bluuebunny/crossref_metadata_2025 huggingface dataset:
LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in Arabidopsis
Authors: Kiyosue Tomohiro , Wada Masamitsu
Published: March 2003

In plants, light is not only an energy source but also a very important signal that modulates development and differentiation. Here, we report a putative photo‐regulatory factor sequence in LKP1 (LOV kelch protein 1). LKP1 cDNA encodes a protein of 610 amino acids and with a molecular weight of 65 905 with an LOV domain and kelch repeats. LOV domains are present in a number of sensor proteins involved in the detection of light, oxygen or voltage. The LKP1 LOV is very similar to the LOV domains in NPH1, a plasma membrane‐associated blue light receptor kinase that regulates phototropism (Huala, E., Oeller, P.W., Liscum, E., Han, I‐S., Larsen, E. & Briggs, W.R. (1997) Science, 278, 2120–2123). LKP1 mRNA accumulates in roots, stems, flowers and siliques. It is most abundant in leaves, and least abundant in seeds. Transgenic plants with a β‐glucuronidase (GUS) reporter gene driven by a 1.5 kb LKP1 promoter display strong GUS activity in leaves. Transgenic plants with a 35S::LKP1 cDNA gene overexpress LKP1 mRNA. These plants have elongated hypocotyls and petioles with elongated cells, and exhibit distinct cotyledon movement during the day. Expression of 35S::LKP1 in transgenic Arabidopsis promotes late flowering in plants grown under long‐day, but not under short‐day conditions. Vernalization does not affect the late flowering phenotype of the 35S::LKP1 plants. Transgenic plants possessing the 35S::GFP‐LKP1 construct also have long hypocotyles and petioles, and a late flowering phenotype, suggesting that the GFP‐LKP1 fusion protein is active. The GFP‐associated fluorescence in 35S::GFP‐LKP1 plants is observed in nuclei and cytosol, indicating that LKP1 is a new nucleo‐cytoplasmic factor that influences flowering time in the long day pathway of Arabidopsis.
Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells
Authors: Bouvier Florence , Suire Claude , D'Harlingue Alain , Backhaus Ralph A. , Camara Bilal
Published: March 2003

The nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non‐secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy‐xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non‐green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.
Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato
Authors: Weller James L. , Schreuder Mariëlle E.L. , Smith Harry , Koornneef Maarten , Kendrick Richard E.
Published: March 2003

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white‐light‐grown wild‐type or phyA‐deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de‐etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high‐irradiance R, and, surprisingly, that the phyA‐dependent low‐irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far‐red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low‐irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.
Indole‐3‐glycerol phosphate, a branchpoint of indole‐3‐acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana
Authors: Ouyang Jian , Shao Xiang , Li Jiayang
Published: March 2003

The phytohormone indole‐3‐acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp‐independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole‐3‐glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2‐1 and trp3‐1. Antisense transgenic plants display some auxin deficient‐like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild‐type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3‐1 and trp2‐1 plants. These results suggest that indole‐3‐glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp‐independent IAA de novo biosynthetic pathway.
A new hyperrecombinogenic mutant of Nicotiana tabacum
Authors: Gorbunova Vera , Avivi‐Ragolski Naomi , Shalev Gil , Kovalchuk Igor , Abbo Shahal , Hohn Barbara , Levy Avraham A.
Published: March 2003

We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six‐ to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non‐homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma‐irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up‐regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.
Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4‐reductase
Authors: Johnson Eric T. , Ryu Sunhyo , Yi Hankuil , Shin Byongchul , Cheong Hyeonsook , Choi Giltsu
Published: March 2003

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4‐reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin‐type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate‐binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.
Maize protein kinase CK2: regulation and functionality of three β regulatory subunits
Authors: Riera Marta , Peracchia Giovanna , De Nadal Eulàlia , Ariño Joaquín , Pagès Montserrat
Published: March 2003

