bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves
Authors: Yang Yinong , Li Rugang , Qi Min
Published: March 2003

A convenient, Agrobacterium‐mediated transient expression assay has been evaluated for rapid analysis of plant promoters and transcription factors in vivo. By simple infiltration of Agrobacterium cells carrying appropriate plasmid constructs into tobacco leaves in planta, reproducible expression assays could be conducted in as little as 2–3 days without using expensive equipment (e.g. biolistic gun or electroporation apparatus) or complicated procedures (e.g. preparation of protoplasts). Biotic and abiotic treatments could be applied to the intact plant to examine their influence on promoter activity and gene expression. Using this method, we have tested the stress‐responsive as‐1 and heat shock elements, yeast GAL4 transactivation system, two promoters of pathogenesis‐related (PR) genes as well as a heat shock promoter. Through deletion analyses of tobacco PR1a promoter and a novel myb1 promoter, we have also successfully identified the cis‐regulatory regions in these promoters that are responsive to salicylic acid treatment or tobacco mosaic virus infection. Together, our results demonstrate that Agrobacterium‐mediated transient expression is a simple and efficient method for in vivo assays of plant promoters and transcription factors.
Efficient insertion mutagenesis of Arabidopsis by tissue culture‐induced activation of the tobacco retrotransposon Tto1
Authors: Okamoto Hiroyuki , Hirochika Hirohiko
Published: March 2003

The tobacco retrotransposon Tto1 is one of a few active retrotransposons in plants. Its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. Here we demonstrate that Tto1 introduced in Arabidopsis is also activated by tissue culture. Transcription of Tto1 was induced by tissue culture and driven by its LTR promoter. Transposed copies of Tto1 were observed in almost all of the plants regenerated from the explants cultured for only 1 week. A total of 255 independent regenerated lines have been produced, and the average copy number of transposed Tto1 in these lines is estimated to be 3.2. Sequences flanking Tto1 were amplified by thermal asymmetric interlaced (TAIL)‐PCR. Of 165 independent amplified products, 123 showed significant homology to known genes or hypothetical protein genes. The insertion sites of Tto1 are spread over all chromosomes and the target site sequence shows moderate consensus. Taken together, these results indicate that Tto1 can be used as a tool for efficient insertion mutagenesis of Arabidopsis which is especially suitable as a reverse genetics system.
Analysis of the G‐overhang structures on plant telomeres: evidence for two distinct telomere architectures
Authors: Riha Karel , McKnight Thomas D. , Fajkus Jiri , Vyskot Boris , Shippen Dorothy E.
Published: March 2003

Telomeres are highly conserved structures essential for maintaining the integrity of eukaryotic genomes. In yeast, ciliates and mammals, the G‐rich strand of the telomere forms a 3′ overhang on the chromosome terminus. Here we investigate the architecture of telomeres in the dicot plants Silene latifolia and Arabidopsis thaliana using the PENT (primer extension/nick translation) assay. We show that both Arabidopsis and Silene telomeres carry G‐overhangs longer than 20–30 nucleotides. However, in contrast to yeast and ciliate telomeres, only half of the telomeres in Silene seedlings possess detectable G‐overhangs. PENT reactions using a variety of primers and reaction conditions revealed that the remaining fraction of Silene telomeres carries either no overhangs or overhangs less than 12 nucleotides in length. G‐overhangs were observed in Silene seeds and leaves, tissues that lack telomerase activity. These findings suggest that incomplete DNA replication of the lagging strand, rather than synthesis by telomerase, is the primary mechanism for G‐overhang synthesis in plants. Unexpectedly, we found that the fraction of telomeres with detectable G‐overhangs decreased from 50% in seedlings to 35% in leaves. The difference may reflect increased susceptibility of the G‐overhangs to nuclease attack in adult leaves, an event that could act as a precursor for the catabolic processes accompanying leaf senescence
Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis‐elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G‐box
Authors: Ezcurra Inés , Wycliffe Paul , Nehlin Lilian , Ellerström Mats , Rask Lars
Published: March 2003

