n CEP 18618-000, Botucatu (SP),
Brazil; ²Departamento de Genética, Escola Superior de Agricultura “Luiz de
Queiroz” (ESALQ), Universidade de São Paulo (USP), Avenida Pádua Dias, 11
CEP 13418-900, Piracicaba (SP), Brazil; ³Empresa Suzano Papel e Celulose SA,
Av. Dr. José Lembo, 1010, CEP 18207-780, Itapetininga (SP), Brazil
E-mail: mceciliapf@yahoo.com.br

*BMC Proceedings* 2011, **5**(Suppl 7):P65

**Background:** *Eucalyptus* genetic improvement is time-consuming mainly because its long time to reach reproductive maturity along with its mixed mating system [6]. As a result, the production of pure lines as in maize is impracticable. The heterozygous status hides deleterious recessive genes that, when in homozygosity can reduce seedling survival due to physiological changes, resulting in considerable loss in seedling production [6]. Therefore, it would be important to identify the gene(s) associated with inbreeding depression. There are very few studies involving the characterization of deleterious recessive genes in Eucalyptus. We detected an anomaly in the offspring obtained from a controlled cross of *Eucalyptus grandis*. The anomaly appears in a ratio of one abnormal to three normal seedlings, suggestive of a Mendelian segregation. Based on the segregation ratio, we hypothesized that the character is controlled by a single gene, with a homozygous genotype for the recessive allele showing abnormal seedlings. The parents showed normal phenotype and they have no relationship, excluding the possibility of inbreeding depression due to

identity by descent. The abnormal seedlings die in a few months and show different characters: high shoot branching, height reduction, leaf area reduction, and changes in leaf shape (Figure 1). In this scenario, we aimed to characterize the genomic and genetic causes of the observed anomaly.

**Methods:** The full-sib individuals generated at Suzano Papel and Celulose SA company were divided into normal and abnormal seedlings. The ratio between normal and abnormal seedlings was tested by chi-square analysis. DNA was extracted according to Ferreira and Grattapaglia [1]. The DNA concentration was quantified using a spectrophotometer (Thermo Scientific NanoDrop™ 1000), and subjected to electrophoresis in 1% agarose gel and stained with ethidium bromide. DNA from ten normal individuals were randomly selected and mixed, creating the normal bulk, and the same was performed to create the abnormal bulk. The bulks were amplified with random primers (kits AD, AE, AF, AJ, and AK – Operon Technologies, Inc.). Amplicons were submitted to electrophoresis in 1% agarose gel stained with ethidium bromide. Polymorphic markers detected by RAPD were converted to SCAR (Sequence Characterized Amplified Regions) markers. DNA fragments from polymorphic marker were purified from agarose gel using the illustra GFX PCR DNA (GE Healthcare), cloned into vector pGEM-T Easy (Promega) and inserted into UltraMAX DH5α-FT competent *Escherichia coli* cells (Life Technologies). DNA was sequenced in an ABI3100 Genetic Analyzer (Applied Biosystems). SCAR primers were designed based upon the above mentioned sequencing and further used to amplify DNA fragments from bulks and six individuals: three anomalous, one individual from normal bulk; and two normal individuals of unrelated populations (BAC and Brasuz). Amplicons of the six individuals were purified, cloned into vector pGEM-T Easy (Promega) and inserted into DH5α-competent *E. coli* cells (Life Technologies). Six colonies from each individual were amplified with the SCAR primers. Purified product of amplification was

<figure><img src="image_35.png" /><figcaption>Figure 1(abstract P65) Normal (left) and abnormal (right) seedlings.</figcaption></figure>
sequenced by ABI3100 Genetic Analyzer (Applied Biosystems). SCAR derived sequences were compared among themselves and with the eucalypt genome (Phytozome v7.0 database - http://www.phytozome.net/) using BLAST. The genomic region identified was analyzed by FGENESH tool (Softberry, Inc. - http://www.softberry.com) to find predicted genes in the region. Predicted genes were translated by EXPASY translate tool (http://expasy.org), and the protein analyzed by PFAM database (http://pfam.sanger.ac.uk) for domain identification.

**Results:** Chi-square analysis confirmed Mendelian segregation of 3 normal: 1 anomalous seedlings in the tested progeny. The data indicate that the anomaly is a monogenic character that manifests when the deleterious recessive allele is in homozygosity. Among the random primers tested only one showed polymorphism with the segregation pattern. This marker appears in all abnormal and in 31% of normal individuals. The RAPD marker was converted into a SCAR marker and its segregation pattern confirmed. All abnormal individuals and 22.2% of normal individuals showed the marker. Sequencing of the SCAR marker was done to identify the possible genomic region where it is linked and detected a polymorphism of two base pair between anomalous (mutant) and normal individuals. Our *in silico* analysis showed the polymorphism in an intergenic region, localized between two protein-coding with a Bet v1-type domain belonging to the PR10 family (Pathogenesis-related protein 10). In general, this is associated with plant defense function in response to biotic and abiotic stresses [3-5]. However, there are reports that PR10 has important roles in biological processes [4,5].

**Conclusions:** According to our results, we propose that the anomalous character might be caused by a major effect gene. The marker found is related with the recessive; therefore, normal individuals with the marker are likely heterozygous. The polymorphism detected in the SCAR sequence, suggests that the mutation might be related to the two base substitution observed. However, at this point, it is unclear whether these base substitutions are involved affecting close genes (such as the identified PR10 proteins) or any other unknown genomic region. Expression analysis of the PR10-coding genes might provide clues to answer this biological question.

## References

1. Ferreira ME, Grattapaglia D: Introdução ao uso de marcadores moleculares em análises genéticas. Brasília: Embrapa-Cenargen, 3 1998, 220.

2. Hoffmann-Sommergruber K, Vanek-Krebitz M, Radauer C, Wen J, Ferreira F, Scheiner O, Breiteneder H: Genomic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-related proteins share intron positions. *Gene* 1997, 197:91-100.

3. Poupard P, Brunel N, Leduc N, Viémont J-D, Strullu D-G, Simoneau P: Expression of a Bet v 1 homologue gene encoding a PR 10 protein in birch root: induction by auxin and localization of the transcripts by *in situ* hybridization. *Aust. J. Plant Physiol* 2001, 28:57-63.

4. Walter MH, Liu J-W, Wünn J, Hess D: Bean ribonuclease-like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression. *Eur. J. Biochem* 1996, 239:281-293.

5. Zobel B, Talbert J: Applied forest tree improvement. New York: John Wiley and Sons 1984, 505.

# P66
## Apparent coordination of isocitrate dehydrogenase and glutamate decarboxylase expression in early stages of tree development

Juan Jesús Molina-Rueda¹, María Belén Pascual²,
Lara Silvia Jiménez-Bermúdez³, Francisco Miguel Cánovas²,
Fernando Gallardo²*

¹Present address: Rutgers University, Dpt Biological Sciences, Newark, New Jersey, USA; ²Universidad de Málaga, Dpt. Biología Molecular y Bioquímica, Málaga, Spain; ³Universidad de Málaga, SCAI, Servicio de Cultivo de Plantas, Málaga, Spain

E-mail: fgallardo@uma.es

BMC Proceedings 2011, 5(Suppl 7):P66

The biosynthesis of 2-oxoglutarate and glutamate are key steps in the biosynthesis of nitrogen compounds and plant development. The reaction catalyzed by cytosolic isoenzyme of NADP+-linked isocitrate dehydrogenase (IDH) is also considered as the main route in the production of 2-oxoglutarate. According to its expression pattern during development, IDH is also involved in other, yet unknown, processes [1,2].

In addition to the importance of glutamate in the biosynthesis of nitrogen compounds, glutamate also serves as precursor of GABA, a molecule that is currently considered as a signal in higher plants. GABA is produced by the action of glutamate decarboxylase (GAD), a cytosolic enzyme that is regulated by Ca2+/calmodulin and pH. In contrast to IDH, that it is encoded by just one gene in most of plant genomes [2], GAD is encoded by a small family of nuclear genes [3]. The expression of IDH and GAD has been investigated during the differentiation of hypocotyl and stem in tree species. Our results indicate a coordination of the expression of IDH and GAD in developmental processes suggesting a role for 2-oxoglutarate supply and GABA synthesis during early stages of organ differentiation in trees.

## References

1. Palomo J, Gallardo F, Suárez MF, Cánovas C: Purification and characterization of NADP+-linked isocitrate dehydrogenase from Scots pine. *Plant Physiol* 1998, 118:617-626.

2. Pascual MB, Molina-Rueda JJ, Cánovas FM, Gallardo F: Spatial distribution of cytosolic NADP+-isocitrate dehydrogenase in pine embryos and seedlings. *Tree Physiol* 2008, 28:1773-1782.

3. Molina-Rueda JJ, Pascual MB, Cánovas FM, Gallardo F: Characterization and developmental expression of a glutamate decarboxylase from maritime pine. *Planta* 2010, 232:1471-1483.

# P67
## NAC regulation of embryo development in conifers

Emma Larsson¹, Folke Sitbon, Jens Sundström, Sara von Arnold

Swedish University of Agricultural Sciences, Department of Plant Biology and Forest Genetics, P.O. Box 7080, 75007 Uppsala, Sweden

E-mail: emma.larsson@vbsg.slu.se

BMC Proceedings 2011, 5(Suppl 7):P67

**Background:** In most gymnosperms the cotyledons develop as a crown surrounding the incipient shoot apical meristem (SAM), which maintains the radial symmetry of the plant throughout embryogenesis. This is in contrast to the *Arabidopsis* embryo in which a symmetry-breaking event (from radial to bilateral symmetry) is associated with the emergence of the SAM and the two cotyledons [1]. We have previously shown that the differentiation of cotyledons and the establishment of the SAM and the root apical meristem (RAM) in somatic embryos of Norway spruce (*Picea abies*) is dependent on polar auxin transport (PAT) [2]. In *Arabidopsis* PAT restricts the expression of several genes belonging to the large plant specific NAC family of transcription factors [3-5], which in turn induce developmental processes involved in bootouthoot and root formation. Members of the NAC family have a conserved DNA-binding domain in the N-terminal region known as the NAC domain, and a more diverse domain in the C-terminal region, proposed to be responsible for the trans-activation capacity of the protein [6]. Despite the diversity of the NAC gene family, and the importance of NAC proteins in fundamental processes, no NAC genes have to our knowledge been characterized in conifers.

**Methods:** Phylogenetic analyses of NAC domain containing genes from *Picea glauca*, *Arabidopsistaliana*, *Medicago truncatula* and *Phycomitrella patens* were performed using PAUP* and MrBayes. Full-length cDNAs of two NAC genes were isolated from maturing somatic embryos of Norway spruce and cloned using standard protocols. The promoters were amplified using the Genome Walker™ kit (Clontech). The motif discovery tool MEME was used to find conserved motifs in the C-terminal regions and in the promoters of the NAC sequences from different species. The expression pattern of the two isolated NAC genes was analyzed in Norway spruce somatic embryos using quantitative real-time PCR. Their expression was also analyzed in embryos that had been treated with the PAT inhibitor NPA as described earlier [2]. Promoter::GUS fusions were inserted into embryogenic cultures of Norway spruce using *Agrobacterium* mediated transformation.

**Results and discussion:** We identified several different NAC sequences from white spruce in GenBank and deployed them to different sub-classes within the NAC gene family. Two full-length cDNAs (*gene 1* and *gene 2*) were cloned from Norway spruce. Both genes have elements in their promoter region which are significant for specific NAC genes. Furthermore, *gene 1* harbors previously characterized motifs that have been shown to have functional importance in NAC genes in *Arabidopsis*.
The relative expression of *gene 1* increased as early embryos differentiated, and remained at a high level until separated cotyledons were clearly visible. However, the up-regulation was blocked in embryos that formed fused cotyledons and lacked distinct SAM and RAM after being treated with NPA. The relative expression of *gene 2* was high in proembryogenic masses and early differentiating embryos but decreased as the embryos developed and matured. Interestingly the down-regulation occurred at about the same time as the cotyledons were delineated. The expression of *gene 2* was not dependent on PAT. Spatial expression analysis using promoter::GUS fusions revealed that the spruce genes have specific expression patterns, distinct from the expression patterns of their most closely related *Arabidopsis* counterparts.

**Conclusion:** Taken together, our results show that NAC genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approximately 300 million years ago. However, through evolution the expression of closely related genes has diversified leading to induction of distinct developmental programs in angiosperms and gymnosperms.

**References**

1. Chandler J: Cotyledon organogenesis. *J Exp Bot* 2008, 59:2917-2931.

2. Larsson E, Sitbon F, Ljung K, von Arnold S: Inhibited polar auxin transport results in aberrant embryo development in Norway spruce. *New Phytol* 2008, 177:356-366.

3. Aida M, Vernoux X, Furutani M, Traas J, Tasaka M: Roles of PIN-FORMED1 and MONOPTEROS in pattern formation of the apical region of the *Arabidopsis* embryo. *Development* 2002, 129:3965-3974.

4. Furutani M, Vernoux T, Traas J, Kato T, Tasaka M, Aida M: PIN-FORMED1 and PINOID regulate boundary formation and cotyledon development in *Arabidopsis* embryogenesis. *Development* 2004, 131:5021-5030.

5. Xie Q, Frugis G, Colgan D, Chua N-H: *Arabidopsis* NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. *Genes Dev* 2000, 14:3024-3036.

6. Taoka K, Yanagimoto Y, Daimon Y, Hibara K, Aida M, Tasaka M: The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins. *Plant J* 2004, 40:462-473.

**Background:** Bolting is regarded as the initiation of reproduction stage in the growth and development of an Arabidopsis plant, when a set of floral integrator genes activate the expression of floral meristem identity genes *LFY* and *AP1* to initiate flowering transition[1,2]. However, how the expression of key genes, such as *AP1*, responds of diverse signals during flower development remains largely unknown. Here we have obtained an inbred line exhibiting an abnormal stem/inflorescence and flower development.

**Methods:** The morphological variations were carefully observed visually or with a Zeiss SteREO Discovery V8 stereomicroscope. Hybridization of GeneChip arrays was done in an Affymetrix Hybridization Oven 640 following the manufacturer's protocol (Affymetrix) and expression levels in seedlings and flower buds were calculated from Affymetrix intensity data. Real-time PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Shanghai, China) with SYBR GreenPCR Master Mix (Applied Biosystems) as the fluorescence source.

**Results:** The inbred line exhibits a flower phenotype similar to the *ap1*, such as homeotic conversion of sepals (first whorl) to leaf-like, petals often absent (second whorl) and complete flower-to-inflorescence conversions (Figure 1, B). However flower meristem was replaced by emerging inflorescence meristems thus leading to a stacked stem/ inflorescences before final flowering (Figure 1, D). Position and degree of stacked stem/inflorescences are varied differently (Figure 1, E and F). Microarray and quantitative real-time PCR analysis revealed that the expression of *AP1* was significantly reduced, while the expression of its interacting genes *TERMINAL FLOWER 1* (*TFL1*), *OVEREXPRESSION OF CONSTANS (SOC1)*, *AGAMOUS*-like 24 (*AGL24*), *SEPALLATA* (*SEP*) and upstream genes *FLOWERING LOCUS M* (*FLM*) were increased in flower buds (Figure 2).

*AP1* sequence analysis showed that the promoter, coding sequences and intron splice sites of *AP1* genomic sequence in this inbred line were unchanged comparing to that in wildtype, suggesting the complexity in the regulation of *AP1* in the line. Therefore these synthetic contributions caused the development of this unique phenotype. For instance, *TFL1* was found to be highly expressed, and this gene can negatively regulate *AP1* and specify inflorescence meristem identity leading to a delay of floral meristem formation[3]. On the other hand, the low levels of *AP1* in flower buds cannot repress expression of *AGL24* and *SOC1*, which promote inflorescence fates rather than flower formation in the meristem and result in more abundant and longer inflorescences[4]. This expression variation of these genes is subjected to a threshold[4], leading to an ON/OFF expression pattern of the master regulatory gene(s) (like *AP1*) to specify a floral or a stem/inflorescence meristem.

**Conclusions:** The inbred line identified in this study is phenotypically similar to *ap1* mutants with noticeable deviations. The abnormal stem/ inflorescence of the inbred line was mainly caused by significant

**P68**

An *Arabidopsis thaliana* (Ler) inbred line exhibiting stacked stem/ inflorescences mainly due to the reduced *AP1* expression

Xiaoli Qi¹, Yao Jiang², Fang Tang², Shu-Tang Zhao², Meng-Zhu Lu²*

¹College of Life Sciences, Jiamusi University, Jiamusi 154007, China; ²State

Key Lab of Forest Genetics and Tree Breeding, Chinese Academy of Forestry,
Beijing 100091, China

E-mail: lumz@caf.ac.cn

BMC Proceedings 2011, 5(Suppl 7):P68

<figure><img src="image_36.png" /><figcaption>**Figure 1(abstract P68)** Phenotype of Wt and An inbred line.A, C: Wt; B, D-F: An inbred line).</figcaption></figure>
<figure><img src="image_37.png" /><figcaption>**Figure 2 (abstract P68)** Expression of flowering regulatory genes in flower buds using qRT-PCR.</figcaption></figure>

downregulation of *AP1*, but also is attributable to crosstalk among key genes like *TFL1*, *AGL24*, *SOC1*, etc. to control the transition of vegetative growth to the flowering phase. The inbred linemerits further molecular characterization to understand better the regulatory molecular network.

**Acknowledgements:** Supported by the national Natural Science Foundation of China (30771697), the Research Foundation of Jiamusi University (S2010-53) and the Education Department of Heilongjiang Province (12513091).

References

1. Blazquez M, Weigel D: Integration of oral inductive signals in *Arabidopsis*. *Nature* 2000, **404**:889-892.

2. Borner R, Kampmann G, Chandler J, Glei ner R, Wisman E, Apel K, Melzer S: *A MADS domain gene involved in the transition to flowering in Arabidopsis*. *Plant J* 2000, **24**:591-599.

3. Liljegren S, Gustafson-Brown C, Pinyopich A, Ditta G, Yanofsky M: *Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate*. *The Plant Cell* 1999, **11**:1007-1018.