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2α), thus lacking CK2β regulatory subunits. To investigate the existence and functionality of CK2β regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full‐length cDNAs encoding CK2β regulatory subunits (CK2β‐1, CK2β‐2 and CK2β‐3) and a cDNA coding for a novel CK2α catalytic subunit, CK2α‐3. The pattern of expression of all these α/β subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2α subunits are constitutive, CK2β subunits are expressed differentially during embryo development. The yeast two‐hybrid system and pull‐down assays have been used to study specific interactions between the different subunits. While CK2α subunits are unable to self‐associate, preferential interactions between α/β isoforms and β/β isoforms can be predicted. Furthermore, we show that maize CK2α/β subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2β subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2β regulatory subunits.
Hyperosmotic stress rapidly generates lyso‐phosphatidic acid in Chlamydomonas
Authors: Meijer Harold J.G. , Arisz Steven A. , Van Himbergen John A.J. , Musgrave Alan , Munnik Teun
Published: March 2003

Plant cells are continuously exposed to environmental stresses such as hyper‐osmolarity, and have to respond in order to survive. When 32P‐labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identified as lyso‐phosphatidic acid (LPA), and was the only lyso‐phospholipid to be accumulated. The increase in LPA was dose‐ and time‐dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non‐salts. At least part of the LPA was generated by phospholipase A2 (PLA2) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid‐signalling pathways in hyperosmotic stress responses is discussed.
A critical role for disproportionating enzyme in starch breakdown is revealed by a knock‐out mutation in Arabidopsis
Authors: Critchley Joanna H. , Zeeman Samuel C. , Takaha Takeshi , Smith Alison M. , Smith Steven M.
Published: March 2003

Disproportionating enzyme (d‐enzyme) is a plastidial α‐1,4‐glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T‐DNA insertion. While d‐enzyme activity is eliminated in the homozygous dpe1–1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1–1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1–1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de‐starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto‐oligosaccharides occurs in dpe1–1 but not in wild type. These data show that d‐enzyme is required for malto‐oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1–1 suggests that malto‐oligosaccharides affect an enzyme that attacks the starch granule, or that d‐enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1–1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto‐oligosaccharides generated during the dark period. We conclude that the primary function of d‐enzyme is in starch degradation.
Cloning and characterization of irregular xylem4 (irx4): a severely lignin‐deficient mutant of Arabidopsis
Authors: Jones Louise , Ennos A. Roland , Turner Simon R.
Published: March 2003

A severe lignin mutant, irx4, has been identified in Arabidopsis thaliana as a result of its collapsed xylem phenotype. In contrast to previously described irx mutants, irx4 plants have 50% less lignin than wild‐type plants, whilst the cellulose and hemicellulose content remained unchanged. These alterations in the composition of irx4 secondary cell walls had a dramatic effect on the morphology and architecture of the walls, which expand to fill most of the cell, and also on the physical properties of irx4 stems. Further analysis indicated that the irx4 mutation occurred in a cinnamoyl‐CoA reductase (CCR) gene within a highly conserved intron splice site sequence of intron 2. As a result, CCR mRNA transcripts were incorrectly spliced. Transgenic plants expressing an IRX3 promoter–CCR cDNA construct were used to generate a series of plants with varying degrees of lignin content in order to assess the role of lignin content in determining the physical properties of Arabidopsis stems.
Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine‐insensitive (coi1) mutation occurs through two distinct mechanisms
Authors: Kloek Andrew P. , Verbsky Michelle L. , Sharma Shashi B. , Schoelz James E. , Vogel John , Klessig Daniel F. , Kunkel Barbara N.
Published: March 2003

A new allele of the coronatine‐insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1‐20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1‐20 plants is correlated with hyperactivation of PR‐1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA‐mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1‐20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA‐mediated defenses are required for restriction of PstDC3000 growth in coi1‐20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1‐20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1‐20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA‐dependent defense pathway; and (ii) an SA‐independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.
Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat
Authors: Engdahl Sheila , Aronsson Henrik , Sundqvist Christer , Timko Michael P. , Dahlin Clas
Published: March 2003