The transcriptional activator ABI3 is a key regulator of gene expression during embryo maturation in crucifers. In monocots, the related VP1 protein regulates the Em promoter synergistically with abscisic acid (ABA). We identified cis‐elements in the Brassica napus napin napA promoter mediating regulation by ABI3 and ABA, by analyzing substitution mutation constructs of napA in transgenic tobacco plantlets ectopically expressing ABI3. In transient analysis using particle bombardment of tobacco leaf sections, a tetramer of the distB ABRE (abscisic acid‐responsive element) mediated transactivation by ABI3 and ABI3‐dependent response to ABA, whereas a tetramer of the composite RY/G complex, containing RY repeats and a G‐box, mediated only ABA‐independent transactivation by ABI3. Deletion of the conserved B2 and B3 domains of ABI3 abolished transactivation of napA by ABI3. The two domains of ABI3 interact with different cis‐elements: B2 is necessary for ABA‐independent and ABA‐dependent activations through the distB ABRE, whereas B3 interacts with the RY/G complex. Thus B2 mediates the interaction of ABI3 with the protein complex at the ABRE. The regulation of napA by ABI3 differs from Em regulation by VP1, in that the B3 domain of ABI3 is essential for the ABA‐dependent regulation of napA.
Three unique mutants of Arabidopsis identify eds loci required for limiting growth of a biotrophic fungal pathogen
Authors: Dewdney Julia , Reuber T. Lynne , Wildermuth Mary C. , Devoto Alessandra , Cui Jianping , Stutius Lisa M. , Drummond Emma P. , Ausubel Frederick M.
Published: March 2003

To identify components of the defense response that limit growth of a biotrophic fungal pathogen, we isolated Arabidopsis mutants with enhanced disease susceptibility to Erysiphe orontii. Our initial characterization focused on three mutants, eds14, eds15, and eds16. None of these is considerably more susceptible to a virulent strain of the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). All three mutants develop a hypersensitive response when infiltrated with Psm expressing the avirulence gene avrRpt2, which activates resistance via the LZ‐NBS/LRR resistance protein encoded by RPS2. The growth of Psm(avrRpt2), while somewhat greater in the mutants than in the wild type, is less than growth of the isogenic virulent strain. These results indicate that resistance mediated via LZ‐NBS/LRR R genes is functional. Analysis of the growth of avirulent Peronospora parasitica strains showed that the resistance pathway utilized by TIR‐NBS/LRR R genes is also operative in all three mutants. Surprisingly, only eds14 and eds16 were more susceptible to Erysiphe cichoracearum. Analysis of the expression profiles of PR‐1, BGL2, PR‐5 and PDF1.2 in eds14, eds15, and eds16 revealed differences from the wild type for all the lines. In contrast, these mutants were not significantly different from wild type in the deposition of callose at sites of E. orontii penetration. All three mutants have reduced levels of salicylic acid after infection. eds16 was mapped to the lower arm of chromosome I and found by complementation tests to be allelic to the salicylic acid‐deficient mutant sid2.
Aquaporin PIP genes are not expressed in the stigma papillae in Brassica oleracea
Authors: Marin‐Olivier Marianne , Chevalier Tony , Fobis‐Loisy Isabelle , Dumas Christian , Gaude Thierry
Published: March 2003