4. Liu C, Xi W, Shen L, Tan C, Yu H: Regulation of floral patterning by flowering time genes. *Dev Cell* 2009, **16**:711-722.

5. Raj A, Rifkin S, Andersen E, Van Oudenaarden A: Variability in gene expression underlies incomplete penetrance. *Nature* 2010, **463**:913-918.

P69

Identification of GPAT acyltransferases in cork oak

Liliana Marum¹, Andreia Miguel, Pinto C Ricardo, Célia Miguel
Instituto de Tecnologia Química e Biológica-Univ. Nova de Lisboa; Instituto
de Biologia Experimental e Tecnológica (ITQB-UNL;IBET), Av. República, EAN,
2780-157 Oeiras, Portugal
E-mail: marum@itqb.unl.pt

BMC Proceedings 2011, **5**(Suppl 7):P69

**Background:** Acyltransferases are enzymes with an important role in the synthesis of both cutin and suberin which are part of the lipophilic barriers, such as epidermis and periderm that protect terrestrial plants against water loss and other external aggressions. During secondary growth in woody plants such as cork oak (*Quercus suber* L.), the epidermis is replaced by a suberized periderm that includes the phellem (cork), phellogen (cork cambium) and phelloderm tissues. In *Q. suber* the successive formation of phellem following removal at periodic intervals (every 9 years) allows for exploitation of cork oak on a sustainable basis. The main component of cork (45-50%) is suberin, a complex polymer comprising both aliphatic and aromatic domains and associated waxes [1,2]. Despite the physiological importance of suberin, its biosynthetic pathway as well as its deposition remains largely unknown. Since cork oak is a unique species among terrestrial plants due to its remarkable capacity for cork production, it is expected that suberin biosynthesis and deposition are tightly controlled mechanisms.

As a first step to start unraveling these control mechanisms we intend to identify and characterize genes coding for the acyltransferases of the GPAT (glycerol-3-phosphate acyltransferase) family, involved in suberin and cutin synthesis in cork oak. Two ESTs highly similar to *GPAT5* (EE 743864 and EE 743865) and one EST (EE743668) highly similar to *GPAT4* shown to be strongly up-regulated in the suberin-rich phellem of cork oak tree (*Q. suber*) were first identified by Soler et al. [3].

**Material and methods:** In this work, phellem tissues from small branches with increasing age (1 to 7 years old) were harvested from cork oak and holm oak (a related but cork non-producing species) at the Instituto Superior de Agronomia (Portugal). Tissues collected during different growth periods were also used for analysis: samples collected during a period of high phellogen activity (April – June, 2009 and 2010) and samples collected during the inactive growth period (January, 2010). Total RNA was successfully extracted from these tissues using a protocol described by Reid et al. [4], with minor modifications. cDNA was synthesized using standard procedures and 5' - and 3' -RACE are being performed in order to determine the full-length of putative GPAT coding sequences from *Q. suber* transcriptome. The expression level of *GPAT4* and *GPAT5* genes was assessed by quantitative RT-PCR in two different seasonal stages (April and June) in periderm cells from 3 year old branches of *Q. suber*. The Cp values were converted into relative quantities, using the formula, $Q=E^{ΔCP}$, where E (the efficiency of the gene amplification for each primer pair) was calculated using the Real-time PCR Miner algorithm.

**Results and conclusions:** A cDNA fragment with 1265bp of the putative *Qs_GPAT5* was obtained. The predicted amino acid sequence displays the glycerol-3-phosphate acyltransferase (PLN02499) conserved domain. Based on a database search we have identified putative orthologs of *AtGPAT5* in *P. trichocarpa* (*Pt_8s_AT*, accession number 002312108; *Pt_10s_AT*, accession number 2315213) and *R. communis* (*Rc_ERGPAT*, accession number 2531580) genome, which show high similarity to the putative *Q. suber* *GPAT5* gene. The *Rc_ERGPAT* is most similar to the *Qs_GPAT5* with an identity of 83% at the amino acid level.

The expression profiles of *GPAT4* and *GPAT5* were successfully analysed during phellem differentiation in periderm collected from cork oak tree. The relative expression level of *GPAT4* gene was similar in April and June. However, the relative expression level of *GPAT5* gene was higher in June which corresponds to a period of higher phellogen activity, when compared to April.

Further information on the expression of these genes in several tissues of cork oak under different developmental stages and stress conditions will also be gathered as a result of the recent effort of the Portuguese research community involved in the transcriptome sequencing of cork oak. With this work we expect to contribute to elucidate basic aspects of the molecular networks involved in cork formation.

**Acknowledgements:** Fundação para a Ciência e Tecnologia (FCT) is acknowledged for support through the project SOBREIRO/0029/2009 and the grant SFRH/BPD/47679/2008.

References

1. Graça J, Pereira H: The periderm development in *Quercus suber*. *IAWA Journal* 2004, **25**:325-335.

2. Silva SP, Sabino MA, Fernandes EM, Correlo VM, Boesel LF, Reis RL: *Cork: properties, capabilities and applications*. *International Materials Reviews* 2005, **50**:345-365.

3. Soler M, Serra O, Molinas M, Huguet G, Fluch S, Figueras M: A Genomic Approach to Suberin Biosynthesis and Cork Differentiation. *Plant Physiol* 2007, **144**:419-431.

4. Reid KE, Olsson N, Schlosser J, Peng F, Lund ST: An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. *BMC Plant Biology* 2006, **6**:27.

P70

Identification of a putative molecular regulator of cork cambium

Andreia Miguel¹*, Pinto C Ricardo¹, Brian Jones², Célia Miguel¹

¹Instituto de Tecnologia Química e Biológica (ITQB-UNL; Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157 Oeiras, Portugal; ²A20 John Woolley Building, 2006 University of Sydney – Australia / UPSC, Sweden

E-mail: andreiamiguel@itqb.unl.pt

BMC Proceedings 2011, **5**(Suppl 7):P70

**Background:** Cork oak (*Quercus suber*) is a long-living species of the Fagaceae family that highly contributes for the economy of several countries such as Portugal. It is the only plant species with a phellogen capable of a sustainable production of cork with properties suited for industry applications. Phellogen, or cork cambium, usually initiates in the
subepidermis and differentiates during the first year of growth [1]. Its meristematic activity gives rise to phellem (cork) cells to the outside and to phelloderm to the inside.

It is likely that genes involved in the regulation of other plant meristems such as shoot apical meristem and vascular cambium are also involved in the regulation of phellogen. Based on its role in other meristems, and on specific expression patterns in poplar stems, we hypothesize that *SHORT-ROOT (SHR)*, a transcription factor from the GRAS family, may be involved in the regulation of phellogen. This gene has been well described in the *Arabidopsis* root where it plays a key role in the radial patterning and in regulating the specification of the root stem cell niche [2-4]. Recently, its involvement in the root vascular system to control patterning processes [5] and in the control of proliferative cell division in developing leaves [6] has also been reported. In this work we report the cloning of *SHR* from cork oak transcriptome and the characterization of *SHR* expression patterns in the stem with the aim of investigating its putative function in the phellogen.

**Materials and methods:** Through a search in public databases we have retrieved putative orthologs of *Arabidopsis* *SHR* gene from species with a sequenced genome including *Populus trichocarpa*, *Vitis vinifera* and *Medicago truncatula*. Based on multiple alignments at the cDNA and deduced aminoacid sequence levels, we have designed degenerate primers in order to clone and characterize *SHR* from cork oak transcriptome. The amplified PCRfragment was inserted into pCR2.1 (Invitrogen) and sequenced to determine the percent identity to known *SHR* gene sequences.

To analyze *SHR* expression patterns poplar is being used as a model tree species. Transgenic poplar plants carrying the *SHR* promoter fused to the *GUS* reporter gene have been previously generated. Stem sections collected from 1-2 year-old transgenic plants have been subject to the histochemical GUS assay and embedded in Technovit 7100 resin for detailed analysis of gene expression localization under the microscope.

Characterization of *SHR* transcript abundance in cork oak developing stems taking into account different developmental stages and collection seasons has also been performed.Transcript abundance was analysed in stem tissues from the first to the third year of growth collected between January and October from a cork oak tree in order to cover periods of active growth and dormancy. Reproduction cork was collected in July which is the period during which the debarking process is performed from cork producer trees. RNA was extracted according to a protocol described by Reid *et al.* (2006) and cDNA was successfully synthesized using the Reproductor High Fidelity cDNA Synthesis Kit (Roche). Quantitative RT-PCR was performed with LightCycler LC480 (Roche).

**Results:** While *Arabidopsis* has one *SHR* gene sequence in its genome, at least two additional *SHR*-like sequences are present in the poplar genome. Also, more than one *SHR*-like sequence seems to be present in the cork oak transcriptome and, at the moment, one of those sequences, *QsSHR1*, consisting of 1455bp and showing strong homology to the *Arabidopsis* *SHR* gene, was identified, and cloned. This putative *SHR*-like gene from cork oak was found to be expressed in stems at different developmental stages as well as in reproduction cork. Furthermore, evidence of specific expression of one of the poplar *SHR*-like sequences has been found in the phellogen and lenticels of stems from transgenic poplar carrying a promoter fusion to the *GUS* reporter gene.

**Conclusions:** *SHR*-like genes may be involved in even more developmental processes than previously thought. It seems plausible that in woody species such as poplar and cork oak, additional copies of *SHR* may have evolved to acquire new functions. We expect that these results, as well as results from further ongoing studies, will contribute to elucidate the putative involvement of *SHR* in the regulation of phellogen. Additionally, these studies may provide tools to be used in future strategies aiming at the improvement of cork production from cork oak.

**Acknowledgements:** Financial support provided by FCT through project PTDC/AGR-GPL/098369/2008 and grant SFRH/BD/44474/2008.

## References

1. Graça J, Pereira H: The Periderm Development in *Quercus suber*. *IAWA Journal* 2004, 25(3):325-335.

2. Benfey PN, *et al*: Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis. *Development* 1993, 119:57-70.

3. Helariutta Y, *et al*: The SHORT-ROOT Gene Controls Radial Patterning of the *Arabidopsis* Root through Radial Signalling. *Cell* 2000, 101(5):555-567.

4. Nakajima K, *et al*: Intercellular movement of the putative transcription factor *SHR* in root patterning. *Nature* 2001, 413:307-311.

5. Yu N, Lee SA, *et al*: Characterization of SHORT-ROOT Function in the *Arabidopsis* Root Vascular System. *Mol Cells* 2010, 30:113-119.

6. Dhondt S, Coppens F, *et al*: SHORT- ROOT and SCARECROW Regulate Leaf Growth in Arabidopsis by Stimulating S-Phase Progression of the Cell Cycle. *Plant Physiology* 2010, 154:1183-1195.

## P71

### Molecular characterization of pine embryogenesis: pursuing the role of a putative non-specific lipid-transfer protein

Marta Simões, Andreira Rodrigues, José de Vega Bartol, Raissa Santos, Célia Miguel*

IBET / ITQB-UNL, Av. República (EAN), 2780-157 Oeiras, Portugal

E-mail: cmiguel@itqb.unl.pt

BMC Proceedings 2011, **5**(Suppl 7):P71

**Background:** Embryogenesis in Gymnosperms presents unique characteristics when compared to Angiosperm embryogenesis. However, little is known about the molecular regulation of embryo development in Gymnosperms because most studies have been conducted in Angiosperm model species. Due to the economic relevance of some forest species included in this group of plants, namely pines, efforts have been made worldwide for the establishment of efficient clonal propagation methods, of which somatic embryogenesis is an example. A deeper knowledge of the molecular players regulating zygotic embryo development in pines will likely contribute to a better understanding and control over somatic embryo development *in vitro*.

We have previously performed a global gene expression analysis during maritime pine (*Pinus pinaster*) embryo development [1] and have selected a set of transcripts for further characterization based on their expression profile. One of such transcripts (*PpAAI-LTSS1*) putatively encodes a protein of the Alpha-Amylase Inhibitors (AAI)/Lipid Transfer (LT)/Seed Storage (SS) protein family, non-specific lipid-transfer protein (nsLTP)-like subfamily. The AAI-LTSS1 family of proteins is unique to higher plants and includes cereal-type alpha-amylase inhibitors, lipid transfer proteins, seed storage proteins, and similar proteins. Proteins in this family are known to play important roles, in defending plants from insects and pathogens, lipid transport between intracellular membranes, nutrient storage, as well as in developmental processes. *PpAAI-LTSS1* is up regulated in zygotic embryos at a pre-cotyledonary stage of development [2]. The aim of this work was to characterize in detail the expression patterns of *PpAAI-LTSS1* along embryo development and its upstream regulatory sequence.

**Materials and methods:** Zygotic embryos were isolated from immature cones and separated into five groups according to the developmental stage/collection date. The embryo staging was based on the system of Pullman and Webb [3] as described by Gonçalves *et al.* [4]. In each collection date, one or several consecutive embryo developmental stages were pooled as follows: Day 0 (stages T0, T1 and T2); Day 5 (stages T3 and T4); Day 11 (stage T4B); Day 15 (stage T5) and Day 25 (stage T7). Quantitative real-time RT-PCR (RT-qPCR) was performed using a iQ5 Real-Time PCR detection system (BioRad) to quantify *PpAAI-LTSS1* transcripts in each of the five embryo groups to confirm differential expression along embryo development and developmental stage showing maximum up-regulation. Cloning of the upstream genomic region putatively corresponding to the promoter region, as well as localization of this transcript within the embryo tissues using in situ hybridization, are also being pursued.

**Results:** Confirming previous results using microarrays, *PpAAI-LTSS1* transcript showed a peak of expression in developmental stages corresponding to pre-cotyledonary and early cotyledonary embryos decreasing onwards until embryo maturation. Preliminary *in situ* localization results point to specific expression of this gene in suspensor tissues but additional experiments are still running to confirm these observations. In addition, a 900bp genomic DNA fragment that putatively belongs to the promoter region of this gene has been cloned. The analysis of this DNA sequence revealed the presence of several conserved promoter motifs and no significant homology to any sequence present in public databases has been found. Further experiments will allow us to isolate the complete promoter sequence to be used in functional analysis studies.

**Conclusion:** The transcript *PpAAI-LTSS1* putatively coding for a protein of the Alpha-Amylase Inhibitors (AAI)/Lipid Transfer (LT)/Seed Storage (SS) protein family may play an important role during embryo development in maritime pine, specifically at the pre-cotyledonary/early cotyledonary
stage, as supported by quantitative gene expression data. The ongoing experiments for cloning the promoter region and for localizing specific expression in embryo tissues will provide additional clues for functional characterization.

**Acknowledgements:** Fundação para a Ciência e a Tecnologia (FCT) is acknowledged for financial support, through projects PTDC/AGR-GLP/102877/2008 and P-KBBE/AGR-GPL/0001/2009, and grant SFRH/BD/32037/2006 (MS)

**References**

1. Simões M, Lorenz W, Maroco J, Dean J, Miguel C: Genome-wide expression analysis in developing pine embryos. *Molecular aspects of plant development International Conference* Vienna, Austria 2010.

2. Gonçalves S, Cairney J, Maroco J, Oliveira MM, Miguel C: Identification of Genes Differentially Expressed During Embryogenesis in Maritime Pine (*Pinus pinaster*). Silva Lusitana 2005, 13(2):203-216.

3. Pullman GS, Webb DT: An embryo staging system for comparison of zygotic and somatic embryo development. *Proceedings of the TAPPI R&D division biological sciences symposium* Minneapolis, MN. Technical Association of the Pulp and Paper Industry Press, Atlanta, GA 1994, 31-34.

4. Gonçalves S, Cairney J, Maroco J, Oliveira MM, Miguel C: Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta 2005, 222(3):556-563.

P72

**In search for the role of thermospermine synthase gene in poplar vascular development**

Ana Milhinhos¹*, Andreira Matos¹, Francisco Vera-Sirera², Miguel Blazquez², Célia Miguel¹

¹Instituto de Tecnologia Química e Biológica-Univ. Nova de Lisboa; Instituto de Biologia Experimental e Tecnológica (ITQB-UNL)BET) Av. República, EAN, 2780-157 Oeiras, Portugal; ²Instituto de Biologia Molecular y Celular de Plantas (CSIC-UPV) 46022 Valencia, Spain

E-mail: milhinho@itqb.unl.pt

BMC Proceedings 2011, 5(Suppl 7):P72

**Background:** Plant polyamines are preferentially detected in actively growing tissues and have been implicated in growth and developmental processes such as embryogenesis, floral developmental, fruit ripening, senescence and stress responses [1]. Recently it has been established a link between polyamines and vascular development as it was found that, in *Arabidopsis*, the loss-of-function mutants of *ACAULIS5* (*ACLS5*) gene, encoding thermospermine synthase, exhibit a severe dwarf phenotype, suggesting that thermospermine acts as a regulator of stem elongation [2,3]. However, in trees, no studies have yet been reported. Due to the relevance of vascular development in wood formation we are investigating the role of thermospermine in vascular tissues of poplar.

**Materials and methods:** A search for *ACLS5*-like sequences in *Populus trichocarpa* genome allowed us to identify three putative *ACLS5* orthologous genes. Based on the degree of sequence similarity, we have selected one of them, *PtACLS5*, to generate transgenic plants bearing the constructs for overexpression and silencing of this gene in poplar.

**Results:** High expression levels of *PtACLS5* in overexpression transgenic lines have been found to be correlated to higher thermospermine content in leaves and young stems, but not to a higher level of other polyamines, suggesting that *PtACLS5* encodes a thermospermine synthase in poplar, and it is most probably an ortholog of *ACLS5* in poplar. Interestingly, these plants display altered and arrested shoot development in the early stages following p35S::PtACLS5 transformation, as well as severe dwarfism. Anatomical changes associated to the lack of elongation include arrested development of the root system and no elongation of the stem from the first internode onwards. Because *Arabidopsisacls5* loss-of-function mutants show accelerated vessel cell death and *ACLS5* expression is confined to xylem vessel elements [4], we further looked for alterations in the vascular pattern of the poplar stem, and observed the development of a wider stem in dwarf plants, composed of primary vascular tissues only, lower number of metaxylem cells and with no secondary growth.