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro‐expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide‐binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co‐factor is essential for proper membrane association. POR protein carrying a mutation in the substrate‐binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein–membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.
Construct design for efficient, effective and high‐throughput gene silencing in plants
Authors: Wesley S. Varsha , Helliwell Christopher A. , Smith Neil A. , Wang MingBo , Rouse Dean T. , Liu Qing , Gooding Paul S. , Singh Surinder P. , Abbott David , Stoutjesdijk Peter A. , Robinson Simon P. , Gleave Andrew P. , Green Allan G. , Waterhouse Peter M.
Published: March 2003

Post‐transcriptional silencing of plant genes using anti‐sense or co‐suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self‐complementary ‘hairpin’ RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti‐sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron‐containing constructs (ihpRNA) generally gave 90–100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co‐suppression or anti‐sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high‐throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large‐scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
Identification of 4‐coumarate:coenzyme A ligase (4CL) substrate recognition domains
Authors: Ehlting Jürgen , Shin Jane J. K. , Douglas Carl J.
Published: March 2003

4‐coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4‐coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate‐forming enzymes such as firefly luciferases, non‐ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4‐coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate‐forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.
Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell‐specific gene expression
Authors: Plesch Gunnar , Ehrhardt Thomas , Mueller‐Roeber Bernd
Published: March 2003

Due to their unique structure and function, guard cells have attracted much attention at the physiological level. Very little, however, is known about the molecular events involved in the determination and maintenance of guard cell specificity. The KST1 gene encodes a K+ influx channel of guard cells in potato, and was therefore chosen as a model to study regulation of guard cell‐specific gene expression. Transgenic potato plants carrying a fusion between the KST1 promoter and the E. coli uidA (β‐glucuronidase) reporter gene revealed promoter activity in guard cells and in flowers. A detailed dissection of the KST1 promoter led to the discovery of two independent small TATA box‐proximal regulatory units, each of which was sufficient to direct guard cell‐specific gene transcription. Both fragments contain the sequence motif, 5′‐TAAAG‐3′, which is related to known target sites for a novel class of zinc finger transcription factors, called Dof proteins. Block mutagenesis of these Dof target sites in the context of different promoter constructs dramatically reduced guard cell promoter activity. A Dof gene, StDof1, was cloned and shown to be expressed in epidermal fragments highly enriched for guard cells. In gel retardation experiments, the StDof1 protein interacted in a sequence‐specific manner with a KST1 promoter fragment containing the TAAAG motif. These results provide evidence that TAAAG elements are target sites for trans‐acting Dof proteins controlling guard cell‐specific gene expression. Our data will add to the design of tailor‐made guard cell promoters as a further tool in molecular engineering of guard cell function and, hence, control of stomatal carbon dioxide (CO2) uptake and water loss in crop plants.
Evidence for expression level‐dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants
Authors: Hofius Daniel , Herbers Karin , Melzer Michael , Omid Ayelet , Tacke Eckhard , Wolf Shmuel , Sonnewald Uwe
Published: March 2003