The pollen grains of angiosperms are usually desiccated at maturity. Following pollination, pollen hydrates on the stigma surface before germination takes place. Rehydration is an essential step for the success of pollination and depends on the movement of water from the stigmatic cells. This water flow has been shown to be biologically regulated, and components of both pollen and stigma surfaces have been demonstrated to play a role in the control of pollen hydration. Regulation of water transport between animal or plant cells involves membrane proteins, designated aquaporins, which possess water‐channel activity. Such molecules may be candidates for controlling pollen hydration, and consequently we investigated whether aquaporins are present in the pollen and stigma cells in Brassica oleracea. Here, we report the identification of two new aquaporin genes, Bo‐PIP1b1 and Bo‐PIP1b2, which are highly homologous to PIP1b from Arabidopsis thaliana. Both Bo‐PIP1b1 and Bo‐PIP1b2 proteins are active water channels when expressed in Xenopus oocytes. Expression of Bo‐PIP1b1 and Bo‐PIP1b2 was observed in reproductive organs as well as in vegetative tissues. Interestingly, the use of a Bo‐PIP1b2 cDNA probe revealed that PIP1‐like transcripts were not present in the pollen grains or in the stigma papillae, but were present in the stigma cell layers underlying the papillar cells. This observation suggests that water flow between the pollen and stigma papillae may be dependent on aquaporins expressed in cells that are not directly in contact with the pollen grain.
Dynamics of ionic activities in the apoplast of the sub‐stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness
Authors: Felle Hubert H. , Hanstein Stefan , Steinmeyer Ralf , Hedrich Rainer
Published: March 2003

Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub‐stomatal apoplastic activities of K+, Cl–, Ca2+ and H+ were continuously monitored by inserting ion‐selective micro‐electrodes through the open stomata of intact Vicia faba leaves. In light‐adapted leaves, the mean activities were 2.59 mm (K+), 1.26 mm (Cl–), 64 µm (Ca2+) and 89 µm (H+). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9–10 min, in which Ca2+ and H+ activities transiently decreased, and Cl– and K+ activities transiently increased. Thereafter, Ca2+, H+ and Cl– activities completely recovered, while K+ activity approached an elevated level of around 10 mm within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl– and Ca2+ activities recovered more slowly. Addition of ABA to dark‐adapted leaves evoked responses of Cl– and Ca2+ similar to those observed in the light. K+ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mm, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K+ kinetics in the sub‐stomatal cavity. We thus conclude that the increase in K+ activity mainly resulted from K+ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl– and Ca2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca2+, a process which activates depolarization‐activated K+ release channels.
Regulation of HEMA1 expression by phytochrome and a plastid signal during de‐etiolation in Arabidopsis thaliana
Authors: McCormac Alex C. , Fischer Andrea , Kumar A. Madan , Söll Dieter , Terry Matthew J.
Published: March 2003

The synthesis of 5‐aminolevulinic acid (ALA) is the rate‐limiting step for the formation of all plant tetrapyrroles, including chlorophyll and heme, and regulation of ALA synthesis is therefore critical to plant development. Glutamyl‐tRNA reductase (GluTR) is the first committed enzyme of this pathway and is encoded by a small family of nuclear HEMA genes. Here, we have used transgenic Arabidopsis (Arabidopsis thaliana L. Col) lines expressing chimeric HEMA1 promoter:gusA fusion genes, combined with RNA gel blot analyses, to characterise the light‐mediated regulation of the Arabidopsis HEMA1 gene during de‐etiolation. HEMA1 was expressed strongly, but not exclusively, in photosynthetic tissues and was shown to be light regulated at the transcriptional level by the phytochrome family of photoreceptors acting in both the far‐red high irradiance and low fluence response modes. The HEMA2 gene, which is expressed only in roots of seedlings, was not light regulated. Analysis of truncated HEMA1 promoter constructs demonstrated that a −199/+252 promoter fragment was sufficient to confer full light‐responsiveness to gusA expression. This fragment contained GT‐1/I‐box and CCA‐1 binding sites that are implicated as the light‐responsive cis elements. Both the full‐length and truncated HEMA1 promoters required the presence of intact chloroplasts for full expression, consistent with previous indications that light and plastid factor signals converge to co‐ordinately regulate expression of photosynthesis‐related nuclear genes. These results provide the most comprehensive analysis to date of the light‐regulation of a tetrapyrrole biosynthetic gene and support a direct link between regulation of HEMA1 transcription and chlorophyll accumulation during seedling de‐etiolation.
Defective cell proliferation in the floral meristem of alloplasmic plants of Nicotiana tabacum leads to abnormal floral organ development and male sterility
Authors: Farbos Isabelle , Mouras Armand , Bereterbide Agnes , Glimelius Kristina
Published: March 2003