**Conclusions:** Overall, our results suggest that thermospermine has a regulatory role in xylem differentiation/maturation in poplar. Although a feedback control of thermospermine synthesis seems to be present in *Arabidopsis*, in our transgenic poplar the high levels resulting from overexpression of thermospermine synthase gene seem to overcome any

turn-over that might be occurring of the excess thermospermine being produced. Currently we are pursuing the spatial localization of the *ACLS5* transcript in poplar plants through *in situ* hybridization, and by taking advantage of the generated transgenic lines we hope to understand the role of thermospermine in the vascular tissues formation in this woody species.

**Acknowledgements:** This work is supported by the FCT project PTDC/AGR-GPL/098369/2008 and FCT PhD grant SFRH/BD/30074/2006 (A.M.). Dr. Max Cheng for providing *P.trichocarpa* Nisqually-1 clone and Dr. Brian Jones for T89 hybrid clone.

**References**

1. Kumar A, Taylor M, Altabella T, Tiburcio AF: Recent advances in polyamine research. *Trends Plant Sci* 1997, 2:124-130.

2. Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G, Komeda Y: *ACAULIS5*, an *Arabidopsis* gene required for stem elongation, encodes a spermine synthase. *The EMBO J* 2000, 19:4248-4256.

3. Knott JM, Römer P, Sumper M: Putative spermine synthases from *Thalassiosira pseudonana* and *Arabidopsis thaliana* synthesize thermospermine rather than spermine. *FEBS Lett* 2007, 581(16):3081-3086.

4. Muñiz L, Minguet EG, Singh SK, Pesquet E, Vera-Sirera F, Moreau-Courtois CL, Carbonell J, Blázquez MA, Tuominen H: *ACAULIS5* controls *Arabidopsis* xylem specification through the prevention of premature cell death. *Development* 135:2573-2582.

P73

**A direct stimulatory role of mobile gibberellin in *Arabidopsis* hypocotyl xylem expansion**

Kaisa Nieminen¹, Laura Ragni, David Pacheco-Villalobos, Richard Sibout, Christian S Hardtke

Department of Plant Molecular Biology, University of Lausanne, Switzerland

E-mail: Kaisa.Nieminen@unil.ch

BMC Proceedings 2011, 5(Suppl 7):P73

**Background:** Can Arabidopsis research contribute to our understanding about wood development? Does the function of vascular cambium in a herbaceous wood resemble that of a tree? Despite its diminutive size as compared to a tree, Arabidopsis still displays cambial driven secondary thickening in several organs, including the hypocotyl. Hypocotyl is a good model organ for wood development studies, as in this organ the radial secondary development can be uncoupled from the apical primary growth. This is due to the fact that the hypocotyl elongates only for five days after germination; thus, the radial secondary growth starts only after the elongation has ceased. This is in contrast to the other Arabidopsis organs displaying cambial growth, where it is accompanied by the simultaneous activity of the shoot and root meristems.

Two phases can be identified in the hypocotyl secondary development: 1) an early phase of proportional radial growth, where the cambium produces both xylem and phloem at a similar rate, and 2) a later xylem expansion phase, where more xylem than phloem is produced (Fig 1A) [1]. Notably, the composition of xylem is different between these two phases: the xylem produced during the first phase consists of xylem vessels and parenchyma cells, and of xylem vessels and fibers during the second phase. Especially the later phase, characterized by extensive wood formation, resembles the secondary growth in tree species.

We have previously shown that in Arabidopsis hypocotyl the transition from the first to the second phase is triggered through the onset of flowering, when the identity of shoot apical meristem changes from vegetative to reproductive [1]. Upon this transition, instead of new leaves, an inflorescence stem emerges from the middle of rosette leaves. What could be the nature of this signal [2]?

**Results and discussion:** Does flower differentiation trigger the onset of xylem expansion? To study this, we analyzed Arabidopsis null mutants for key flowering regulators. We were able to confirm that xylem expansion took place in all our mutants, suggesting that neither floral specification nor bolting (the emergence and elongation of the inflorescence stem from the rosette) are required for this process.

Thus, our signal appeared to be upstream of flowering; possibly the same signal activated both flowering and xylem expansion? Since gibberellin hormone (GA) has been shown to be an important regulator of flowering initiation, we wondered what effect it would have on the secondary growth. Indeed, both xylem expansion and flowering were initiated upon
<figure><img src="image_38.png" /><figcaption>**Figure 1(abstract P73)** A) Cross-section of Arabidopsis hypocotyl showing the two phases of secondary development. B) In Arabidopsis grafting, young seedlings are cut into two through hypocotyl, and the hypocotyl of shoot (scion) part and the root (stock) part are then re-attached in the desired combination. C) Schematic picture of the *ga1-3* vs wild-type (WT) grafting experiment. GA from the WT scion was able to rescue xylem expansion in the hypocotyl of the GA deficient *ga1-3* stock.</figcaption></figure>

GA treatment. To study if GA biosynthesis was required for xylem expansion, we analyzed *ga1-3*, a null mutant for the GA REQUIRING 1 gene encoding a key GA biosynthesis enzyme. As expected, these plants displayed strongly reduced xylem expansion.

Next we wanted to study, if not only the GA content, but the actual level of GA signaling, was important for the regulation of xylem expansion. We observed that the transgenic and mutant lines with elevated GA signaling displayed increased and the lines with impaired GA signaling accordingly reduced xylem expansion, thus confirming the rate-limiting role of GA in this process.

Does GA signaling stimulate xylem expansion at the cambium, or does it function at the shoot apex to launch the production of the mobile signal? To study this, we performed grafting experiments (Fig 1B). We could see that the wild-type level of GA signaling in either part of the graft could not rescue the xylem expansion if the other part was dominantly inhibited in GA signaling. Accordingly, elevated GA signaling level enhanced xylem expansion only locally; the effect was not graft-transmissible to the wild-type part of the graft. Thus, GA signaling cascade appears to act as a local regulator of cambial activity, downstream of the mobile signal.

Could the GA hormone itself be mobile? We studied this by reciprocally grafting the GA deficient *ga1-3* mutant with a wild-type plant. Strikingly, in these plants, the wild-type scion (shoot part) restored xylem expansion in the hypocotyl of the *ga1-3* stock (root part) (Fig 1C). To sum up, an impaired GA signaling did not affect xylem expansion systemically, suggesting that it acts downstream of the mobile cue. By contrast, the GA effect was graft transmissible, confirming that GA itself is the mobile shoot-derived signal.

**Conclusions:** Our study shows that GA acts as a mobile signal that activates the onset of extensive xylem production in Arabidopsis hypocotyl. It would be interesting to study if a similar GA driven process takes place also in tree species upon the seasonal or age-related activation of their cambial growth. We think that Arabidopsis research has the potential to contribute novel hypotheses into secondary development research, and that it can complement tree studies in some areas of this research field. However, due to the sheer extend of secondary development present in tree species, and their other special characteristics (the seasonal activity-dormancy cycle etc.), many processes of secondary growth remain to be best studied in trees.

**References**

1. Sibout R, Plantegenet S, Hardtke CS: Flowering as a condition for xylem expansion in Arabidopsis hypocotyl and root. *Curr Biol* 2008, 18(6):458-463.

2. Ragni L, Nieminen K, Pacheco-Villalobos D, Sibout R, Schwechheimer C, Hardtke CS: Mobile gibberellin directly stimulates Arabidopsis hypocotyl xylem expansion. *Plant Cell* 2011, 23(4):1322-1336.

P74

Deep sequencing combined with microarray hybridization to identify novel and conserved microRNAs during somatic embryogenesis of hybrid yellow-poplar (*Liriodendron chinense* (Hemsl.) Sarg. *X L. tulipifera* Linn.)

Li Tingting, Chen Jinhui*, Shi Jisen*, Xu Jin*

The key laboratory of forest genetics and gene engineering of the ministry of education of China Nanjing Forestry University Nanjing 210019, China

E-mail: xjinhsh@njfu.edu.cn

BMC Proceedings 2011, 5(Suppl 7):P74

**Background:** Hybrid yellow-poplar was a man-made cross between *Liriodendron tulipifera* Linn.x *L. chinense* (Hemsl.) Sarg.. Heterosis was strong both on growth and adaptation, no matter what, the crosses or the reciprocals. During the last 12 years, somatic embryogenesis systems were successfully established in our lab, in order to meet the needs of planting materials for expanding plantations in Southern China. Synchronous development of high quality embryos was always required both for the study of embryo development, and the supply of commercial emblings. The functions of plant microRNAs (miRNAs) in post-transcriptional gene regulation by targeting mRNAs for degradation, or the translational repression by hybridizing to complementary sequences within target mRNA molecules were well reported [1]. One of the major roles of miRNAs in regulating plant development has been obtained from a number of studiesbut molecular mechanisms regulating the development of embryos are not well understood, especially during the early stages of somatic embryos [2]. Here we present some results on the discovery of novel and conserved miRNAs during somatic embryogenesis of hybrid yellow-poplar using Illumina sequencing and miRNA microfluidic chip in order to
understand how the embryogenic somatic mass is developed into embryos and plants.

**Materials and methods:** The pre-embryo mass (PEM) and different stages of somatic embryos (SE) (Figure 1) were collected sequentially from the hybrid yellow-poplar somatic embryogenesis system. All the callus and embryo tissues were staged by microscope and immediately forzen in liquid nitrogen and stored at -80C until used. Total RNA was isolated from different developmental stages of somatic embryogenesis using Total RNA Purification Kit (Norgen Biotek Corporation, Canada). All the RNA samples from different somatic embryo tissues were mixed equally to form a single RNA pool. Following the instruction of LC Science, small RNA sample was prepared and then sequenced using Illumina Technology. Processing and analysis of raw data was done following Sunkar et al [3,4]. Conserved and candidate novel miRNAs in hybrid yellow-poplar were identified. A mixed small RNA microarray hybrid was used to validate the sequencing result and detect new miRNAs in hybrid yellow-poplar; 299 sequences detected by sequencing and 1,450 plant miRNAs sequences in miRBase 15.0 were ordered to serve as probes. A well-developed stem-loop strategy [5]and SYBR Green master mix were used to perform real time PCR to validate the miRNAs expressed in both sequencing and microarray analysis. The potential targets of miRNAs were predicted using the psRNATarget program (http://bioinfo3.noble.org/psRNATarget/) with default parameters. The biology and molecular functions of these predicted targets were categorized by the Gene Ontology program (http://www.Geneontology.org/).

**Results:** A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNA extracted from all stages of somatic embryogenesis tissues. Hybrid yellow-poplar has a complex small RNA population and the length of small RNAs varied. The 24-nt and 21-nt are the predominant length for the majority of the small RNAs. Combining deep sequencing and bioinformatics analysis there were 82 sequences, with perfectly matched known miRNAs from 32 conserved miRNAs families and 273 species-specific candidate miRNAs whose precursors were generated from *Liriodendron tulipifera* ESTs and form good hairpin structure. Among these miRNAs, both miR156 and miR166 which have been reported to be important during plant early embryonic patterning, had a large family members expressed in this process. Microarray hybridization and qRT-PCR technology were used to demonstrate that most conserved and species-specific miRNAs expressed in hybrid yellow-poplar. In addition, another set of 149 miRNAs included in 29 conserved families was found, which were not discovered by deep sequencing analysis but have obvious detectable signals in the microarray. Potential targets of biological processes and molecular functions of these miRNAs were predicted by blasting with *Arabidopsis thaliana* and analyzing with Gene Ontology. These predicted target genes participate in many metabolic and biological processes such as transport, signal transduction, response to stress, cell organization and many other cellular or developmental processes.

**Conclusions:** By combining Illumina deep sequencing with microarray hybridization we discovered 231 miRNAs from 61 conserved miRNA families and 273 species-specific candidate miRNAs. The potential miRNA target functions related to various biological and metabolic processes. These results indicate that microRNAs play wide-ranging and important roles during all stages of somatic embryogenesis in hybrid yellow-poplar. Additional experiments on the functional verification of interesting candidate miRNAs are now under consideration.

## References

1. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. *Cell* 2004, 116:281-297.

2. Oh TJ, Wartell RM, Cairney J, Pullman GS: Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (*Pinus taeda*). *New Phytologist* 2008, 179:67-80.

3. Sunkar R, Girke T, Jain PK, Zhu JK: Cloning and characterization of microRNAs from rice. *Plant Cell* 2005, 17:1397-1411.

4. Sunkar R, Zhou X, Zheng Y, Zhang W, Zhu JK: Identification of novel and candidate miRNAs in rice by high throughput sequencing. *BMC Plant Biol* 2008, 8:25.

5. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Gue-gler KJ: Real-

time quantification of microRNAs by stem-loop RT-PCR. *Nucleic Acids Res* 2005, 33(20):e179.

6. Nodine MD, Bartel DP: MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis. *Gene & Development* 2010, 24:2678-2692.

7. Grigg SP, Galinha C, Kornet N, Canales C, Scheres B, Tsiantis M: Repression of apical homeobox genes is required for embryonic root development in Arabidopsis. *Curr Biol* 2009, 19:1485-1490.

## P75

### Phytohormone targeting in plant tissues

Miroslav Strnad*, Ondrej Novak, Jakub Rolcik, Danuse Tarkowska, Jiri Gruz, Karel Dolezal
Laboratory of Growth Regulators, Faculty of Science, Palacky University & Institute of Experimental Botany AS CR, Slechitelu 11, Olomouc, CZ-783 71, Czech Republic
E-mail: miroslav.strnad@upol.cz
*BMC Proceedings* 2011, **5**(Suppl 7):P75

**Background:** The identification and quantification of plant hormones in plant tissues are necessary for physiological studies of their metabolism and mode of action. The major problem associated with plant hormone analysis is that the amount of phytohormones present endogenously in plant tissues is very low, usually in the range of fmol to pmol/g fresh weight.

**Methods:** Homogenization and extraction with organic solvents was done in one microcentrifuge tube and accelerated by crushing the plant material in a vibration mill. The extracts from minute amounts of fresh plant material were immediately purified using a solid-phase extraction (C18, C8, Oasis™ HLB cartridges)in combination with ion-exchange and/or immunoaffinity purification. A fast chromatography technique, the ultra performance liquid chromatography (Acquity™ UPLC, Waters) was coupled to triple quadrupole mass spectrometer (Xevo™ TQ MS, Waters) equipped with an electrospray interface (ESI) and the unique performance of collision cell – ScanWave™. The mass spectrometric conditions were optimized for each analyte and quantification was obtained by multiple reaction monitoring (MRM) of precursors and the appropriate product ions.

**Results and discussion:** We found that a combination of different sorbents, reverse phases and ion-exchange phases, was the best tool in the one-step purification, giving a total extraction recovery ranging between 50-80% for all studied biologically active compounds. In MRM mode, the detection limit for most of phytohormones (cytokinins [1,2], auxins [3], abscisic acid [4], gibberellins, brassinosteroids) as well as phenolic acids [5] and mammalian steroids [6] was close to 1 fmol and achieved linear range was at least five orders of magnitude. Use of our procedures can allow the quantification of plant hormones and their derivatives (in total 145 compounds) in very limited amounts of material, ca. 100 mg FW. The methods provide substantial improvements in terms of robustness, sensitivity, selectivity, convenience, through-put and cost-effectiveness over previous methods published.

**Conclusions:** The application of new analytical approaches based on UPLC separation makes possible a new direction in plant hormone research. We believe that UPLC-ESI(+)MS/MS technology can be used for fast and sensitive quantitative analysis showing reproducibility in the plant hormones profiling in different tree extracts.

## References

1. Novak O, Hauserova E, Amakorova P, Dolezal K, Strnad M: Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. *Phytochem* 2008, 69(11):2214-2224.

2. Tarkowski P, Vaclavikova K, Novak O, Pertry I, Hanus J, Whenham R, Vereecke D, Sebela M, Strnad M: Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry. *Anal. Chim. Acta* 2010, 680(1-2):86-91.

3. Pencik A, Rolcik J, Novak O, Magnus V, Bartak P, Buchtik R, Salopek-Sondi B, Strnad M: Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. *Talanta* 2009, 80(2):651-655.

4. Tureckova V, Novak O, Strnad M: Profiling ABA metabolites in *Nicotiana tabacum* L. leaves by ultra-performance liquid chromatography–electrospray tandem mass spectrometry. *Talanta* 2009, 80(1):390-399.
5. Gruz J, Novak O, Strnad M: Rapid analysis of phenolic acids in beverages by UPLC-MS/MS. *Food Chem* 2008, 111(3):789-794.

6. Simersky R, Novak O, Morris DA, Pouzar V, Strnad M: Identification and quantification of several mammalian steroid hormones in plants by UPLC-MS/MS. *J. Plant Growth Regul* 2009, 28(2):125-136.

## P76
### Ectopic expression of C-terminal tubulin variants alters wood composition and structure in Populus

Prashant Swamy¹, Feng Long¹, Batbayar Nyamdari¹, Jeng-Der Chung²,
Shawn Mansfield³, Scott Harding¹, Chung-Jui Tsai¹*

¹University of Georgia, USA; ²Taiwan Forestry Research Institute, Taiwan;

³University of British Columbia, Canada

E-mail: cjsai@uga.edu

BMC Proceedings 2011, 5(Suppl 7):P76

Cortical microtubules are cytoskeletal components that are relevant to the bioenergy and forest products industry due to their postulated role in orchestrating cellulose microfibril deposition during cell wall formation. The microtubule component proteins α- (TUA) and β-tubulins (TUB) are encoded by multi-gene families with very high overall sequence homology across species. To advance our initial characterization studies (Oakley et al., 2007), we have developed a suite of transgenic *Populus* that exhibit perturbed *TUA* to *TUB* transcript ratios, or that express tubulin PTM mimics. Most of the construct combinations resulted in abnormal organogenesis and vascular development, and failed to produce viable plants. Only three of the combinations led to whole-plant regeneration, and interestingly, all three featured the C-terminal variants. This is significant because the C-terminus is the post-translational modification (PTM) hot-spot in animal tubulins. One of the PTM mimics that we developed lacks a C-terminal tyrosine, thought to be the target of a tyrosine cleavage and re-ligation cycle important for tubulin regulation in animal cells. The transgenic trees appeared morphologically normal, but exhibited a range of epinasty and twisting phenotypes in mature leaves. Bark color was noticeably lighter in the transgenics. Microfibril angle, wood density, lignin content, lignin structure and metabolic profiles were altered in the transgenic wood. The results are consistent with a function of microtubules and microtubule PTMs for plant development and cell wall biogenesis in *Populus*, and offer novel strategies to manipulation of wood properties.