High‐level constitutive expression of the cell‐to‐cell movement protein from the phloem‐restricted potato leafroll virus (PLRV‐MP17) in transgenic tobacco plants leads to growth retardation and severe phenotypic changes of source leaves paralleled by a drastic accumulation of soluble sugars and starch (Herbers et al., 1997). To investigate whether the MP17‐induced alteration in carbon metabolism is related to the targeting and modification of specific plasmodesmata (Pd) or is rather due to pleiotropic effects caused by high MP17 protein amounts, non‐phenotypic tobacco plants expressing a MP17:GFP fusion protein were obtained and compared with previously described MP17 transgenic lines. Confocal laser scanning microscopy and immunogold labelling studies revealed an overall affinity of MP17 to Pd in vascular and non‐vascular tissue of source leaves, whereas in sink leaves GFP fluorescence was restricted to Pd of trichomes. In source leaves, plasmodesmal size exclusion limits of mesophyll cells were likewise increased by MP17 and MP17:GFP independent from steady‐state levels of the protein amount and phenotypic alteration. Conversely, carbohydrate contents in source leaves strictly correlated with quantified MP17 protein levels. Low expression of MP17 and MP17:GFP decreased soluble sugars and starch contents in leaves possibly due to changes in plasmodesmal permeability while increasing MP17 protein levels led to carbohydrate accumulation and a stunted growth. Infection of transgenic lines with the unrelated potato virus Y (PVY)N revealed an expression level‐dependent mode of MP17‐mediated resistance. Phenotypic changes and carbohydrate‐mediated defence responses as indicated by elevated levels of PR‐protein transcripts were crucial for increased viral resistance, whereas plasmodesmal targeting and modification by MP17 per se had either no effect or even increased susceptibility to PVY. Thus, our results implicate that the absolute level of expression needs to be critically considered when elucidating the effect of MPs on carbon metabolism, biomass allocation and virus resistance.
Control of flowering time by FLC orthologues in Brassica napus
Authors: Tadege Million , Sheldon Candice C. , Helliwell Chris A. , Stoutjesdijk Peter , Dennis Elizabeth S. , Peacock W. James
Published: March 2003

FLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. We isolated five FLC‐related sequences from Brassica napus (BnFLC1–5). Expression of each of the five sequences in Arabidopsis delayed flowering significantly, with the delay in flowering time ranging from 3 weeks to more than 7 months, relative to the flowering time of 3 weeks in untransformed Ler. In the reciprocal experiment, expression of Arabidopsis FLC (AtFLC) in an early flowering B. napus cultivar delayed flowering by 2–6 weeks, confirming the requirement of this gene for floral repression. In B. napus, we show that late flowering and responsiveness to vernalization correlate with the level of BnFLC mRNA expression. The different BnFLC genes show differential expression in leaves, stems and shoot tips, but expression is not detectable in roots. Vernalization dramatically reduces the level of BnFLC transcript and restores early flowering in the winter cultivar Colombus. We conclude that BnFLC genes confer winter requirement in B. napus and account for the major vernalization‐responsive flowering time differences in the different cultivars of B. napus in a manner analogous to that of AtFLC in Arabidopsis ecotypes.
Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease‐1 (FEN‐1), and their distribution in mitotic and meiotic cell cycles
Authors: Kimura Seisuke , Suzuki Tadzunu , Yanagawa Yuki , Yamamoto Taichi , Nakagawa Hiroki , Tanaka Ichiro , Hashimoto Junji , Sakaguchi Kengo
Published: March 2003

The biochemical and cell cycle‐dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease‐1 (OsFEN‐1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN‐1 and increased the flap‐endonuclease activity of OsFEN‐1 by 2.5‐fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN‐1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN‐1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN‐1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10–20 intense spots at leptotene. The number of spots decreased to 1–5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN‐1 in mitotic and meiotic cell cycles are discussed.
Infra‐red thermography revealed a role for mitochondria in pre‐symptomatic cooling during harpin‐induced hypersensitive response
Authors: Boccara Martine , Boué Christine , Garmier Marie , De Paepe Rosine , Boccara Albert‐Claude
Published: March 2003

The establishment of Erwinia amylovora harpin‐induced hypersensitive response (HR) in Nicotiana sylvestris was followed by infra‐red thermography (IRT). Three to four hours after elicitation, the temperature decreased in the harpin‐infiltrated zone associated to stomatal opening. The marked drop in temperature which reached 2°C and preceded necrosis symptoms for several hours, is thus likely caused by higher transpiration. Neither of these effects was observed in a respiratory mutant, affected in complex I structure and function and over‐expressing alternative oxidase, indicating that they are directly or indirectly mediated by mitochondrial function. However, as the HR establishment was similar in both wild type and mutant, cell death was either uncorrelated with the observed epidermal changes or occurred by a different signalling pathway in the two genotypes. IRT revealed a novel aspect of plant–pathogen interactions and could be applied to screen for mutants affected in elicitor signalling and/or for respiratory mutants.
Chromosome painting in Arabidopsis thaliana
Authors: Lysak Martin A , Fransz Paul F , Ali Hoda B. M , Schubert Ingo
Published: March 2003