Flowers of an alloplasmic male‐sterile tobacco line, comprised of the nuclear genome of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, develop short, poorly‐pigmented petals and abnormal sterile stamens that often are fused with the carpel wall. The development of flower organ primordia and establishment of boundaries between the different zones in the floral meristem were investigated by performing expression analysis of the tobacco orthologs of the organ identity genes GLO, AG and DEF. These studies support the conclusion that boundary formation was impaired between the organs produced in whorls 3 and 4 resulting in partial fusions between anthers and carpels. According to the investigations cell divisions and floral meristem size in the alloplasmic line were drastically reduced in comparison with the male‐fertile tobacco line. The reduction in cell divisions leads to a discrepancy between cell number and cell determination at the stage when petal and stamen primordia should be initiated. At the same stage expression of the homeotic genes was delayed in comparison with the male‐fertile line. However, the abnormal organ development was not due to a failure in the spatial expression of the organ identity genes. Instead the aberrant development in the floral organs of whorls 2, 3 and 4 appears to be caused by deficient floral meristem development at an earlier stage. Furthermore, defects in cell proliferation in the floral meristem of the alloplasmic male‐sterile line correlates with presence of morphologically modified mitochondria. The putative causes of reduced cell number in the floral meristem and the consequences for floral development are discussed.
Physical interactions between ABA response loci of Arabidopsis
Authors: Nakamura Shingo , Lynch Tim J. , Finkelstein Ruth R.
Published: March 2003

Genetic and physiological studies have shown that the Arabidopsis thaliana abscisic acid‐insensitive (ABI) loci interact to regulate seed‐specific and/or ABA‐inducible gene expression. We have used the yeast two‐hybrid assay to determine whether any of these genetic interactions reflect direct physical interactions. By this criterion, only ABI3 and ABI5 physically interact with each other, and ABI5 can form homodimers. The B1 domain of ABI3 is essential for this interaction; this is the first specific function ascribed to this domain of the ABI3/VP1 family. The ABI5 domains required for interaction with ABI3 include two conserved charged domains in the amino‐terminal half of the protein. An additional conserved charged domain appears to have intrinsic transcription activation function in this assay. Yeast one‐hybrid assays with a lacZ reporter gene under control of the late embryogenesis‐abundant AtEm6 promoter show that only ABI5 binds directly to this promoter fragment.
Elimination of POR expression correlates with red leaf formation in Amaranthus tricolor
Authors: Iwamoto Kuninori , Fukuda Hiroo , Sugiyama Munetaka
Published: March 2003

Amaranthus tricolor L. tricolor cv. Earlysplendor, an ornamental amaranth, generates red leaves instead of green leaves in late summer to early autumn. Red leaf formation was promoted under short‐day conditions and delayed by night‐break treatments. Red leaves were characterized by lower levels of chlorophyll accumulation rather than higher levels of red pigment (betacyanin) accumulation. However, the metabolic activity toward the production of Mg‐protoporphyrin, an intermediate in the biosynthesis pathway for chlorophyll, was detected in red leaves as well as in green leaves.