## P77
### Isolation and characterization of hybrid poplar galactinol synthases

Faride Unda¹*, Shawn Mansfield²

¹Wood Science Department -University of British Columbia, Canada; ²Wood Science Department- University of British Columbia, Canada

E-mail: farideu@interchange.ubc.ca

BMC Proceedings 2011, 5(Suppl 7):P77

The raffinose family of oligosaccharides (RFOs) likely fulfill at least few major physiological roles in plants, translocation of carbon in the phloem, storage in sink tissues, and as putative biological agents to combat both abiotic and biotic stress [1-3]. The synthesis of galactinol from *myosinol* + UDP-galactose, by galactinol synthase (GolS), is considered a key regulatory step in RFO synthesis [4]. To investigate the functional roles of this class of compounds in trees, two cDNAs that encode galactinol synthase (GolS), were identified and cloned from hybrid poplar (*P. alba* × *grandidentata*). Phylogenetic analyses of the *Populus* GolS isoforms with other known galactinol synthases suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. The predicted protein sequences of both isoforms (PaxgGolSI and PaxgGolSII) showed characteristics of galactinol synthases from other species, including a serine phosphorylation site at position 266 and the pentapeptide hydrophobic domain ASAP [5]. Kinetic analyses of recombinant PaxgGolSI and PaxgGolSII resulted in $K_m$ values for UPD-galactose of 0.79 and 0.65 mM and $V_{max}$ values of 660.4 and 1245 nM min⁻¹, respectively. PaxgGolSI inherently possessed broader pH range and temperature sensitivity when compared to PaxgGolSII. Interestingly, spatial and temporal expression analyses revealed that *PaxgGolSII* transcript levels varied seasonally, while *PaxgGolSI* did not, thereby

implying a temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. Based on this evidence, we suggest that PaxgGolSI may be involved in basic metabolic activities (i.e. storage), while PaxgGolSII is likely involved in seasonal mobilization of carbohydrates.

### References

1. Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, et al: Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in *Arabidopsis thaliana*. *Plant Journal* 2002, 29(4):417-426.

2. Panikulangara TJ, Eggers-Schumacher G, Wunderlich M, Stransky H, Schoffl F: Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in *Arabidopsis*. *Plant Physiology* 2004, 136(2):3148-3158.

3. Philippe RN, Ralph SG, Mansfield SD, Bohlmann J: Transcriptome profiles of hybrid poplar (*Populus trichocarpa* × *deltoides*) reveal rapid changes in undamaged, systemic sink leaves after simulated feeding by forest tent caterpillar (*Malacosoma disstria*). *New Phytologist* 2010, 188(3):787-802.

4. Keller F, Pharr DM: Metabolism of carbohydrates in sinks and sources: Galactosyl-sucrose oligosaccharides. *Photoassimilate distribution in plants and crops* New York: Marcel Dekker: Zamski E, & Schaffer AA 1996, 115-184.

5. Sprenger N, Keller F: Allocation of raffinose family oligosaccharides to transport and storage pools in *Ajuga reptans*: The roles of two distinct galactinol synthases. *Plant Journal* 2000, 21(3):249-258.

## P78
### Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

Daniel Vestman¹*, Emma Larsson¹, Daniel Uddenberg¹, John Cairney²,

David Clapham¹, Eva Sundberg¹, Sara von Arnold¹

¹Swedish University of Agricultural Sciences, Sweden; ²NanoBiotechnologies

E-mail: daniel.vestman@slu.se

BMC Proceedings 2011, 5(Suppl 7):P78

**Background:** Although most morphogenic events in plants occur in the sporophyte following seed germination, the embryonic phase is crucial as it is then that the meristems are specified and the shoot-root body pattern of the plant is established. We are using somatic embryogenesis in Norway spruce (*Picea abies*) as a model system for studying embryology in conifers. A deeper understanding of the genetic regulation can provide clues on how to improve the culture conditions in order to propagate economically important conifers via somatic embryos. The model system includes a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages [1]. Here, we have extended the study of early events during embryogenesis in Norway spruce, by using microarray slides spotted with 12,536 cDNA clones from loblolly pine cDNA libraries. We have focused on the first stages of embryogenesis: the differentiation of early embryos from proembryogenic masses (PEMs) and the beginning of the development of late embryos. Few such studies have been undertaken and the work, which we describe here, is the first comprehensive analysis of gene expression of a conifer species during early stages of somatic embryogenesis. The analysis revealed the importance of previously unknown molecular events regulating putative processes associated with pattern formation.

**Methods:** The developmental stages of somatic embryogenesis in Norway spruce have been described previously [2]. The stages used in this study included proliferating PEMs on medium containing PGRs, early embryos one week after withdrawal of PGRs and developing late embryos after one week on medium containing ABA. Alteration in gene expression pattern during embryo differentiation and development was analyzed by comparing gene expression of samples from sequential developmental stages. In addition, to identify rapidly occurring transient changes, gene expression in proliferating PEMs was compared with gene expression in PEMs 24 hours after withdrawal of PGRs. Amplified RNA molecules were labeled and expression profiling was conducted using the TIGR loblolly pine microarray [3] with 12,536 validated cDNA clones originating from whole megagametophytes and zygotic and somatic embryos from different stages of development.
**Results:** Testing for significant differential expression identified 106, 208 and 464 mRNAs that showed different abundance 24 hours after withdrawal of PGRs, during differentiation of early embryos and development of late embryos, respectively. 53 mRNAs were present in more than one set, giving a total of 720 unique transcripts. The reliability of the microarray results was confirmed by qRT-PCR.

The relative abundances of Gene Ontology (GO) terms within the sets of differentially expressed genes were compared against the GO distribution of all clones on the array. We observed a general over-representation of genes involved in response to stress, both during the transition from proliferation to differentiation of early embryos and from differentiation of early to development of late embryos. During transition from early to late embryogeny, genes involved in catabolic processes, in carbohydrate metabolic processes and in gibberellin (GA) -mediated signaling were over-represented, while genes involved in metabolic processes were under-represented.

Our data show that already 24 hours after withdrawal of PGRs, the elimination of cells by PCD has been initiated as PCD-related genes like *METACASPASE 9* (MC9, AT5G04200) were up-regulated.

During differentiation of early somatic embryos, the first week after withdrawal of PGRs, genes related to cell wall modifications were down regulated. An increase in transcript accumulation was observed for genes coding for developmental regulators like MATERNAL EFFECT EMBRYO ARREST 49 (MEE49, AT4G01560), and a decrease of transcripts coding for LOB-DOMAINS containing proteins, indicating that pattern formation has started. The analysis revealed a putative increase in the content of both auxin and gibberellin.

At the developmental switch to late embryogenesis, several genes of importance for cell organization, developmental processes, and other biological processes were up- or down-regulated. The relative transcript abundance of *L1L*, *ROXY1* (AT3G02000) and *WRKY6* (AT1G62300) decreased while the abundance of transcripts such as *ABI3*, *ABI4* (AT2G40220) and *GROWTH-REGULATING FACTOR 1* (GRF1, AT2G22840) increased. In addition, genes coding for the LEA proteins were up-regulated. These changes in transcript levels suggest that pattern formation continued and that maturation had initiated at this developmental stage. The transcript levels of genes encoding a fatty acid elongase, FIDDLEHEAD (FDH, AT2G26250), which regulates epidermal cell differentiation, and for an EXTRACELLULAR DERMAL GLYCOPROTEIN (EDGP, AT1G03230) decreased. Transcripts encoding SUR1, MYB77 (AT3G50060), the auxin-induced protein INDOLE-3ACETIC ACID INDUCIBLE 11 (IAA11, AT4G28640), the auxin receptor TRANSPORT INHIBITOR RESPONSE 1 (TIR1, AT3G62980) and a positive regulator of brassinosteroid signalling (AT1G78700) were up-regulated, indicating that the auxin-responsive machinery was up-regulated. Furthermore, genes encoding the GA signaling repressors were up-regulated, while a GA induced and a gene encoding for a GA inactivating enzyme were down-regulated.

**Discussion:** By examining changes in global gene expression, we have been able to determine the timing of molecular events regulating putative processes during embryogenesis based on the assumption that the conifer genes are homologous to their Arabidopsis counterparts. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm and programmed cell death. Our analyses of genes and putative processes that take place during differentiation of early embryos and development of late embryos in a conifer can now serve as a basis for further studies of the processes.

## References

1. Bozhkov PV, von Arnold S: Polyethylene glycol promotes maturation but inhibits further development of Picea abies somatic embryos. *Physiologia Plantarum* 1998, 104:211-224.

2. Filonova LH, Bozhkov PV, von Arnold S: Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking. *J Exp Bot* 2000, 51:249-64.

3. Rensink WA, Buell CR: Microarray expression profiling resources for plant genomics. *Trends Plant Sci* 2005, 10:603-9.

# S5. BIOTIC AND ABIOTIC INTERACTIONS

P79

## Identification of genes that contribute to drought stress tolerance in Populus

Muhammad Arshad*, Kamal Biswas, Jim Mattsson, Sherryl Bisgrove
Aine Plant Department of Biological Sciences, Simon Fraser University,
Burnaby, BC, Canada, V5A 1S6

E-mail: maa44@sfu.ca

BMC Proceedings 2011, **5**(Suppl 7):P79

**Background:** The cultivation of poplars (*Populus* spp.) is favored for forestry and reclamation purposes all over the northern hemisphere where they represent a commercially important resource. Poplars may become a component of programs to optimize carbon sequestration however; poplars are generally regarded as drought sensitive. The patterns of episodic drought over the last decade suggest that the development of drought tolerant poplar genotypes could be a useful tool to achieve sustained forest productivity [1]. Previous reports have shown that expression of hundreds of poplar genes changes in response to drought, presenting a problem in the identification of genes that are more important than others in counteracting the harmful effects of drought [2,3].

The genus *Populus* contains many fast growing hybrids that show varied drought tolerance according to genotype [4]. Hence, there is genetic variation among poplar hybrids that can be used to identify genes that contribute to drought stress tolerance. Despite extensive physiological and morphological descriptions of the response of Populus to drought, little work has been undertaken to explain genotype differences at the gene level. Therefore, this research has been undertaken and its major objective is to identify the genes that contribute to drought stress tolerance in poplar by correlating the physiological responses to gene expression. These genes may potentially be used as molecular markers in the drought tolerance breeding programs.

**Materials and methods:** Hardwood cuttings of 9 poplar hybrids (Green Giant, Assiniboine, AP-36, Canam, Katepwa, Hill, Walker, WP-69 and WP-86) were obtained from Dr. Barb Thomas (Alberta Pacific Forest Industries) and Bill Schroeder (Agriculture & Agri Food Canada, Indian Head, Saskatchewan). Cuttings were established in the greenhouse and plants were obtained for the drought stress trial. At the start of the trial, water was withheld from plants used for drought experiment whereas control plants were watered regularly. During the drought stress trial, a plastochron index was established that helped to collect the data from similar leaves of all drought stressed and control plants. Data were collected from 9 different poplar hybrids at 3 time points (mild stress, severe stress and recovery). Split plot design was used to collect the data from drought stressed and control plants using genotypes as main plots (9) and time points as sub-plots (3). Physiological data were collected for height growth, new leaf formation, water potential (Ψ) and relative water content (RWC) from drought stressed and control plants. Young leaves were also collected for gene expression analysis from both stressed and control plants. Quantitative polymerase chain reaction (Q-PCR) was used to analyze the expression of several candidate drought responsive genes.

**Results:** Drought significantly affected the growth of the trees (Figure 1). Data from 3 drought stress trials in the greenhouse showed that different hybrids responded drought stress differently. Some hybrids behaved as tolerant whereas others as sensitive and some hybrids showed intermediate response to drought. By statistical analysis of physiological data, we ranked these 9 poplar hybrids according to their drought tolerance ability. Statistical analysis showed that Walker maintained significantly better height growth, leaf formation, ψ and RWC during mild, severe stress and recovery as compared to all other hybrids; therefore Walker was identified as the most drought tolerant hybrid genotype. On contrary, Green Giant showed poor maintenance of physiological parameters at all time points and hence was regarded as the least drought tolerant hybrid genotype. An intermediate response of all other hybrids was recorded during 3 drought stress trials in the greenhouse. Further, we focused only on 2 genotypes (the most and the least drought tolerant) to analyze the expression of several candidate drought responsive genes, which were obtained from microarray studies in poplar. Q-PCR results revealed that selected 2 genotypes had no significant effect
on the stress response of some genes, however, other genes showed marked and significant differences in expression between the two genotypes.

**Conclusions:** Three trials in the greenhouse showed that physiological response to drought varies among different poplar hybrids.

Walker was identified as the most whereas Green Giant as the least drought tolerant genotype among a group of poplar hybrids.

Q-PCR analysis showed that among a set of candidate drought responsive genes, some genes showed differential expression between the most (Walker) and the least (Green Giant) drought tolerant genotypes.

The genes showing differential expression in the most and the least drought tolerant genotypes might be playing an important role in drought tolerance of poplar.

We have identified Populus genes those expression correlate with drought tolerance, providing candidate genes for drought tolerance breeding.

These genes may also be used as molecular markers for drought tolerance in Populus.

**Acknowledgements:** We are thankful to Natural Sciences and Engineering Research Council of Canada and Higher Education Commission of Pakistan for providing the funding for this research.

## References

1. Rood S, Braatne J, Hughes F: Ecophysiology of riparian cottonwoods: stream flow dependency, water relations and restoration. *Tree physiol* 2003, 23:1113-1124.

2. Plomion C, Lalanne C, Claverol S, Meddour H, Kohler A, Bogeat-Triboulot M, Barre A, Provost G.L, Dumazet H, Jacob D, Bastien C, Dreyer E, Daruvar A, Guehl J, Schmitter J, Martin F, Bonneu M: Mapping the proteome of poplar and application to the discovery of drought-stress responsive proteins. *Proteomics* 2006, 6:6509-6527.

3. Street N, Skogstrom O, Sjodin A, Tucker J, Acosta MR, Nilsson P, Jansson S, Taylor G: The genetics and genomics of the drought response in Populus. *The Plant J* 2006, 48:321-341.

4. Marron N, Villar M, Dreyer E, Delay D, Boudouresque E, Petit J, Delmotte F, Guehl J, Brignolas F: Diversity of leaf traits related to productivity in 31 Populus deltoides × Populus nigra clones. *Tree Physiol* 2005, 25:425-435.

# P80
## Are there any Pinus pinaster trees resistant to Bursaphelenchus xylophilus? Studies implemented in Portugal to address this question

Rita Costa¹*, Patricia Ribeiro¹, Isabel Evaristo¹, Bruno Ribeiro¹, Alexandre Aguiar¹, Isabel Carrasquinho¹, Carla Santos², Marta Vasconcelos²

¹Instituto Nacional dos Recursos Biológicos I.P. L-INIA – Quinta do Marquês, Av. da República, 2780-159 Oeiras, Portugal; ²CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal

E-mail: ritalourencocosta@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P80

The Pinewood nematode *Bursaphelenchus xylophilus* (PWN) was found to be responsible for maritime pine (*Pinus pinaster*) death for the first time in Europe in 1999, in a Portuguese forest south of Lisbon. It is the causal agent of the Pine Wilt Disease (PWD) and a quarantine organism in the European Union with approximately €80 million spent from 1999 – 2009 for attempting its eradication, which must also be added to direct losses from tree mortality. During this period, important progress has been made in understanding the relationships between PWN, its insect vector and the host tree/environmental factors that result in pine wilt in all countries affected but, especially, in Portugal.

Although PWN can cause devastating tree mortality, there is also evidence that some tree species or individual trees within a species are either tolerant (i.e. support the nematode, but are not killed immediately) or resistant to the nematodes. In order to investigate the susceptibility of *Pinus pinaster* to *Bursaphelenchus xylophilus* as a first step for the establishment of methodologies for the improvement of its resistance, a research program was implemented recently in Portugal for this species, combining genomic and quantitative genetics approaches. Until now, more than 500 trees were phenotypically selected in a highly affected stand and monitored during two years, regarding their phytossanitary status. The percentage of mortality observed in the first year was about 10%, with this value decreasing to less than 5% in the second year.

Around 200 trees (including 150 of the selected trees) were genotyped for 6 microsatellite loci and three combinations of AFLP markers. The genotyping results show a high diversity of genotypes, which may open good prospects for the selection of resistant plants to *Bursaphelenchus xylophilus*.

At the same time, genomic and transcriptomic studies have been initiated with the final goal to discover candidate genes, loci and molecular markers related with disease resistance to PWN. With regards to the effects of the disease at a transcriptional level, the SSH technique was utilised to identify ESTs in *P. pinaster* and *P. pinea* when inoculated with PWN. ESTs were isolated, cloned, sequenced and identified using BlastN and BlastX, and clearly indicated that at an initial stage of the disease there is activation of a defence response at a molecular level, mainly related to oxidative stress, production of lignin and ethylene and post-transcriptional regulation of nucleic acids. 58% of the isolated sequences are not yet described, which shows the lack of genomic information currently available for pine. The results obtained are presented and discussed.