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome‐specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C−1). Pools of contiguous 113–139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.
Cloning and characterization of rice (Oryza sativa L) telomerase reverse transcriptase, which reveals complex splicing patterns
Authors: Heller‐Uszynska Katarzyna , Schnippenkoetter Wendelin , Kilian Andrzej
Published: March 2003

Plant chromosomes terminate in telomeres as in other eukaryotes. Telomeres are vital to genome stability and their malfunctioning is lethal. One of the core components of the telomere complex is telomerase. The enzyme activity depends on RNA (TER) and reverse transcriptase (TERT) subunits. We describe here the isolation, sequencing and characterization of the telomerase reverse transcriptase catalytic subunit from the monocot plant Oryza sativa L. (OsTERT). A single copy of this gene is present in the rice genome. The protein predicted from the OsTERT sequence has all the signature motifs of the TERT family members. Our data indicate that rice telomerase activity is developmentally regulated and is high in in vitro tissue and cell culture. However, steady‐state transcript levels of the TERT gene do not seem to correlate with enzyme activity. Northern and RT–PCR analyses of the OsTERT gene transcript profile show multiple differentially spliced transcripts in both telomerase‐positive and telomerase‐negative tissues. Based on quantitative analysis of these transcripts, we speculate that the overall balance between the quantities of particular alternatively spliced transcripts may determine whether the TERT protein(s) is active or not. The diversity of splicing variants detected suggests that, as recently discovered for mammalian TERT proteins, rice TERT protein variants may perform functions other than telomere maintenance.
Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1‐dependent in cpr5
Authors: Clarke Joseph D. , Aarts Nicole , Feys Bart J. , Dong Xinnian , Parker Jane E.
Published: March 2003

The systemic acquired resistance (SAR) response in Arabidopsis is characterized by the accumulation of salicylic acid (SA), expression of the pathogenesis‐related (PR) genes, and enhanced resistance to virulent bacterial and oomycete pathogens. The cpr (constitutive expressor of PR genes) mutants express all three SAR phenotypes. In addition, cpr5 and cpr6 induce expression of PDF1.2, a defense‐related gene associated with activation of the jasmonate/ethylene‐mediated resistance pathways. cpr5 also forms spontaneous lesions. In contrast, the eds1 (enhanced disease susceptibility) mutation abolishes race‐specific resistance conferred by a major subclass of resistance (R) gene products in response to avirulent pathogens. eds1 plants also exhibit increased susceptibility to virulent pathogens. Epistasis experiments were designed to explore the relationship between the cpr‐ and EDS1‐mediated resistance pathways. We found that a null eds1 mutation suppresses the disease resistance phenotypes of both cpr1 and cpr6. In contrast, eds1 only partially suppresses resistance in cpr5, leading us to conclude that cpr5 expresses both EDS1‐dependent and EDS1‐independent components of plant disease resistance. Although eds1 does not prevent lesion formation on cpr5 leaves, it alters their appearance and reduces their spread. This phenotypic difference is associated with increased pathogen colonization of cpr5 eds1 plants compared to cpr5. The data allow us to place EDS1 as a necessary downstream component of cpr1‐ and cpr6‐mediated responses, but suggest a more complex relationship between EDS1 and cpr5 in plant defense.
Spatial analysis of plant metabolism: Sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns
Authors: Borisjuk Ljudmilla , Walenta Stefan , Rolletschek Hardy , Mueller‐Klieser Wolfgang , Wobus Ulrich , Weber Hans
Published: March 2003