RNA gel blot analysis was performed to assess the expression of nine genes encoding eight enzymes involved in chlorophyll biosynthesis. Among these enzymes, red‐leaf‐specific reduction of gene expression was observed only for NADPH‐protochlorophyllide oxidoreductase (POR), a key enzyme catalyzing a later step of chlorophyll biosynthesis. In addition, immunoblot analysis showed no accumulation of POR protein(s) in red leaves. These data indicate that the repression of POR gene expression and resultant loss of chlorophyll synthesis activity plays a role in red leaf formation of A. tricolor.
A fast neutron deletion mutagenesis‐based reverse genetics system for plants
Authors: Li Xin , Song Yujuan , Century Karen , Straight Shelly , Ronald Pamela , Dong Xinnian , Lassner Michael , Zhang Yuelin
Published: March 2003

A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.
High‐level expression of a synthetic red‐shifted GFP coding region incorporated into transgenic chloroplasts
Authors: Reed M. L. , Wilson S. K. , Sutton C. A. , Hanson M. R.
Published: March 2003

We describe here a synthetic red‐shifted variant of GFP that can be introduced into tobacco plastid genomes and is highly expressed in regenerated plants that appear normal and fertile. The variant contains the S65G and S72A mutations which shift the absorption maximum from the 395 nm of wild‐type GFP closer to 488 nm, a wavelength emitted by a laser commonly used in confocal microscopy. In addition to enhanced fluorescence, the removal of significant absorption below 450 nm will potentially facilitate double‐labelling experiments. The variant GFP encoded by the synthetic gene can be expressed at a high level, forming approximately 5% of total leaf protein.
Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity
Authors: Hanfrey Colin , Sommer Susanne , Mayer Melinda J. , Burtin Daniel , Michael Anthony J.
Published: March 2003

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non‐enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site‐directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head‐to‐tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre‐protein monomer.
Light‐dependent gene expression for proteins in the respiratory chain of potato leaves
Authors: Svensson Å. Staffan , Rasmusson Allan G.
Published: March 2003

Expression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L. cv. Desiree) leaves. The recently characterized nda1 and ndb1 genes, homologues to genes encoding the non‐proton pumping respiratory chain NADH‐dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton‐pumping NADH dehydrogenase (complex I). As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone‐insensitive NADH oxidation. The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged. A variation in transcript level, specific for nda1, is seen at different times of the day with highest expression in the morning. This variation also influences the apparent developmental induction. Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re‐induction when plants are returned to light. Corresponding immunodetected NDA protein is specifically decreased in mitochondria isolated from dark‐treated plants, accompanied by a lower capacity for internal rotenone‐insensitive NADH oxidation. Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins. Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence. In addition to the specific change in nda1, a general, slower down‐regulation in darkness was seen for the other NAD(P)H dehydrogenase genes. The nda1 expression during development, and in response to light, indicates a specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.
The role of SPY and its TPR domain in the regulation of gibberellin action throughout the life cycle of Petunia hybrida plants
Authors: Izhaki Anat , Swain Stephen M. , Tseng Tong‐seung , Borochov Amihud , Olszewski Neil E. , Weiss David
Published: March 2003

SPY acts as a negative regulator of gibberellin (GA) action in Arabidopsis, but its mode of action and regulation are still unknown. SPY over‐expression in transgenic petunia plants affected various GA‐regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA‐induced gene, GIP. A similar phenotype was obtained when wild‐type petunia plants were treated with the GA‐biosynthesis inhibitor, paclobutrazol. The N‐terminus of SPY contains tetratricopeptide repeats (TPR). TPR motifs participate in protein–protein interactions, suggesting that SPY is part of a multiprotein complex. To test this hypothesis, we over‐expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant‐negative SPY mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. We cloned the petunia SPY homologue, PhSPY, and showed that its mRNA level is not affected by GA or ABA. The results of this study support the role of SPY as a negative regulator of GA action, suggest that the TPR domain is required for the interaction with other proteins to form an active complex and indicate that different plants use similar mechanisms to transduce the GA signal.
Opposing roles of phytochrome A and phytochrome B in early cryptochrome‐mediated growth inhibition
Authors: Folta Kevin M. , Spalding Edgar P.
Published: March 2003