# P81
## Isolation, characterization and genes expression analysis of three dehydrin genes during cold acclimation of *Eucalyptus globulus*

Marta Fernández¹*, Sofia Valenzuela²*¹

¹Genómica Forestal S.A., Chile; ²Universidad de Concepción, Facultad de Ciencias Forestales. Centro de Biotecnología. Genómica Forestal S.A., Chile

E-mail: sofvalen@udec.cl

BMC Proceedings 2011, 5(Suppl 7):P81

*Eucalyptus globulus* is an important species for pulp production in Chile; however it has a high sensitivity to frost. During the last few years, many studies have directed their efforts to elucidate the molecular mechanisms that regulate plant response to cold stress through the analysis of gene expression. This work reports the isolation and characterization of coding and non coding sequence of three dehydrin genes of *E. globulus* which allowed comparing the gene expression of these genes during cold acclimation, the type of dehydrin and the presence of regulatory elements in their promoters which provided valuable information about the possible signaling pathways and regulation of these genes. The three dehydrins identified in *E. globulus* showed a high transcript accumulation in stem and leaf tissue of acclimated plants, compared to non-acclimated, and the highest transcript accumulation was observed after the exposition of plants to night frosts (-2°C). Furthermore, the freezing resistant genotype exhibited a higher transcript accumulation after frost exposition compared to the sensitive genotype. These results support the idea that dehydrin proteins have an important role during cold acclimation and frost tolerance in *E. globulus*.

# P82
## Induced resistance and associated defence gene responses in *Pinus patula*

Katrin Fitza¹*, AA Myburg¹, Emma Steenkamp², Kitt Payn³, Sanushka Naidoo¹

¹Department of Genetics, University of Pretoria, Pretoria 0002, South Africa;
²Department of Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; ³Mondi South Africa, P.O. Box 12, Hilton, 3245, South Africa

E-mail: katrin.fitza@fabi.up.ac.za

BMC Proceedings 2011, 5(Suppl 7):P82

**Background:** Plants are able to incite a type of broad spectrum resistance against pathogens upon pre-treatment with biological or chemical inducers. Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR) are two types of induced resistance which lead to the accumulation of specific pathogenesis-related (PR) proteins. Non-pathogenic rhizobacteria are inducing agents for ISR and increased levels of ethylene (ET) and jasmonate (JA) are associated with this pathway, whereas SAR is associated with an increase in salicylic acid (SA) levels [1]. *Pinus patula* and *P. radiata* are commercially planted conifer species in South Africa, but are both highly susceptible to the causal agent of pitch canker, *Fusarium circinatum*. Annually, the forestry sector suffers substantial economic losses due to this disease which affects 20-30% of
<table><caption>**Table 1(abstract P82) Disease progression represented as percentage live stem in 6 month old *P. patula* seedlings during an eight week period post inoculation with *F. circinatum*. The significance levels are relative to the relevant control**</caption><thead><tr><th>Week</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th></tr></thead><tbody><tr><td>Chitosan (1mg/ml)</td><td>89.73</td><td>82.31</td><td>76.75</td><td>71.43</td><td>60.81</td><td>61.16</td><td>36.10</td></tr><tr><td>Chitosan (10mg/ml)</td><td>91.12*</td><td>84.89*</td><td>80.60*</td><td>72.80*</td><td>64.28*</td><td>60.32</td><td>44.18</td></tr><tr><td>Control</td><td>89.23</td><td>82.10</td><td>74.48</td><td>69.04</td><td>58.65</td><td>55.15</td><td>34.53</td></tr></tbody></table>

the planting stock. Bonello *et al.* (2001) showed that repeated mechanical inoculation of *P. radiata* with *F. circinatum* activated induced resistance, enhancing the protection of the tree against subsequent pathogen challenge [2].

Detailed knowledge of the molecular mechanisms underlying induced resistance may be useful to develop strategies to control diseases of pine trees. This study aimed to compare the efficiency of ten biological and chemical inducers in inciting resistance against *F. circinatum*. Additionally the molecular basis of this induced resistance was investigated by analyzing the response of selected putative defence response genes.

**Methods:** Ten activators of induced resistance (Bion®, Messenger®, Chitin, MeJA, Fusarium oxysporum, Pseudomonas fluorescens, SA, Kannar, Ralstonia solanacearum and potassium phosphate monobasic) were compared. A set of 80 *P. patula* seedlings were used per treatment. Inducers were applied at four and six months of age and *F. circinatum* spores (1x10⁴) were used to challenge the seedlings a week after the booster application (six months). Disease severity was assessed six weeks after inoculation by comparing the size of the lesions on treated plants to water control plants. Three inducers that curved symptoms most successfully were selected for further analysis. A set of 116 plants per treatment were screened weekly for eight weeks. Aerial parts of the six month old plants were harvested for RNA isolation at 24 hrs after the second application. For each treatment, three replicates, with 12 plants per replicate, were harvested. Subsequently, RNA was extracted for the reverse transcriptase quantitative PCR (RT-qPCR) analysis, where four putative defence genes were profiled using the Roche LightCycler® 480 instrument.

**Results and discussion:** MeJA, Messenger and chitin treatment resulted in the reduction in symptom severity (results not shown). MeJA, Messenger and the deacetylated version of chitin, chitosan were then tested under stringent inoculation conditions with *F. circinatum* to verify the effectiveness of the inducers. The most promising treatment was chitosan at a concentration of 10 mg/ml, which resulted in a significant reduction in lesion length over a period of 6 weeks. Lesion lengths were converted to percentage live stem (calculated as lesion length divided by plant height multiplied by 100) and are displayed in Table 1.

The defence response elicited by chitosan application was investigated. Four putative genes, representing the onset of SAR and ISR were analyzed (Table 2). Phenylalanine ammonia lyase (PAL) had a three-fold accumulation in transcript expression in comparison to uninduced plants (Table 2). The expression level of the 1-deoxy-d-xylulose-5-phosphate synthase (DXS) gene, which catalyses the methyl erythriol-phosphate pathway, was down-regulated in comparison to the control. This pathway is important for the production of terpenes, which are building blocks for resin [3]. In previous studies, PAL and DXS were both shown to be responsive to chitosan. Using the PAL gene as a diagnostic marker of the phenylpropanoid pathway, the up-regulation of the transcript suggests that chitosan treatment induces the phenylpropanoid pathway which is

known to lead to the production of secondary metabolites to elicit resistance.

**Conclusion:** The potential of priming *P. patula* to defend itself against pathogen attack was explored. We tested the application of ten different inducers to enhance tolerance to *F. circinatum*. The application of chitosan reduced pitch canker symptoms. Reduced lesion length was observed for a period of six weeks, indicating the activation of induced resistance. Further molecular analysis suggests that the treatment may activate the phenylpropanoid pathway, which is involved in the production of secondary metabolites that have antifungal properties [4]. Entire defence response pathways influenced by chitosan application in *P. patula* will be investigated in subsequent expression profiling assays.

**References**

1. Gurr S, Rushton P: Engineering plants with increased disease resistance. *Trends Biotechnol* 2005, 23:275-282.

2. Bonello P, Gordon T, Storer A: Systemic induced resistance in Monterey pine. *Forest Pathol* 2001, 31:99-106.

3. Kim Y, Kim S, Kang M: Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes. *Tree physiol* 2009, 5:1-13.

4. Ferrer J, Austin M, Stewart J: Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. *Plant Physiol* 2008, 46:356-370.

P83

**Local and systemic defense response in Aspen clones: contrasting defense response to biotrophic and necrotrophic pathogens**

Carl Gunnar Fossdal¹*, Nadeem Yaqoob¹, Halvor Solheim¹, Jan Karlsson², Benedicte Riber Albrectsen²

¹The Norwegian Forest and Landscape Institute, Norway; ²Umeå Plant Science Centre, Sweden

E-mail: foc@skogoglandskap.no

BMC Proceedings 2011, **5**(Suppl 7):P83

Trees are exposed to a variety of pathogenic fungi. The defense response toward a biotroph may require a different strategy that toward a necrotroph. To understand the key processes of defense responses toward pathogenic fungi in aspen (*Populus tremulae*) at the transcript level we inoculated clones of this species with a foliar rust on the leaves and a necrotroph in the bark. Leaf samples were collected from above the inoculation site to examine the long distance (systemic) defense responses and bark tissue around the site of inoculation examined for the local response as early as day1 post treatments. We performed microarray experiments on the biotrophic and necrotrophic interaction and between healthy controls of two SwAsp clones. Selected candidate genes were also examined in more detail by qRT-PCR and chemical analysis for phenols and tannins was also performed. We found that the two clones

<table><caption>**Table 2(abstract P82) Log2 expression levels of putative defence response genes in *P. patula* 24 hrs after booster treatment with 10 mg/ml chitosan. Three biological replicates were used to calculate significance using the t-test**</caption><thead><tr><th>Gene symbol</th><th>Gene Name</th><th>Log2 Expression</th><th>P-value (t-test)</th></tr></thead><tbody><tr><td>PAL</td><td>Phenylalanine ammonia lyase</td><td>1.773</td><td>0.028</td></tr><tr><td>DXS</td><td>I-deoxy-d-xylulose-5-phosphate synthase</td><td>-1.142</td><td>0.041</td></tr><tr><td>FMO</td><td>Flavin-dependent monoxygenase</td><td>0.486</td><td>0.173</td></tr><tr><td>PR-3</td><td>Chitinase</td><td>-0.997</td><td>0.199</td></tr></tbody></table>

<footnote>*p<0.05, Kruskal-Wallis test.</footnote>
respond in a very different in fashion at the transcriptional level to both the biotrophic and necrotrophic pathogen. The more resistant clone responded systemically within 24 hours while little response at the transcriptional level was detected in the more susceptible clone in response to the biotroph, while indications of suppression in response to the necrotroph was found.

P84

The reaction zone is a unique plant defense found in trees: differentially expressed genes and cell wall changes

Carl Gunnar Fossdal¹, Nina Nagy, Hietala Ari, Igor Yakovlev, Halvor Solheim Norwegian Forest and Landscape Institute, Norway

E-mail: foc@skogoglandskap.no
BMC Proceedings 2011, 5(Suppl 7):P84

Heterobasidion annosum senso lato is the most devastating pathogen of conifers such as Norway spruce in Europe. This pathogen enter Norway spruce trees trough the roots or wounds and colonizes the tree from within, growing as a saprophyte when established within the dead heartwood and acting as a necrotroph when in contact with living host tissue. We have examined the host response in Norway spruce at the molecular level as well as the responses of the pathogen.

We have studied the defense reactions toward pathogenic fungi in the ecological and economic important conifer Norway spruce from both a molecular and anatomical perspective. We have studied the host responses of the tree as well as the attack modes and genes induced by its pathogens. The disease caused by this *H. annosum* s.l. is complex as it is can act both a necrotroph and saprotroph as well as a broad host range. Twenty percent of the trees in Norwegian spruce stands tend to be infected by this pathogen and *H. annosum* s.l. typically colonize and decay the economically important wood inside the trunk. However, Norway spruce have defences against this and other pathogens and the attack can be fought off by the living bark and sapwood but not the hearthwood being composed of dead zylem.

The bark has effective defense reactions that can be induced and we have seen indications of systemic and primed defense responses but also the sapwood has defensive capabilities. The tree has a unique defense against this internal attack by forming a reaction zone; in this case the host defense is directed inwardly by the still living sapwood toward the central colonized wood. We have studied the host defense at the transcriptional level, changes in phenols and lignification and found that the speed of the host response appear to be crucial in fending off the pathogen.

P85

Transformed Hairy Roots of the actinorhizal shrub *Discaria trinervis*: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Leandro Imanishi¹, Alice Vayssières², Claudine Franche²*, Didier Bogusz², Luis Wall¹, Sergio Svistoonoff²

¹Programa Interacciones Biológicas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes R. Sáenz Peña 352, B1876BXD Bernal, Argentina; ²Groupe Rhizogenèse, Unité Mixte de Recherche Diversité Adaptation et Développement des Plantes (DIADE), Institut de Recherche pour le Développement (IRD), 911 avenue Agropolis, BP 5045, 34394 Montpellier Cedex 5, France

E-mail: claudine.franche@ird.fr
BMC Proceedings 2011, 5(Suppl 7):P85

**Background:** Nitrogen is a major limiting factor of plant growth in many ecosystems. Root nodule symbiosis (RNS) is one of the most efficient adaptations allowing plants to cope with nitrogen deficiency by establishing a symbiotic association with diazotrophic bacteria able to produce ammonium from atmospheric nitrogen. Nevertheless RNS is restricted to two groups of plants: legumes and Parasponia (*Celtidaceae*), that interact with a group of gram-negative proteobacteria collectively called rhizobia, and actinorhizal plants, a group of 220 species, mostly shrubs and trees distributed in the orders Fagales, Cucurbitales and Rosales, that interact with gram-positive actinomycetes of the genus *Frankia* [1]. All these plants belong to the Rosid I clade, suggesting a common origin for the ability to establish RNS [2].

In recent decades a strong research effort focused on model legumes lead to the identification of key molecular actors involved in nodulation, including the bacterial signalling molecules, the Nod factors and several genes involved in the symbiotic signalling pathways [3]. Much less is known in non model legumes and actinorhizal plants, particularly in species that are not infected like model legumes through root hairs but show more ancestral infection mechanisms like crack entry or intercellular infection. Yet important cues regarding the diversity and evolution of RNS are being found precisely in these more primitive non-model systems [4,5].

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized [6,7]. Intercellular infection has been described in *Discaria trinervis*, an actinorhizal shrub belonging to the Rosales order [8]. To decipher the molecular mechanisms underlying intercellular infection with *Frankia*, we set up an efficient genetic transformation protocol for *D. trinervis* based on *A. rhizogenes*.

**Methods:** We analyzed the susceptibility of *D. trinervis* to two strains of *A. rhizogenes*: A4RS, and ARqua1; both strains contained a pHKN29 plasmid with a 35S::GFP fusion [9]. The classic *in-vitro* inoculation was compared to an *ex-vitro* method reported to be successful in several plant species [10]. The functionality of the symbiosis was tested on composite plants by performing nodulation tests and acetylene reduction assays. Using this technique, we introduced the promoter of *MtEnod11*, a nodulin gene from *M. truncatula* widely used as a marker for early infection-related symbiotic events in model legumes [11].

**Results:** Transgenic roots showing strong levels of GFP were obtained for all treatments. The *ex-vitro* method using Arqua1 was the best compromise to obtain a good co-transformation efficiency while minimizing the impact on root system architecture. Co-transformed roots were specifically and efficiently nodulated with *Frankia* and the resulting nodules were undistinguishable from non-transgenic nodules in terms of developmental timing, anatomy, nitrogen fixation and feedback control by nitrogen. The expression of reporter genes such as *GUS* and *GFP* could be easily detected within transgenic *D. trinervis* root systems. The promoter of *MtEnod11* retained its symbiotic activation in transgenic *D. trinervis* nodules. Similar results were obtained in *C. glauca* [12].

**Conclusions:** These findings open new avenues to study the genetic mechanisms of intercellular root invasion and single cell infection, allowing detailed characterization of genes involved in *D. trinervis* nodulation and a better understanding of the most ancestral infection pathways leading to RNS. In addition, because *D. trinervis* belongs to the Rosales order, evolutionary comparisons can be made with plants belonging to the same clade but unable to nodulate (most *Rosaceae*), or with *Parasponia* sp., the only non-legume able to enter RNS with rhizobia. The transformed roots in *D. trinervis* with appropriate reporter genes would be a powerful tool to explore signaling mechanisms in symbioses with this ancestral infection mode [13].

**References**

1. Vessey JK, Pawlowski K, Bergman B: Root-based N 2-fixing symbioses: legumes, actinorhizal plants, *Parasponia* sp. and *cycads*. *Plant Soil* 2005, **266**:205-230.

2. Soltis DE, Soltis PS, Morgan DR, Swensen SM, Mullin BC, Dowd JM, Martin PG: Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms. *Proc. Natl. Acad. Sci. U.S.A* 1995, **92**:2647-2651.

3. Kouchi H, Imaizumi-Anraku H, Hayashi M, Hakoyama T, Nakagawa T, Umehara Y, Suganuma N, Kawaguchi M: How many peas in a pod? Legume genes responsible for mutualistic symbioses underground. *Plant Cell Physiol* 2010, **51**:1381-1397.

4. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre JC, Jaubert M, Simon D, Cartieaux F, Prin Y: Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. *Science* 2007, **316**:1307-1312.

5. Op den Camp R, Streng A, De Mita S, Cao Q, Polone E, Liu W, Ammiraju JSS, Kudrna D, Wing R, Untergasser A, Bisseling T, Geurts R: LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume *Parasponia*. *Science* 2011, **331**:909-912.

6. Sprent JI: Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation. *New Phytol* 2007, **174**:11-25.

7. Wall: The Actinorhizal Symbiosis. *J. Plant Growth Regul* 2000, **19**:167-182.

8. Valverde C, Wall LG: Time course of nodule development in the *Discaria trinervis* (Rhamnaceae)-*Frankia* symbiosis. *New Phytol* 1999, **141**:345-354.
9. Kumagai H, Kouchi H: Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. *Mol. Plant Microbe Interact* 2003, 16:663-668.

10. Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG: Ex vitro composite plants: an inexpensive, rapid method for root biology. *Plant J* 2005, 43:449-457.

11. Journet EP, El-Gachtouli N, Vernoud V, de Billy F, Pichon M, Dedieu A, Arnould C, Morandi D, Barker DG, Gianinazzi-Pearson V: Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells. *Mol. Plant-Microbe Interact* 2001, 14:737-748.

12. Svistoonoff S, Sy M-O, Diagne N, Barker DG, Bogusz D, Franche C: Infection-specific activation of the medicago truncatula Enod11 early nodulin gene Promoter During actinorhizal root nodulation. *Mol. Plant-Microbe Interact* 2010, 23:740-747.

13. Imanishi L, Vayssières A, Bogusz D, Franche C, Wall LG, Svistoonoff S: Transformed Hairy Roots of Discaria trinervis: A valuable tool for studying actinorhizal symbiosis in the context of intercellular infection. *Mol Plant Microbe Interact* 2011 in press.