During legume embryogenesis the differentiation of the cotyledons proceeds gradually in a wave‐like manner. The process is metabolically and genetically controlled and regulated by sugars. In order to perform a spatial and temporal analysis of the sugar distribution pattern a new method was developed to specifically measure sucrose directly in tissues via bioluminescence and single photon counting. This enabled a quantitative sucrose imaging with a resolution close to the single cell level. The procedure was applied on sections of Vicia faba cotyledons covering the main stages of histodifferentiation. Young embryos before the storage phase contained moderate levels of sucrose, which were evenly distributed. At the onset of maturation high concentrations were present within a tissue layer covering the outwarded half of the coytledons. This layer was directly underneath the epidermis expressing a sucrose transporter gene indicating that epidermal transporters caused the high sucrose accumulation in the underlying tissue. At that stage the sucrose gradient was inversely oriented compared with cell size and the starch content. Cells within the interior were larger, contained starch but low sucrose. Thus, the sucrose pattern is controlled by uptake activity and permeability within the parenchyma. However, during the main storage phase actively elongating and starch accumulating cells contain highest sucrose concentrations indicating that differences in growth and starch accumulation also affect intracotyledonary sugar distribution. High sucrose concentrations were correlated with transcript levels of sucrose synthase and ADP‐Glc pyrophosphorylase indicating a signaling function for sucrose to induce starch biosynthesis on the gene expression level. Carbon flux through the sucrose synthase pathway towards starch increased when hexoses levels decreased.
Gene transfer from mitochondrion to nucleus: novel mechanisms for gene activation from Cox2
Authors: Daley Daniel O. , Adams Keith L. , Clifton Rachel , Qualmann Svenja , Millar A. Harvey , Palmer Jeffrey D. , Pratje Elke , Whelan James
Published: March 2003

The evolutionarily recent transfer of the gene for cytochrome c oxidase subunit 2 (cox2) from the mitochondrion to the nucleus in legumes is shown to have involved novel gene‐activation steps. The acquired mitochondrial targeting presequence is bordered by two introns. Characterization of the import of soybean Cox2 indicates that the presequence is cleaved in a three‐step process which is independent of assembly. The final processing step takes place only in the mitochondria of legume species, and not in several non‐legume plants. The unusually long presequence of 136 amino acids consists of three regions: the first 20 amino acids are required for mitochondrial targeting and can be replaced by another presequence; the central portion of the presequence is required for efficient import of the Cox2 protein into mitochondria; and the last 12 amino acids, derived from the mitochondrially encoded protein, are required for correct maturation of the imported protein. The acquisition of a unique presequence, and the capacity for legume mitochondria to remove this presequence post‐import, are considered to be essential adaptations for targeting of Cox2 to the mitochondrion and therefore activation of the transferred gene in the nucleus.
Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis
Authors: Franke Rochus , Hemm Matthew R. , Denault Jeff W. , Ruegger Max O. , Humphreys John M. , Chapple Clint
Published: March 2003

The end products of the phenylpropanoid pathway play important roles in plant structure and development, as well as in plant defense mechanisms against biotic and abiotic stresses. From a human perspective, phenylpropanoid pathway‐derived metabolites influence both human health and the potential utility of plants in agricultural contexts. The last known enzyme of the phenylpropanoid pathway that has not been characterized is p‐coumarate 3‐hydroxylase (C3H). By screening for plants that fail to accumulate soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. We have now shown that the ref8 mutant is defective in the gene encoding C3H. Phenotypic characterization of the ref8 mutant has revealed that the lack of C3H activity in the mutant leads to diverse changes in phenylpropanoid metabolism. The ref8 mutant accumulates p‐coumarate esters in place of the sinapoylmalate found in wild‐type plants. The mutant also deposits a lignin formed primarily from p‐coumaryl alcohol, a monomer that is at best a minor component in the lignin of other plants. Finally, the mutant displays developmental defects and is subject to fungal attack, suggesting that phenylpropanoid pathway products downstream of REF8 may be required for normal plant development and disease resistance.
The WUSCHEL gene promotes vegetative‐to‐embryonic transition in Arabidopsis
Authors: Zuo Jianru , Niu Qi‐Wen , Frugis Giovanna , Chua Nam‐Hai
Published: March 2003

Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4‐dichlorophenoxyacetic acid (2,4‐D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative‐to‐embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical‐inducible activation‐tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high‐frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol‐inducible XVE‐WUS transgene can phenocopy pga6‐1 and pga6‐2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative‐to‐embryogenic transition and/or maintaining the identity of the embryonic stem cells.
DNA binding and dimerization specificity and potential targets for the TCP protein family
Authors: Kosugi Shunichi , Ohashi Yuko
Published: March 2003

The TCP domain is a plant‐specific DNA binding domain found in proteins from a diverse array of species, including the cycloidea (cyc) and teosinte branched1 (tb1) gene products and the PCF1 and PCF2 proteins. To understand the role in transcriptional regulation of proteins with this domain, we have analysed the DNA binding and dimerization specificity of the TCP protein family using rice PCF proteins, and further evaluated potential targets for the TCP protein. The seven PCF members including five newly isolated proteins, were able to be grouped into two classes, I and II, based on sequence similarity in the TCP domain. Random binding site selection experiments and electrophoretic mobility shift assays (EMSAs) revealed the consensus DNA binding sequences of these two classes to be distinct but overlapping; GGNCCCAC for class I and GTGGNCCC for class II. The TB1 protein from maize, which belongs to class II, had the same specificity as the rice class II proteins, suggesting the conservation of binding specificity between TCP domains from different species. The yeast 2‐hybrid assay and EMSA revealed that these proteins tend to form a homodimer or a heterodimer between members of the same class. We searched predicted 5′ flanking sequences of Arabidopsis genes for the consensus binding sequences and found that the consensus sites are distributed in the genome at a considerably lower frequency. We further analysed eight promoters containing the class I consensus TCP sites. The transcriptional activities of six promoters were decreased by a mutation of the TCP binding site, which is consistent with the observation that the class I TCP site can confer transactivation function on a heterologous promoter. These results suggest that the two classes of TCP protein are distinct in DNA binding specificity and transcriptional regulation.
Expression of Arabidopsis SR‐like splicing proteins confers salt tolerance to yeast and transgenic plants
Authors: Forment Javier , Naranjo Miguel Ángel , Roldán Marta , Serrano Ramón , Vicente Oscar
Published: March 2003

Searching for novel targets of salt toxicity in eukaryotic cells, we have screened an Arabidopsis thaliana cDNA library to isolate genes conferring increased tolerance to salt stress when expressed in the yeast Saccharomyces cerevisiae. Here we show that expression of the ‘alternating arginine‐rich’ (or RS) domains of two different SR‐like, putative splicing proteins from Arabidopsis allows yeast cells to tolerate higher lithium and sodium concentrations. Protection against salt stress appears to require the in vivo phosphorylation of these plant polypeptides, since the yeast SR protein kinase Sky1p, which was able to phosphorylate in vitro at least one of them, also proved to be essential for the observed salt tolerance phenotype. In addition, a clone encoding the U1A protein, a previously characterised Arabidopsis splicing factor, was also isolated in the screening. No significant decrease in the intracellular concentration of lithium was observed in yeast cells incubated in the presence of LiCl upon expression of any of the Arabidopsis proteins, suggesting that their effects are not mediated by the stimulation of ion transport. In support of the general significance of these data, we also show that the expression of the RS domain of one of the SR‐like proteins in transgenic Arabidopsis plants increases their tolerance to LiCl and NaCl. These results point to an important role of pre‐mRNA splicing and SR‐like proteins in the salt tolerance of eukaryotic cells, offering a novel route to improve this important trait in crop plants.