The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1‐dependent membrane depolarization, which is caused by the activation of plasma‐membrane anion channels within seconds of blue light treatment. High‐resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1‐mediated growth inhibition, which is causally linked to the preceding anion‐channel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1‐dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the first few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This finding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light‐dependent inhibitory and promotive influences. It appears that phyB opposes the cry1/phyA‐mediated inhibition by promoting growth during at least the first 120 min of blue light treatment.
ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis
Authors: Signora Laurent , De Smet Ive , Foyer Christine H , Zhang Hanma
Published: March 2003

The formation of lateral roots (LR) is a major post‐embryonic developmental event in plants. In Arabidopsis thaliana, LR development is inhibited by high concentrations of NO3–. Here we present strong evidence that ABA plays an important role in mediating the effects of NO3– on LR formation. Firstly, the inhibitory effect of NO3– is significantly reduced in three ABA insensitive mutants, abi4‐1, abi4‐2 and abi5‐1, but not in abi1‐1, abi2‐1 and abi3‐1. Secondly, inhibition by NO3– is significantly reduced, but not completely abolished, in four ABA synthesis mutants, aba1‐1, aba2‐3, aba2‐4 and aba3‐2. These results indicate that there are two regulatory pathways mediating the inhibitory effects of NO3– in A. thaliana roots. One pathway is ABA‐dependent and involves ABI4 and ABI5, whereas the second pathway is ABA‐independent. In addition, ABA also plays a role in mediating the stimulation of LR elongation by local NO3– applications.
Analysis of surface growth in shoot apices
Authors: Dumais Jacques , Kwiatkowska Dorota
Published: March 2003

A salient feature of shoot meristem growth is the maintenance of distinct anatomical and morphological features despite a continuous flux of cells. To investigate how meristem organization is self‐perpetuated, we developed a protocol for the analysis of meristem growth in 3‐D. Our protocol uses a non‐destructive replica method to follow the pattern of cell expansion and cell divisions on the meristem surface over several days. Algorithms to reconstruct the meristem surface and compute its curvature and rate of extension were implemented. We applied this approach to the shoot apical meristem of Anagallis arvensis and showed that a subcellular resolution of extension rates can be achieved. This is the first detailed quantitative analysis of meristem geometry and surface expansion in 3‐D. This new approach will be useful to connect cellular activities such as cell expansion, cell division, and differential gene expression with overall meristem morphogenesis.
HFR1, a phytochrome A‐signalling component, acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana
Authors: Kim Young‐Mi , Woo Je‐Chang , Song Pill‐Soon , Soh Moon‐Soo
Published: March 2003

HFR1, a basic helix–loop–helix protein, is known to be required for a subset of phytochrome A (phyA)‐dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA‐dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1‐triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light‐inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1‐mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA‐dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.
Post‐transcriptional mechanisms control catalase synthesis during its light‐induced turnover in rye leaves through the availability of the hemin cofactor and reversible changes of the translation efficiency of mRNA
Authors: Schmidt Matthias , Dehne Silvia , Feierabend Jürgen
Published: March 2003