P86
DNA methylation and adaptive response in forest tree species

Angeles Guevara¹, Enrique Sáez¹, Luis-Manuel Díaz¹, Tamara Herranz¹, Carmen Barbero¹, Dolóres Vélez¹, Carmen Collada², David Sánchez-Gómez³, Ismael Aranda¹, Teresa Cervera¹*

¹Dpto. Ecología y Genética Forestal, CIFOR-INIA, Ctra. de La Coruña km 7, 28040 Madrid, Spain; ²Dpto. de Biotecnología. ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain; ³Dpto. Silvopasticultura. ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain

E-mail: cervera@inia.es

BMC Proceedings 2011, 5(Suppl 7):P86

Progressive increase of temperatures as well as longer seasonal drought periods revealed by climate studies correspond to fast environmental changes that forest species face with their actual genetic background. Natural selective processes cannot develop an adaptive response within this time frame. Thus the capability of forest tree species to adapt to the new environments will depend on their genetic background, but also rely on their phenotypic plasticity. Several reports have shown the involvement of epigenetic modifiers as the basis of the phenotypic plasticity, and in particular to the adaptation to abiotic stresses. DNA methylation (methylation of cytosine residues) is one the most important epigenetic modification in eukaryotes. It is involved in specific biological processes such as gene transcription regulation, gene silencing, mobile element control or genome imprinting. Therefore, there is a great interest in analyzing cytosine methylation levels and distribution within the genome.

In order to analyze methylation-sensitive anonymous CCGG restriction sites we used MSAP technique (Methylation-Sensitive Amplified Polymorphism), an AFLP-based technique for the analysis of cytosine methylation. The technique is based on the use of isochizomers that show differential cleavage sensitivity to cytosine methylation. *Hpall* and *Mspl* are isochizomers that are frequently used to detect cytosine methylation. *Hpall* cannot cleave if one or both cytosines are fully methylated (in both strands), whereas *Mspl* cleaves C⁵⁻⁷CGG but not ⁵⁻⁷CCGG sequences. For each sample, MSAP analysis is performed using both *EcoRI/Hpall* and *EcoRI/Mspl* digested samples. Comparative analysis between *EcoRI/Hpall* and *EcoRI/Mspl* fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that are associated with genetic variability and will show common *EcoRI/Hpall* and *EcoRI/Mspl* profiles among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with epigenetic variability and detected as amplified fragments differing in their presence or absence or in their intensity between *EcoRI/Hpall* and *EcoRI/Mspl* patterns of the same sample. Thus, full methylation of the internal cytosine at the assayed CCGG sites will be associated with fragments detected in *EcoRI/Mspl* pattern which are absent or less intense in *EcoRI/Hpall* profile. Hemi-methylation of the external cytosine will be associated with fragments observed in *EcoRI/Hpall* pattern which are absent in *EcoRI/Mspl* profile.

We have optimized DNA methylation analysis for two forest tree species, including both angiosperm (Fagus sp.) and gymnosperm (Pinus sp.) species. The set-up of the MSAP technology has allowed study intra-specific variability of DNA methylation in *Pinus pinea* L. a specie which is

characterized by a low genetic variability and high level of phenotypic plasticity. Representative populations spanning the whole distribution of this specie within Spanish geography were chosen for this study, including coastal and interior populations. MSAP patterns from vegetative propagated trees were compared among and within populations. Percentage of methylation-sensitive polymorphisms was calculated and selective markers were identified.

To test a possible role of DNA methylation in the plasticity of *Fagus sylvatica* L. in response to water deficit we analyzed two populations of beech from Spain and Sweden which differ in the response to water stress. Patterns of DNA methylation clearly differed between populations. Although the rate of DNA methylation was similar between irrigated and non-irrigated trees in each polulation after AMOVA, some specific MSAPs associated with water stress response were identified.

P87
Analysis of adaptive responses of *Pinus pinaster* to changing environmental conditions in the Mediterranean region

Marina de Miguel¹, Angeles Guevara¹, Nuria de Maria¹, Enrique Sáez¹, Luis-Manuel Díaz¹, Carmen Collada², Alvaro Soto³, Pedro Perdiguero⁴, José-Antonio Cabezas⁵, David Sánchez-Gómez⁶, Ismael Aranda¹, Teresa Cervera¹*¹
¹Dpto. Ecología y Genética Forestal, CIFOR-INIA, Ctra. de La Coruña km 7, 28040 Madrid, Spain; ²Dpto. de Biotecnología, ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain; ³Dept. Silvopasticultura, ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain; ⁴Dept. de Silvopasticultura, ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain; ⁵Dpto. Investigación Agroalimentaria, IMIDRA, Finca El Encín. Ctra A-2, Km38.200, 28800 Alcalá de Henares, Spain; ⁶Dpto. Silvopasticultura, ETSIM (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain

E-mail: cervera@inia.es

BMC Proceedings 2011, 5(Suppl 7):P87

Recent climate evolution studies highlight the progressive temperature increase and prevalence of seasonal drought, with specially incidence in the Mediterranean region. Although conifers are very important species regarding forest conservation, sustainability and productivity, given the large forest surface they cover in Spain and their active role in preventing soil erosion and desertification, we know little about the molecular mechanisms which control adaptation in this ancient taxonomic group. The work is focused on the study of adaptive responses of maritime pine (*Pinus pinaster* Ait.), one of the most important gymnosperm species in the Mediterranean region. To better understand these processes, we designed a strategy which integrates several approaches:

- Phenotypic characterization in response to drought, evaluating functional (physiological) and morphological parameters in a full-sib family originated from a controlled cross between two progenitors showing an *a priori* contrasting response to drought. In order to improve the phenotypic estimates, clonal material was generated by vegetative propagation and several ramets per clone were analyzed per experiment. Two experiments have been already developed: a) Plants from a complete full-sib family were exposed to severe drought for a short time; b) Selected clones showing contrasting behavior in their response to drought were subjected to a moderate drought for long time. The latest experiment was designed to better understand the functional mechanisms of drought tolerance. In 2011 a third experiment will be developed to evaluate survival capacity.

- Molecular characterization of the adaptive response, including:

1. Transcriptome analysis. ESTs collections associated with different tissues and growth conditions. We developed: a) Subtractive cDNA libraries to study the response of clonal material subjected to controlled hydric stress. A total of 386 unigenes have been identified from the SSH library (Sanger sequencing), 351 of them presumably corresponding to nuclear sequences. We have selected a set of 67 reliable candidate genes significantly upregulated by PEG-induced water stress, according to a microarray analysis: 45, 29 and 29 from roots, stems and needles, respectively. Functional classification of the unigenes, based on the BLAST homology analysis, showed that the largest group corresponds to genes involved in metabolism (16%). Up to 42% of these are related with carbohydrate metabolism, consistently with the role played by sugar accumulation in drought tolerance. b) cDNA library made of mRNAs extracted from buds collected from January to April, to dissect bud burst in this species. A total of 9,000 ESTs were sequenced by Sanger (CT574594
- CT583294) in collaboration with C. Plomion, UMR BIOGECO. c) A cDNA library made of mRNAs from multiple tissues (needles, stems and roots) and growing conditions (including drought stress and hormone treatments - auxin and cytokinin) collected from full-sib progeny individuals (collaboration with C. Díaz-Sala, UAH). The cDNA library was constructed by EVROGEN and used as template for Roche GS-FLX Titanium high throughput sequencing (Lifesequencing). A total of 22,427 isotigs/contigs were assembled from 1,218,000 reads. 15,722 isotigs/contigs were annotated and a total of 9,085 proteins detected.

2. Characterization of full-length cDNAs (FLcDNA). Complete and accurate annotation of functional genes rely largely on the information of FL-cDNAs since eukaryotic genes often have differential splicing of introns and unpredictable transcription starting sites, making gene prediction less accurate. In order to characterize FLcDNAs, a pool of approximately 5.000 PCR amplified inserts from selected cDNA clones (based on cDNA length versus annotation) from different EST libraries was generated. This pool of inserts was sequenced using Roche GS-FLX Titanium. A total of 3,259 isotigs from 573,242 aligned reads were obtained. This work was carried out in collaboration with S. Fluch (ARC) and C. Plomion (UMR BIOGECO).

3. SNP identification and analysis. Two sources of SNPs were used: 1) SNPs detected in a set of re-sequenced candidate genes (in vitro SNPs) used to design a 384 and a 1536 Golden Gate arrays for diversity analysis (to be used in association studies) and construction of genetic maps, and 2) SNPs detected from the third cDNA library described in the transcriptome analysis section, to design a 1536 Golden Gate array for construction of genetic maps as well as a 7600 Infinium assay for diversity and mapping purpose (the latest in collaboration with C. Plomion).

4. Construction of genetic linkage maps using dominant (SAMPLs), as well as co-dominant (microsatellites, ESTPs and SNPs) markers. Two INIA and two INRA mapping progenies will be used to construct species genetic map of *Pinus pinaster*. For this purpose, a set of common markers including SSRs, ESTPs and SNPs from three Illumina assays (384 Vera Code; 1536 Golden Gate; 7600 Infinium assays) will be performed to genotype 100 individuals per mapping progeny. Comparative mapping with other conifers will also be addressed.

5. Association studies. Association analyses are in progress by looking for association between phenotypes (growth, drought stress response) and genotypes (SNP genotypes) of 22 populations (each represented by 20 to 30 trees), spanning the natural distribution of the species (collaboration with C Plomion, UMR BIOGECO) and a metapopulation from Central Spain which consists of 5 populations (each represented by 20 to 30 trees; collaboration with C. Plomion, UMR BIOGECO and Reiner Finkeldey, UGO). QTL analysis is carried out to identify genome regions involved in the genetic control of maritime pine response to hydric stress, growth and biomass production based on association studies between genotypes of mapping populations and their corresponding phenotypes.

6. Maritime pine genome sequence. De novo sequencing of *Pinus pinaster* genome using haploid DNA will be carried out using current next-generation DNA sequencing technologies and a shotgun strategy. This approach includes: 1) >30x genome coverage based on paired-end (short-insert libraries) and additional >10x genome coverage base on mate-pairs (long-insert libraries) using HiSeq2000 sequencing; 2) use of mate-pairseqming with GS FLX Titanium to construct longer scaffolds; 3) use of transcriptomics data described in sections "a" including FLcDNAs (section "2") to confirm results; and 4) genome assembly and annotation. This research results from the collaborative work between Spanish groups from UMA, PAB, UV, UPM, CNAG, BSC, Lifesequencing and CIB-CSIC coordinated by CIFOR-INIA and UAH and it is supported by the Spanish Ministry of Science and Innovation.

P88

The effect of heterologous VHb expression to the functioning of stress-
related genes in hybrid aspen lines exposed to biotic stress

Hely Häggman¹*, Suvi Sutela¹, Soile Jokipii-Lukkari¹, Tiina Ylijoa², Risto Kasanen³,
Anna-Kaisa Anttila¹, Marko Suokas¹, Estelle Dumont¹, Pauli T Kallio⁴

¹Department of Biology, University of Oulu, Finland; ²Finnish Forest Research Institute, Suonenjoki Research Unit, Finland; ³Department of Forest Sciences, University of Helsinki, Finland; ⁴Institute of Microbiology, ETH-Zürich, CH-8093 Zürich, Switzerland

E-mail: hely.haggman@oulu.fi

BMC Proceedings 2011, 5(Suppl 7):P88

**Background:** The hemoglobin produced by *Vitreoscilla* bacterium, VHb, is capable to promote the respiratory activity and ATP production under hypoxic conditions [1]. In micro-organisms grown under oxygen-limited conditions, the heterologous *vhb* expression improves the production of primary and secondary compounds, growth as well as stress tolerance [2,3]. VHb has also been shown to protect plants against deleterious effects of nitric oxide (NO), the important signalling molecule produced during pathogen challenges [4]. We have previously studied the *vhb* expressing hybrid aspen lines (*Populus tremula* L. x *tremuloides* Michx.) under elevated UV-B illumination and showed that the VHb lines had increased secondary metabolite production and also enhanced accumulation of starch in chloroplasts when grown in standard greenhouse conditions [5]. In order to reveal the effect of VHb on the expression of stress-related genes in hybrid aspen exposed to biotic stress, we conducted experiments with pathogenic fungus and larvae of insect herbivore.

**Materials and methods:** In the herbivory experiment, two-year-old VHb line and non-transgenic hybrid aspen line were exposed to the feeding of chestnut moths larvae (*Conistra vaccinii* L.) for 24 hours. Four VHb hybrid aspen lines and two non-transgenic hybrid aspen lines were infected with pathogenic fungus *Venturiatremulae*, *in vitro*. Samples for the RNA and protein extractions were collected after 10 and 21 days of the pathogen inoculation. In both experiments, the transcriptome changes were studied with microarrays and real-time RT-PCR. The cDNA slides contained 8256 stress-related oligos of *Populus euphratica*. The relative expression of selected genes were conducted with the LightCycler® 2.0 (Roche) and LightCycler® 480 instruments according to the manufacturer's chemistry. The amount of VHb protein during pathogen fungus experiment was analysed by Western blots.

**Results and conclusions:** The consumption of the leaves by the chestnut moth larvae was comparable on the non-transgenic and the VHb line. The effect of herbivory to the gene expression levels of wounded and systemic leaves was similar in the VHb and non-transgenic hybrid aspen lines, but in the non-transgenic line, the detected changes were more severe than in the VHb hybrid aspen line. The genes encoding bark storage proteins (BSPs), copper chaperone (CCH), and nitrate transporter (NRT) were among the limited number of genes which showed different expression changes between the VHb and non-transgenic hybrid aspen line.

The hybrid aspen lines infected with *V. tremulae* showed symptoms of shoot blight i.e. blackened stems and leaves, but the amount of VHb protein did not correlate with the severity of the disease. At the transcriptome level, the studied lines responded to the pathogen treatment individually in the first sampling (10 days) whereas the expression profiles of the lines resembled each other in the second sampling (21 days). The functional grouping of the genes with known mode of action resulted most representatives in protein metabolism, photosynthesis, stress as well as RNA metabolism groups.

In general, the transcriptome profiling of fungus- and herbivory-induced responses did not reveal any biological or molecular process altered in all of the VHb lines due to the biotic interactions. Moreover, based on the real-time RT-PCR results, the *vhb* expression did not show induction after the 24-hour herbivore treatment or the inoculation with the *V. tremulae*.

References

1. Kallio PT, Kim D-J, Tsai PS, Bailey JE: Intracellular expression of *Vitreoscilla* hemoglobin alters *Escherichia coli* energy metabolism under oxygen-limited conditions. *Eur. J. Biochem* 1994, 219:201-208.

2. Frey AD, Kallio PT: Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. *FEMS Microbiol. Rev* 2003, 27:525-545.3.

3. Jokipii-Lukkari S, Frey AD, Kallio PT, Häggman H: Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants. *J. Exp. Bot* 2009, 60:409-422.

4. Mur LAJ, Carver TLW, Prats E: NO way to live; the various roles of nitric oxide in plant-pathogen interactions. *J. Exp. Bot* 2006, 57:489-505.

5. Häggman H, Frey AD, Ryynänen L, Aronen T, Jalkunen-Tiitto R, Tiimonen H, Pihakaski-Maunsbach K, Jokipii S, Chen X, Kallio PT: Expression of *Vitreoscilla* haemoglobin in hybrid aspen (*Populus tremula* x *tremuloides*). *Plant Biotechnol. J* 2003, 1:287-300.
P89

Increased *Bacillus thuringiensis* δ-endotoxin Cry3Aa toxicity against longhorned beetle by fusing to peptide specifically binding to beetle Cx-cellulase

Shu-Tang Zhao¹*, Chang-Hua Guo², Jian-Jun Hu¹, Xiao-Jiao Han², Jun Chen¹, Meng-Zhu Lu¹

¹State Key Laboratory of Forest Genetics and Tree Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, PR China;

²International Centre for Bamboo and Rattan, Beijing 100102, PR China

E-mail: shutang@caf.ac.cn

BMC Proceedings 2011, 5(Suppl 7):P89

**Background:** *Bacillus thuringiensis* (Bt) Cry toxins have specific toxicity to susceptible insects. They are being used in transgenic plants or spray to control insect pests in agriculture [1, 2, 3]. Cry3A toxins are used extensively for biological control of coleopteran larvae [4, 5]. A Bt886-Cry3Aa gene that exhibited a high activity against Coleoptera insects isolated Our laboratory. Insect bioassay performed on *Anoplophora glabripennis* Motsch and *Apriona germari* Hope showed that the mortality of larvae fed with the product of this gene was over 60% [6]. However, both transgenic poplar with native *Cry3Aa* and withmodified-*Cry3Aa* by using poplar-preferred codons did little effects on longhorned beetles probably due to its low expression level in poplar.A peptide (LPPNPTK) named PCx that specifically bind to cellulase from midgut of longhorned beetle larvae was screened out from a phage display library previously in our laboratory[7].

**Materials and methods:** Fused Cry3Aa genes with PCx coding sequence at 5' or 3'-end were amplified using pET-30a(+-)Cry3Aa [6] as a template and designed primers, and, used to construct three recombinant plasmids (pET-30(+)-PCx-Cry3Aa, pET-30(+)-Cry3Aa-PCx and pET-30(+)-Cry3Aa). Target proteins were characterized by Western Blot, ELISA and LC-MS/MS methods. To analyze the activity of PCx-Cry3Aa, Cry3Aa-PCx and Cry3Aa proteins against longhorned beetle larvae, bioassays were performed on *A. germari*Hope larvae by artificial feed with toxins. The retaining time of target proteins in midgut and the cellulase activity of longhorned beetle larvae were measured in order to elucidate the potential mechanism of the fused toxins of PCx and Cry3Aa against longhorned beetle larvae.

**Results:** Expression products contained target proteins were characterized by SDS-PAGE, Western-blot and ELISA after induced by IPTG. The target bands were digested and analyzed by LC-MS/MS to further confirm them as Cry3Aa proteins. The bioassay showed that the mortality of larvae fed with the two fused Cry3Aa proteins (PCx-Cry3Aa and Cry3Aa-PCx) was up to three times higher than that fed with Cry3Aa (Figure 1). Retaining time analysis was performed on excretas that

collected at different times after feeding. The result showed that the fused Cry3Aa was concentrated in excreta collected at 6 h, whereas Cry3Aa at 4 h. Meanwhile, the Cry3Aa concentration in midgut juice after fed with fused Cry3Aa was higher than that with Cry3Aa alone. This indicated that the retaining time of fused PCx-Cry3Aa in midgut of larvae is longer compared to that of Cry3Aa alone (Figure 2). In addition, we also analyzed the cellulase activity when bond with fused Cry3Aa or Cry3Aa alone and showed that the fused protein did not affect the activity of cellulase. Therefore, the remaining time of fused Cry3Aa is prolonged after binding with cellulase, thus the enhanced toxicity of fused Cry3Aa is due to the prolonged retaining time.