The enzyme catalase is light‐sensitive. In leaves, losses caused by photoinactivation are replaced by new enzyme and the rate of de novo synthesis must be rapidly and flexibly attuned to fluctuating light conditions. In mature rye leaves, post‐transcriptional mechanisms were shown to control the rate of catalase synthesis. The amount of the leaf catalase (CAT‐1) transcript did not increase with light intensity, but was even higher after dark exposure of light‐grown leaves. Initiation was apparently not limiting translation in the dark, as the association of the Cat1 mRNA with polysomes did not change notably under different light conditions. By analysing the translation of catalase polypeptides in cell‐free systems with poly(A)+ RNA from leaves or with mRNA transcribed from a Cat1‐containing cDNA clone, two mechanisms of post‐transcriptional control were identified. First, translation of catalase depended on the presence of hemin. In leaves, the availability of hemin may signal the extent of catalase degradation as the hemin of the inactivated enzyme is recycled. Second, the translation efficiency of the Cat1 transcripts was reversibly modulated in a dose‐dependent manner by the light intensity to which leaves were exposed, prior to extraction. The Cat1 mRNA from light‐exposed leaves was translated much more efficiently than mRNA from dark‐exposed leaves. The increase of its translation activity in vivo was not blocked by cordycepin but was suppressed by methylation inhibitors, indicating a reversible modification of pre‐existing mRNA by methylation. Translation of in vitro synthesized Cat1 mRNA required a methylated cap (m7GpppG), but was virtually below detection when it contained an unmethylated cap (GpppG).
Isolation, cloning and expression of a multifunctional O‐methyltransferase capable of forming 2,5‐dimethyl‐4‐methoxy‐3(2H)‐furanone, one of the key aroma compounds in strawberry fruits
Authors: Wein Martina , Lavid Noa , Lunkenbein Stefan , Lewinsohn Efraim , Schwab Wilfried , Kaldenhoff Ralf
Published: March 2003

Strawberry fruits contain an uncommon group of key aroma compounds with a 2,5‐dimethyl‐3(2H)‐furanone structure. Here, we report on the methylation of 2,5‐dimethyl‐4‐hydroxy‐3(2H)‐furanone (DMHF) to 2,5‐dimethyl‐4‐methoxy‐3(2H)‐furanone (DMMF) by a S‐adenosyl‐L‐methionine dependent O‐methyltransferase, the cloning of the corresponding cDNA and characterization of the encoded protein. Northern‐hybridization indicated that the Strawberry‐OMT specific transcripts accumulated during ripening in strawberry fruits and were absent in root, petiole, leaf and flower. The protein was functionally expressed in E. coli and exhibited a substrate specificity for catechol, caffeic acid, protocatechuic aldehyde, caffeoyl CoA and DMHF. A common structural feature of the accepted substrates was a o‐diphenolic structure also present in DMHF in its dienolic tautomer. FaOMT is active as a homodimer and the native enzyme shows optimum activity at pH 8.5 and 37°C. It does not require a cofactor for enzymatic activity. Due to the expression pattern of FaOMT and the enzymatic activity in the different stages of fruit ripening we suppose that FaOMT is involved in lignification of the achenes and the vascular bundles in the expanding fruit. In addition, it is concluded that the Strawberry‐OMT plays an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF.
Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation
Authors: K. Mühlbauer Stefan , Lössl Andreas , Tzekova Lilia , Zou Zhurong ,   Hans‐Ulrich Koop
Published: March 2003

Sequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild‐type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.
Expression of a Phytophthora sojae necrosis‐inducing protein occurs during transition from biotrophy to necrotrophy
Authors: Qutob Dinah , Kamoun Sophien , Gijzen Mark
Published: March 2003

Phytophthora sojae is an oomycete that causes stem and root rot on soybean plants. To discover pathogen factors that produce disease symptoms or activate plant defense responses, we identified putative secretory proteins from expressed sequence tags (ESTs) and tested selected candidates using a heterologous expression assay. From an analysis of 3035 ESTs originating from mycelium, zoospore, and infected soybean tissues, we identified 176 putative secreted proteins. A total of 16 different cDNAs predicted to encode secreted proteins ranging in size from 6 to 26 kDa were selected for expression analysis in Nicotiana benthamiana using an Agrobacterium tumefaciens binary potato virus X (PVX) vector. This resulted in the identification of a 25.6‐kDa necrosis‐inducing protein that is similar in sequence to other proteins from eukaryotic and prokaryotic species. The genomic region encoding the P. sojae necrosis‐inducing protein was isolated and the expression pattern of the corresponding gene determined by RNA blot hybridization and by RT‐PCR. The activity of this P. sojae protein was compared to proteins of similar sequence from Fusarium oxysporum, Bacillus halodurans, and Streptomyces coelicolor by PVX‐based expression in N. benthamiana and by transient expression via particle bombardment in soybean tissues. The P. sojae protein was a powerful inducer of necrosis and cell death in both assays, whereas related proteins from other species varied in their activity. This study suggests that the P. sojae necrosis‐inducing protein facilitates the colonization of host tissues during the necrotrophic phase of growth.
Polyribosome loading of spinach mRNAs for photosystem I subunits is controlled by photosynthetic electron transport
Authors: Sherameti Irena , Nakamura Masayaki , Yamamoto Yoshiharu Y. , Pfannschmidt Thomas , Obokata Junichi , Oelmüller Ralf
Published: March 2003