**Conclusions:** These data demonstrate that the cellulase-binding peptide could enhance the toxicity of *Bacillus thuringiensis* Cry3Aa against the longhorned beetle. We also confirmed that the increased lethality in larvae fed with PCx-Cry3Aa or Cry3Aa-PCx was attributable to the ability of the toxin to bind Cx-cellulase, thereby increasing toxin retention in the midgut. These uniquely modified Cry3Aa proteins have potential use for pest control. The significantly enhanced activity of Cry3Aa fused with the Cx-cellulase-binding peptide provides a new strategy for increasing δ-endotoxin efficacy against the longhorned beetle.

<figure><img src="image_39.png" /><figcaption>**Figure 1(abstract P89)** Toxicity of Cry3Aa and the Cry3Aa fusion proteins against *A. germari*Hope larvae. Corrected mortality rates are shown for larvae fed with Cry3Aa, PCx-Cry3Aa, or Cry3Aa-PCx. Both Cry3Aa fusion proteins exhibited increased toxicity toward the larvae, compared with non-modified Cry3Aa.</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>**Figure 2(abstract P89)** Concentration of Cry3Aa in *A. germari* Hope larvae after feeding with Cry3Aa and the Cry3Aa fusion proteins. a, Cry3Aa was measured in larval excretions collected at 2, 4, 6, and 8 h after feeding. The Cry3Aa content peaked at 4 h after feeding with Cry3Aa, whereas the peak was observed at 6 h after feeding with the Cry3Aa fusion proteins. b, Cry3Aa was measured in larval midgut extractions at 4 h after feeding. The concentration of Cry3Aa in the midgut extracts was higher after feeding with the fusion proteins, compared with non-modified Cry3Aa.</figcaption></figure>
P90

Metrology of morphological response of Siberian elm to drought stress:
increased stomatal pore depth

Ki Woo Kim¹, Go Eun Park², Don Koo Lee²

¹School of Ecological and Environmental System, Kyungpook National University, Sangju, 742-711, Korea; ²Department of Forest Environmental Science, Seoul National University, Seoul, 151-742, Korea

E-mail: kiwoo@knu.ac.kr

BMC Proceedings 2011, 5(Suppl 7):P90

**Background:** Structural characteristics of stomata consist of stomatal shape, density, depth, and pore dimension. Commonly called Siberian elm, the species *Ulmus pumila* L. is a fast-growing and small to medium-sized tree. Due to the superb adaptations to the harsh conditions of the Gobi Desert, the trees have been preferentially planted in Mongolia. It is worthwhile to investigate the morphological characteristics of the tree species that are tolerant to drought stresses in such arid areas.

**Materials and methods:** Based on average annual precipitations, two types of leaf specimens were collected from Korea, China, and Mongolia:

(i) leaves under normal environmental conditions and (ii) leaves under arid conditions. Leaf stomatal characteristics of Siberian elm were investigated by electron microscopy and white light scanning interferometry [1].

**Results and conclusions:** Field emission scanning electron microscopy revealed stomata on the lower leaf surface of the tree species. Measured as ca. 30 micrometers in width, the stomata appeared to be oval in shape. In-lens secondary electron imaging by a coaxial annular type detector showed a difference in depth from epidermis to pore between the two types of leaves. Leaf stomata under arid conditions appeared to have higher levels of depth from epidermis to pore than ones under normal conditions (Figure 1). Line profile analysis by white light scanning interferometry allowed for the nondestructive measurement of the stomatal dimension (Figure 2). The depth from epidermis to pore of stomatal complexes under normal conditions was ca. 1.79±0.13 micrometers, whereas that under arid conditions was ca. 2.12±0.08 micrometers. In addition, higher levels of surface roughness were observed in stomata under arid conditions than those under normal conditions. These results suggest that increased stomatal pore depth would be responsible for the adaptations of the tree species to arid conditions. Furthermore, such architectural differences in

<figure><img src="image_41.png" /><figcaption>**Figure 1(abstract P90)** Field emission scanning electron micrographs of stomata of Siberian elm. (A) Stoma under normal conditions. (B) Stoma under arid conditions.</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>**Figure 2(abstract P90)** Three-dimensional surface plot of a stoma under arid conditions by white light scanning interferometry.</figcaption></figure>
stomatal dimension could be quantitatively analyzed by complementary microscopy.

Reference

1. Kim KW, Lee IJ, Kim CS, Lee DK, Park EW: Micromorphology of epicuticular waxes and epistomatal chambers of pine species by electron microscopy and white light scanning interferometry. *Microsc Microanal* 2011, **17**:118-124.

P91

Regulation of the boron transporter EgBor1 in *Eucalyptus globulus*: a plausible model

Hans Pieringer<fnref n="1" />, Fabiola Baltierra<fnref n="1" />, Maria Cecilia Gamboa<fnref n="1" />*, Erwin Krauskopf²

Boron is a micronutrient that plays an important role in plant cell wall biosynthesis. Nevertheless, an excess of boron in the soil causes severe damage to the respiratory tissue of the plant. In *Arabidopsis thaliana*, the *Atbor1* gene encodes a boron transporter that distributes this element throughout the plant.

A cDNA sequence encoding a *bor1* transporter was isolated from a *Eucalyptus globulus* cDNA library. This sequence contains several stop codons within the coding region. Initial bioinformatic analyses suggest that this interruption corresponds to an intron that may generate a truncated protein.

*Egbor1* was overexpressed in *Saccharomyces cereviseae* to assess whether it was capable of restoring the phenotype of a mutant strain that lacks the boron transporter. It was also overexpressed in a wild type strain as a control. In both cases a significant increase in boron tolerance was observed, suggesting that the encoded transporter is functional. Subsequently, a western blot analysis showed that the expressed transporter corresponds to the product of the full-length protein, rather than the truncated protein.

Additional bioinformatic analyses showed that the intron presents several regulatory elements, therefore it may function as a promoter for a small protein encoded in the 3' region of *Egbor1*. Transient expression of GUS under the control of the intron sequence proved its capability to activate gene expression. Thus, we have identified three ORFs in the *Egbor1*

sequence: a full length protein encoded by the spliced mRNA named fragment C, and two smaller proteins encoded by the 5' and the 3' regions of the non spliced mRNA named fragments A and B.

We are currently challenging the yeast mutant strain with all the putative encoded proteins to understand their specific role in boron transport.
Funded by PFB-016 and DI-UNAB.

P92

The expressed genes of Japanese red pine (*Pinus densiflora*) involved in
the pine wilt disease severity

Hiroyuki Kuroda<fnref n="1" />*, Susumu Goto², Etoh Kazumi³, Keiko Kuroda⁴

**Background:** Japanese pine trees have been severely damaged by the pine wilt diseases, and hardly been keeping the productive forest in Japan. The pine wood nematode, *Bursaphelenchus xylophilus*, is a virulent pathogen in Japan, while it is originated and is an endemic one in the United State [1]. The diseases have now been spread not only in East Asia but also in a part of Europe. The Japanese Government has been developed the resistant pine breed varieties which had selected the survived-tree lineages obtained from the damaged pine forests. The developed varieties vary in the disease severity depending on the individual trees even in the same variety, partly because of the wind-pollination. Thus it needs molecular indices to select the resistant individuals without nematode infection.

Shin et al. [2] reported up-regulated genes in a non-selected, or susceptible, Japanese red pine, *Pinus densiflora*, after the nematode inoculation. Defense responses distinct from the susceptible trees may be expressed in the resistant bleed variety against the pine wood nematodes. By comparing the expressed gene profiles between the resistant and susceptible varieties against the diseases, a molecular clue for the resistant response against the diseases will be provided. Here, we have compared gene expression profiles between the susceptible and resistant breed varieties of Japanese red pine at the stage when the defense response had

<figure><img src="image_43.png" /><figcaption>Figure 1. A Schematic Diagram of the Pine Defense.

Triangles show secreta from the nematode</figcaption></figure>

**Figure 1(abstract P92)** A schematic diagram of the pine defense. Triangles show secreta from the nematode.

<footnote n="1">¹Fundacion Ciencia para la Vida, Santiago, Chile; ²Facultad de Ciencias</footnote>

<footnote>Biologicas, Universidad Andres Bello, Santiago, Chile</footnote>

<footnote>E-mail: cecy_gamboa@yahoo.es</footnote>

<footnote>BMC Proceedings 2011, 5(Suppl 7):P91</footnote>

<footnote n="1">¹Institute of Sustainable Science, Kyoto University, Uji, Kyoto, Japan; ²Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan; ³Wood Research Institute, Kyoto University, Uji, Kyoto, Japan; ⁴Graduate School of Agricultural</footnote>

<footnote>Science, Kobe University, Kobe, Japan</footnote>

<footnote>E-mail: hkuroda@rish.kyoto-u.ac.jp</footnote>

<footnote>BMC Proceedings 2011, 5(Suppl 7):P92</footnote>
just established after the nematode inoculation. We also discuss on the
gene components involved in the disease severity or resistance.

**Methods:** Pine wood nematodes were inoculated on 2-year-old seedlings of a non-selected susceptible and one of the strongest resistant breed variety, respectively. The five samples were harvested at every 7 days and counted the nematode numbers of the samples. They are also stored at -80°C for RNA extraction. The nematodes were collected by Baermann funnel technique and counted under a stereomicroscope to judge the resistance. RNAs were extracted from the resistant and susceptible stems judged at the stage when the nematodes had just started to propagate, by Quagen RNeasy Plant Mini Kit with minor modification. The cDNAs obtained from the resistant and susceptible breed varieties, were differentially screened by Megasort beads technology. The 1507 and 1329 cDNAs, or ESTs, from the respective breed varieties, were effectively sequenced and assembled as the contigs. They were subjected to Blast retrieval against various public databases for their assignments. The data sets obtained were analyzed in *silico*.

**Results and conclusions:** A contig sequence is composed of ESTs, and it approximates an expressed gene species, while the numbers of ESTs in a contig approximate the expressed level of the gene. Comprehensive expression analyses show that the contig numbers, i.e. gene species, were more abundant in a resistant breed variety than those in a susceptible one. The contigs expressed more than 4 ESTs, being composed of 803 ESTs, were three times abundant in the resistant variety than in the susceptible one. The defense response might be triggered by the nematode invasion in a resistant variety but not in a susceptible one. Alternatively, it may be caused by the suppressed gene expression in the susceptible one. The latter is more probable because the signal transduction involved in the resistant response had blocked in the susceptible variety (Figure 1).

The flavonoid biosynthesis had been transcriptionally more expressed in the resistant variety than in the case of the susceptible one. One of the authors had suspected that nematidical stilbenoids, which are abundantly localized in pine heartwood, might be involved in the disease resistance [3]. The stilbenoid biosynthesis was activated by methyljasmonate or salicylate application but did not detect in this experiment at the stage when the resistant variety had just established defending response. The phenolic metabolites were more produced in the resistant variety comparing to the case of the susceptible one, supporting the results from the transcriptional observation. Some pathogen related (PR) proteins were not observed in the resistant breed but were up-regulated in the susceptible one, suggesting such proteins may be induced by stress and not directly involved in the defense response. A cluster, being composed of the contigs, approximates a gene family and represents a hot spot in the mutation which may or may not be involved in the defense response. We will also discuss those gene families.

**Acknowledgements:** The authors are deeply expressed their thanks to Dr. Ohhira, M. and Dr. Fujisawa, Y. of Forest Tree Breeding Center, FFPRI, for their kind supply of shoots of a resistant variety after the nematode infection. The study was supported by the grants from ISS, Kyoto University and also from the JSPS for HK.

**References**

1. Pine Wilt Disease. Tokyo Berlin Heidelberg New York: Springer: Zhao, BG., Futai, K., Sutherland, JR., Takeuchi, Y. 2008.

2. Shin H, Lee H, Woo K-S, Noh E-W, Koo Y-B, Lee K-J: Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine. *Tree Physiology* 2009, 29:411-421.

3. Kodan A, Kuroda H, Sakai F, A stilbene synthase from Japanese red pine (*Pinus densiflora*): Implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis. *Proc. Nat. Acad. Sci. USA* 2002, 99:3335-3339.

P93

Occurrence of African cassava mosaic virus (ACMV) and East African cassava mosaic virus – Uganda (EACMV-UG) in *Jatropha curcas*

Rose Ramkat, Alberto Calari, Fatemeh Maghuly, Margit Laimer
Plant Biotechnology Unit (PBU), Dept. Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria
E-mail: m.laimer@iam.boku.ac.at
BMC Proceedings 2011, 5(Suppl 7):P93

**Background:** *Jatropha curcas* is a drought resistant shrub native in tropical America, now widely grown in many tropical and subtropical

regions for biodiesel production [1]. First reports on virus infections in *Jatropha* indicated the occurrence of viruses closely related to *Cassava mosaic virus* in India, reaching a disease incidence from 25 to 47%. This might represent a major constrain to the production of *Jatropha* in large scale [2]. The genome of *Cassava mosaic geminiviruses* (CMG) consist of two components termed DNA A and DNA B (~ 2.7 – 3.0 kb) [3]. Furthermore, *Jatropha* has been described as host of *Cucumber mosaic virus* (CMV) [2].

**Methods:** In this study we attempted to detect and molecularly characterize viruses infecting *Jatropha* in Eastern Africa (Kenya and Ethiopia). Detection methods will be valuable tools for early screening of plant viruses in order to make appropriate decisions and selection of planting material.

A total of 127 *Jatropha* samples from Ethiopia and Kenya (districts: Kakamega, Siaya, Busia and Nakuru showing typical virus symptoms and symptomless plants were used in this study. ELISA was performed to detect the presence of three RNA viruses: CMV, *Cassava common mosaic virus* (CsCMV) and *Cassava brown streak virus* (CBSV). PCR was performed using newly designed primers based on multiple alignments of full length DNA A sequences of geminiviruses available in the NCBI Genbank, reported to infect either *Jatropha* or cassava. This allowed to amplify the variable regions of full length (2800 bp) and shorter sequences (380-1085 bp). PCR products were sequenced. A phylogenetic tree was constructed from multiple alignments by performing a heuristic search. Multiple alignments were analyzed by maximum parsimony with full-length DNA A using Phylogenetic Analysis Using Parsimony (PAUP) and a bootstrap analysis with 1000 replicates.

**Results and conclusions:** None of the *Jatropha* samples analysed was infected with the RNA viruses CBSV, CMV and CsCMV. PCR primers amplifying a 380 bpfragment of AC1, AC2 and AC3 yielded positive results with 75% of the symptomatic samples from Kenya and further detected 20% of asymptomatic samples as positive. Furthermore, 61% of symptomatic *Jatropha* samples from Ethiopia were positive. Full length primers were able to detect 69% symptomatic *Jatropha* samples from Kenya, and also in 67% of asymptomatic samples. PCR analyses of sample K1J5 amplified the expected 2.8 kb of a near full length DNA A component of the *Begomovirus* sequence and an additional shorter fragment.

Complete nucleotide sequences of 34 DNA A components typical of Begomoviruses were determined in the Kenyan samples. Thirty three sequences ranged from 2770 bp to 2816 bp while one (K1J5) consisted only of 1416 bp and termed as a defective (Def) DNA. Phylogenetic analyses indicated that the defective molecule belongs to geminiviruses involved in CMG, representing a Def from DNA A of the bipartite *Begomovirus ACMV*. All viruses characterized in this study grouped with two previously identified Begomoviruses found in cassava in Western Kenya, namely EACMV - UG and ACMV. The Def DNA showed 96.6% sequence identity with the ACMV reference sequence [GenBank NC001467.1].

In this study, we report for the first time the detection of Begomovirus: ACMV and EACMV - UG in *Jatropha* from Kenya. From an evolutionary perspective, the phylogenetic data indicate that the virus isolates from the study were closely related to those isolated previously in Western Kenya from cassava [4,5]. Recombination and synergism that have long occurred in cassava [6,7] could have led to the recent spread of the virus in the field to infect *Jatropha*. Presence of EACMV - UG and ACMV on different *Jatropha* plants in the same field indicates the opportunity for mixed infections, hence offering good opportunities for more recombination to occur. EACMV - UG and ACMV are associated with severe synergistic epidemics on cassava. Synergism lead to a 10 - 50 fold increase in viral DNA accumulation which substantially increases the potential for a higher efficiency of vector transmission to even infect non cassava host plants [6,7]. This explain why EACMV - UG is the predominant virus in *Jatropha*. The deletions occurring in the Def DNA found in the study might affect the replication of the molecule and it might depend entirely on its helper virus for replication.

There is a possibility of *Cassava mosaic virus* in *Jatropha* being more wide spread than anticipated, since we have detected it also in *Jatropha* samples from Ethiopia.

**Acknowledgements:** The financial support of FFG project 820132 and Biplant R & D is kindly acknowledged.

**References**

1. Openshaw K: A review of *Jatropha curcas*: an oil plant of unfulfilled promise. *Biomass Energy* 2000, 19:1-15.
2. Raj SK, Snehi SK, Kumar S, Khan MS, Pathre U: First molecular identification of begomovirus in India that is closely related to *Cassava mosaic virus* and causes mosaic and stunting of *Jatropha curcas* L. *Australasian Plant Dis Notes* 2008, **3**:69-72.

3. Yadava P, Suyal G, Mukherjee SK: Begomovirus DNA replication and pathogenicity. *Current Science* 2010, **98**:360-368.

4. Bull SE, Briddon RW, Ssserubombwe WS, Ngugi K, Markham PG, Stanley J: Genetic diversity and phylogeography of *cassava mosaic viruses* in Kenya. *J Gen Virol* 2006, **87**:3053-3065.

5. Stanley J, Gay MR: Nucleotide sequence of cassava latent virus DNA. *Nature* 1983, **301**:260-262.

6. Zhuo X, Liu Y, Calvert L, Munoz C, Otim-Nape GW, Robinson DJ, Harrison BD: Evidence that DNA -A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination. *J Gen Virol* 1997, **78**:2101-2111.

7. Harrison BD, Zhou X, Otim-Nape GW, Liu Y, Robinson DJ: Role of a novel type of double infection in geminivirus induced epidemic of severe cassava mosaic in Uganda. *Ann App Biol* 1997, **131**:437-448.

8. Legg JP, Fauquet CM: Cassava mosaic geminiviruses in Africa. *Plant Mol Biol* 2004, **56**:585-599.

9. Monde G, Walangululu J, Winter S, Bragard C: Dual infection by cassava begomoviruses in two leguminous species (*Fabaceae*) in Yangambi, North Eastern Democratic Republic of Congo. *Arch Virol* 2010, **155**:1865-1869.

P94

Characterization of responses to flooding and post - flooding recovery in two *Populus deltoides* clones: physiological and biochemical aspects

Maria Emilia Rodriguez¹, Guillermo Doffo¹, Fabio Achinelli², Carlos Bartoli¹, Virginia Luquez¹*

¹Instituto de Fisiología Vegetal (INFIVE), CONICET CCT La Plata - FCAyF UNLP, CC 327, La Plata, Buenos Aires, 1900, Argentina; ²Instituto de Fisiología Vegetal (INFIVE), CONICET CCT La Plata - FCAyF UNLP, CC 327, La Plata, Buenos Aires, 1900, Argentina. CIC-Buenos Aires

E-mail: vluquez@agro.unlp.edu.ar

BMC Proceedings 2011, 5(Suppl 7):P94

**Background:** In Argentina, most poplar plantations are located at the Paraná River Delta. Climate change models indicate an increase of flooding events for the next decades in this area. Therefore, poplar genotypes with a higher flooding tolerance will be needed for the forest activity. Whether this aim is achieved through traditional breeding or biotechnological developments, it will be important to gain a better understanding of poplar responses to flooding at physiological, biochemical and molecular levels.

In a previous work, we characterized the growth and morphological responses of several poplar clones to 35 days of flooding of the root system [1]. From this group, we selected two *Populus deltoides* clones with different growth responses to flooding: Alton and Stoneville 67. In clone Alton, above ground growth is not affected by flooding, while in Stoneville 67 growth is reduced after the second week of flooding. In the present work, our aim was to identify mechanisms underlying the differences in growth responses to flooding between the two clones. We characterized physiological and biochemical responses in roots and leaves, during a flooding period of 28 days, followed by an after flooding period of three weeks.

**Materials and methods:** Two *Populus deltoides* clones were used, Alton and Stoneville 67. Plants were obtained from 20 cm length one year old cuttings. The cuttings were planted in 3.5 L pots filled with a mix of topsoil and sand 50:50, and grown in a greenhouse in a totally randomized design; pots were watered daily to field capacity until the start of the treatment. The flooding treatment was started when the plants reached 50 cm of height. Flooding was imposed by covering the pots with tap water up to 5 cm over the surface soil, during 28 days, after this period flooding was ended and pots previously flooded were removed from the water and left to drain.

Growth in height was measured weekly with a graduate stick. Stomatal conductance was measured in the last expanded leaf with a Decagon SC1 porometer. Electrolyte leakage was measured in root tips. Samples were taken from leaves and roots to measure oxidative damage by thiobarbituric acid reactive substances (TBARs) method and enzymatic

and non-enzymatic antioxidants. Leaf discs were taken from the last expanded leaf to measure protein content using the Bradford method. This destructive sampling was carried on in the following dates: day 0 (before the start of flooding); 2 weeks after the start of flooding, 4 weeks after the start of flooding; 1 day after the end of flooding, and 1, 2 and 3 weeks after the end of flooding.

**Results and discussion:** After 28 days of flooding, growth in height was reduced 14% in flooded plants of Stoneville 67 compared with non-flooded control plants, but it was not affected in Alton. This was a lasting difference; three weeks after the end of flooding the height of previously flooded Stoneville 67 plants still was 15% lower than the control plants. Stomatal conductance was reduced by flooding in both clones, but in Stoneville 67 to a greater extent: 81% compared with only 63% in Alton. The recovery of the stomatal conductance to the control levels after flooding was slow and occurred only after two weeks, and was faster in Alton than in Stoneville 67.

Protein soluble content per unit leaf area of the last expanded leaf increased in flooded plants of both clones, in Alton the increase was two fold, and 2.5 times in Stoneville 67, 28 days after the start of flooding. This increase is due to changes in the structure of the leaves of flooded plants; these leaves have a lower area, and a higher number of stomata and epidermal cells than the control leaves per unit area (Rodriguez et al. unpublished results). Likely, these alterations in the leaf structure will have effects in the photosynthetic activity of leaves.

In several species, the sudden exposure of tissues to oxygen after a period of anoxia can cause oxidative stress; this damage is known as post-anoxic injury [2]. We measured electrolyte leakage to estimate membrane damage. Electrolyte leakage was measured in roots, since these were the organs that were submerged and experienced lack of oxygen. The value was higher in Stoneville 67 than in Alton after 28 days of flooding, but there were no differences between clones or treatments 24 h after the end of flooding and thereafter. Since electrolyte leakage is a general measurement of membrane damage, it cannot be attributed solely to oxidative stress. Therefore, we are measuring TBARs content to confirm if this damage is caused by oxidative stress.

As a preliminary conclusion, it seems that clone Alton is less sensitive to flooding than Stoneville 67 because its leaf stomatal conductance and root membrane integrity are less affected by flooding, and its recovery after the end of the stress period is faster.

**References**

1. Luquez V, Achinelli F, Cortizo S, Guiamét JJ: Responses to flooding of poplar clones planted in the Paraná River Delta, Argentina. *Proceedings of the IUFRO Conference: Adaptation of Forest and forest management to climate change with emphasis on forest health: a review of science, policies and practices* Umeå, Sweden 2008 [http://www.forestadaptation2008.net/en/].

2. Braendle R, Crawford RMM: Plants as amphibians. *Perspectives in Plant Ecology, Evolution and Systematics* 1999, **2**:1/56-78.

P95

Molecular integration of light responses in *Arabidopsis*

Juan Pablo Matte Risopatron¹, Yuqiang Sun², Liping Ke², Errin Johnson¹, Neil Wilson¹, Amandeep Kour¹, Steven Swain³, Goran Sandberg⁴, Brian Jones¹

¹Faculty of Agriculture, Food and Natural Resources, University of Sydney, Australia; ²College of Life and Environmental University, Hangzhou Normal University, China; ³Plant Industry, CSIRO, Black Mountain, Canberra, Australia;

⁴Department of Plant Physiology, Umeå University, Sweden

E-mail: jmatte9966@uni.sydney.edu.au

BMC Proceedings 2011, 5(Suppl 7):P95

Growth habit and developmental phase transitions in plants are acutely responsive to the quantity and quality of incident light. Information about neighbouring plants is primarily perceived by changes in the light environment. Arabidopsis is able to distinguish these changes through the phytochrome (PHYA-E) red light receptors that perceive changes in the red to far red (R:FR) ratio, and the cryptochrome and phototropin blue light receptors. Phytochrome, in its biologically active far-red light-absorbing form (Pfr), mediates its effects by directly binding with the basic helix-loop-helix (bHLH) phytochrome-interacting factors (PIFs). High R:FR ratios initiate phytochrome (Pfr)-PIF binding and PIF protein degradation. Low R:FR ratios photo-convert Pfr into the inactive form Pr
<figure><img src="image_44.png" /><figcaption>Figure 1 (abstract P95)</figcaption></figure>

allowing the gene expression that leads to a range of adaptations, including the shade avoidance responses. PIF1 regulates the expression of a number of genes by directly binding to a G-box element in their promoter. T-DNA insertional mutagenesis of a number of these genes results in pleiotropic effects associated with an altered capacity to respond appropriately to environmental cues, including those signalling changes in soil water status and the ambient light environment. The data show evidence of a perturbation of light regulated response processes, including germination, the shade avoidance responses, stomatal conductance, flowering and the circadian clock perturbation in the PIF-regulated gene mutants. The *pif1,3,4,5* quadruple and one of the PIF-regulated gene mutants fail to show shade avoidance responses in older plants grown under shade inducing conditions. This suggests that the encoded proteins mediate a parallel pathway in the induction of the SAS. The implications of our findings for an understanding of how plants appropriately respond to changes in their environment will be discussed.

significantly to the paper pulp industry due to their favourable wood fibre properties. Eucalypt plantation trees are affected by numerous pathogens during their lifetime, some of which can cause severe losses such as *Phytophthora* spp and *Chrysoporthe* spp. Plant defence mechanisms against pathogens is currently better understood in the model plant *Arabidopsis thaliana* where it has been shown that the salicylic acid (SA) and jasmonic acid (JA) signalling pathways enhance resistance against biotrophic and necrotrophic pathogens, respectively [1]. This process involves the up-regulation of specific defence genes which are considered to be marker (diagnostic) genes for the two signalling pathways [2,3].

**Methods:** The aim of this study was to utilize the draft (8X) *E. grandis* genome sequence that has recently become available (http://eucalyptusdb.bi.up.ac.za) to identify *Eucalyptus* orthologs of defence marker genes (e.g. *PR2*; *PR3*; *PR4*; *PR5*; *LOX2*) specific for the SA and JA signalling pathways [2,3]. Bioinformatics tools were used to identify putative orthologs of these marker genes in *E. grandis* based on sequence information from other plants. This was followed by a co-phylogenetic analysis in which a neighbour-joining tree with 10 000 permutations was constructed to add confidence that the correct orthologs had been identified. In the phylogenetic tree analysis, closely related family members of a particular gene were added to increase the certainty and accuracy of selecting a specific ortholog.

The expression profile of the putative marker genes was assessed via Reverse Transcription quantitative PCR (RT-qPCR) analysis of transcript levels following treatment with various concentrations of the inducers (SA and JA) as well as different time points. This was done to confirm that the putative orthologs respond to the appropriate pathways in *Eucalyptus*. Additionally the expression profile of these putative orthologs was analyzed in response to the causal agent of *Eucalyptus* stem canker, *Chrysoporthe austroafricana*. The defence response of *Eucalyptus* to this nectrotrophic pathogen was investigated in both a tolerant (*EgrTOL*) and
<table><caption>**Table 1(abstract P96) Selected results from the dose response and specificity trial**</caption><thead><tr><th rowspan="2">Marker</th><th colspan="2">SA (5mM)</th><th colspan="2">MeJA (100μM)</th></tr><tr><th>Exp Ratio*</th><th>P - value</th><th>Exp Ratio*</th><th>P - value</th></tr></thead><tbody><tr><td>PR2</td><td>4.05</td><td>0.01</td><td>-0.34</td><td>0.28</td></tr><tr><td>PR3</td><td>-4.09</td><td>0.03</td><td>0.9</td><td>0.05</td></tr><tr><td>PR4</td><td>1.3</td><td>0.25</td><td>1.5</td><td>0.00005</td></tr><tr><td>PR5</td><td>0.5</td><td>0.3</td><td>1.6</td><td>0.09</td></tr><tr><td>LOX2</td><td>-4.6</td><td>0.04</td><td>0.5</td><td>0.17</td></tr></tbody></table>

*Expression ratios are represented as LOG2 values relative to the control samples.

susceptible (EgrSUS) species. Changes in the level of transcript expression of the putative marker genes (PR2; PR4; PR5; LOX3) were assessed at three time points using RT-qPCR.

**Results and discussion:** A dose response experiment of the putative marker genes was conducted with various concentrations to elucidate which would elicit the most paramount response in gene expression. It was found that amongst the tested candidates, 5mM and 100μM displayed the most significant change in gene expression for SA and JA respectively (Table 1). The specificity of the putative markers was also determined by profiling the putative marker genes with material induced by the opposing pathway, i.e SA markers were assessed with MeJA induced material.

A time course experiment was done to investigate how the expression profiles of the genes respond over a period of time. This would shed light on a possible window period that one could focus on for enhancing resistance as the timing of defence is crucial in determining the outcome of a pathogen interaction. For example the PR2 gene, a marker for the SA pathway, was shown to be drastically induced at 24hrs followed by a decline at 48hrs. This could be due to the fact that high levels of SA are toxic to the cell so the plant needs to closely monitor SA levels. On the other hand PR4, a marker for the JA pathway displayed a gradual increase over time beginning at 6hrs and peaking at 48hrs.

When the putative marker genes were assessed in tissue infected with *Chr. austroafricana*, it was observed that role of SA could potentially have a crucial role in determining the outcome of the infection. It is interesting to note that at two weeks there is no significant difference in lesion length between *EgrSUS* and *EgrTOL*. In *EgrTOL*, the expression level of PR2 was significantly up-regulated at two weeks post-inoculation whereas *EgrSUS* had significantly altered levels of expression only at six weeks. However in *EgrSUS*, the level to which PR2 is induced is still lower than in *EgrTOL*. At two weeks and six weeks there is an increase in PR4 transcript levels in *EgrSUS*. This could explain the inability of *EgrSUS* to accumulate SA due to the antagonistic relationship between the two pathways which is in accordance to what is currently known in Arabidopsis [1].

**Conclusion:** The genes identified in this study were tested as a diagnostic tool for the screening of pathogen challenged eucalypt plant to determine which signaling pathway(s) were playing a role in defence against various pathogens. It was found that SA could potential play a role in enhancing resistance to *Chr. austroafricana*. Future work would involve studying the expression profile of these genes in response to various other pathogens as well as to elucidate more putative marker genes. This research provides a platform from which to expand our knowledge of plant defence in *Eucalyptus* and work towards curbing tree diseases.

**References**

1. Pieterse C, Leon-Reyes A, Ent S: Networking by small-molecule hormones in plant immunity. *Nature* 2009, 5:308-316.

2. Boter M, Ruiz-Rivero O, Abdeen A: Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. *Genes Dev* 2004, 18:1577-1591.

3. Mishina T, Zeier J: Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis. *Plant J* 2007, 50:500-513.

P97

**Investigating *Eucalyptus* – pathogen and pest interactions to dissect broad spectrum defense mechanisms**

Sanushka Naidoo*, Ronishree Naidoo, Caryn Oates, Febe Wilken, Alexander Myburg

FABI, University of Pretoria, South Africa

E-mail: sanushka.naidoo@fabi.up.ac.za

BMC Proceedings 2011, **5**(Suppl 7):P97

**Background:** *Eucalyptus* species, hybrids and clones are attacked by various fungal and bacterial pathogens and pests during their life-time. Global climate changes are predicted to create favourable environments for such pathogens and pests and increase incidence of host jumping from other crops, resulting in increased losses to the forestry industry [1]. The use of tolerant or resistant plant varieties as part of an integrated disease management strategy is recognised as a desirable means to curb disease incidence. Vertical resistance mediated by resistance (R) genes, may be easily overcome by a pathogen and is thus not adequate on plantation species such as *Eucalyptus*, which would be exposed to various pathogens during its life-time. Broad spectrum resistance on the other hand, would be desirable to provide resistance against multiple challenges [2].

The aim of this study is to investigate mechanisms involved in host resistance with an emphasis on broad-spectrum resistance. The availability of the complete genome sequence of *Eucalyptus grandis* (http://www.eucagen.org) and the transcriptome sequence of a *E. grandis* X *E. urophylla* (GU) hybrid [3] has provided resources to investigate defense responses in the natural host. When a pathogen attacks a plant, the plant launches a sophisticated defense response involving phytohormones such as salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET). These responses are finely tuned and tailored to the invader [4]. Down-stream of the signalling cascade is the production of pathogenesis related (PR) genes and antimicrobial genes which serve to limit the pathogen and afford protection. PR genes, such as PR-1 and PR-5, are known markers of the salicylic acid defense pathway, while PR-3, PR-4 and the lipoxygenase (LOX) genes are known markers of the MeJA and ET signalling pathways. The discovery of PR genes in *Eucalyptus* is desirable as these genes have previously been shown to afford broad spectrum resistance in other crops. We present our progress in exploiting the *Eucalyptus* genomic and transcriptomic data for the discovery of tree defense genes and explore the application thereof in determining which pathways are activated in response to various pathogens.

**Materials and methods: Infections and infestations:** *E. grandis* clones were treated with *Chrysoporthe austroafricana* in the following manner: wounds were created using a 0.3cm cork borer to expose the cambial tissue and an agar plug containing fungal mycelia were applied to the wound and sealed with parafilm. As a control, plants were mock inoculated. *E. nitens* plants were inoculated with *Phytophthora cinnamomi* using a 0.3cm cork borer, and a mycelial plug applied. The wound site containing the pathogen was sealed with moist cheese cloth and parafilm. A set of plants received no inoculum. *E. grandis* clones were maintained in the FABI nursery and were naturally infested with *Leptocybe invasa*. A set of plants were maintained under similar conditions but were not exposed to the insect pest.

**Expression analysis:** RNA was harvested from from stem tissue (from *C. austroafricana* and *P. cinnamomi* interactions) and leaf tissue (for *L. invasa* interactions) at two time points after challenge in order to detect early and late responses. RNA was isolated using the CTAB method [5] and cDNA synthesized. Reverse transcriptase quantitative PCR (RT-qPCR) was performed using the Roche 480 LightCycler instrument.

**Results and discussion:** Reliable pathosystems were established for *Eucalyptus* with *P. cinnamomi* and *C. austroafricana*. Figure 1A and 1B show the lesions which developed after pathogen challenge compared to mock inoculated plants. Figure 1C shows the results of natural infestation of young leaves of *E. grandis* clones under nursery conditions compared to plants not exposed to the pest. The oviposition sites are evident on the leaf midrib (indicated by the white arrow).

Using a bioinformatic and phylogenetic approach, the putative orthologs for PR-1, PR-2, PR-3, PR-4 and PR-5 were identified. The basal expression level of PR-3 in the GU hybrid transcriptome is indicated in Figure 2 as an example.