In light‐, but not in dark‐grown spinach seedlings, the mRNAs for the nuclear‐encoded photosystem I subunits D, F and L are associated with polyribosomes and this association is prevented by the application of 3‐(3′,4′‐dichlorophenyl)‐1,1′‐dimethyl urea (DCMU), an inhibitor of the photosynthetic electron transport. To identify the cis‐elements which are responsible for this regulation, we generated a series of chimeric PsaD constructs and tested them in transgenic tobacco. The spinach PsaD 5′‐untranslated region is sufficient to confer light‐ and photosynthesis‐dependent polyribosome association onto the uidA reporter gene, while the tobacco PsaD 5′‐untranslated region directs constitutive polyribosome association. These results are discussed with regard to signals from photosynthetic electron flow which control processes in the cytoplasm.
Differential regulation of TGA transcription factors by post‐transcriptional control
Authors: Pontier Dominique , Privat Isabelle , Trifa Youssef , Zhou Jun‐Ma , Klessig Daniel F. , Lam Eric
Published: March 2003

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We show here that specific members from a family of conserved Arabidopsis bZIP transcription factors, the TGA proteins, are regulated in their protein stability by developmental stage‐specific proteolysis. Using GFP fusions of three different Arabidopsis TGA factors that represent members of distinct subclasses of the TGA factor family, we demonstrate that two of these TGA proteins are specifically targeted for proteolysis in mature leaf cells. Using a supershift gel mobility assay, we found evidence for similar regulation of the cognate proteins as compared to the GFP fusion proteins expressed under the cauliflower mosaic virus (CaMV) 35S promoter. Using various inhibitors, we showed that the expression of at least one of these three TGA factors could be stabilized by inhibition of proteasome‐mediated proteolysis. This study indicates that TGA transcription factors may be regulated by distinct pathways of targeted proteolysis that can serve to modulate the contribution of specific members of a multigene family in complex regulatory pathways.
Antisense repression of StubGAL83 affects root and tuber development in potato
Authors: Lovas Ágnes , Bimbó Andrea , Szabó László , Bánfalvi Zsófia
Published: March 2003

StubGAL83 is a potato gene that encodes the β‐subunit of a protein kinase complex similar to the yeast SNF1, and the mammalian AMPK complexes that are modulated by changes in the cellular AMP/ATP ratio and are important regulators of metabolic and stress responses. Here we show that the expression of StubGAL83 in potato foliage is much higher in the dark than in the light and can be repressed by metabolisable sugars in the dark. The amounts of StubGAL83 mRNA are higher in sink than in source leaves. To unravel the role of StubGAL83, transgenic potato plants expressing a part of the StubGAL83 cDNA in antisense orientation under the control of the constitutive CaMV35S promoter were generated. Northern analysis revealed a reduction up to 90–95% in StubGAL83 mRNA accumulation in leaves of seven lines. Five out of these seven lines exhibited a reduction of StubGAL83 mRNA levels also in root and tuber tissues. Independent on the type of repression, the transgenic lines showed a delay in rooting and an increased sensitivity to salt stress. The roots were stunted and possessed less pronounced tap roots than the controls albeit with different severity in the different transgenic lines. The root cells were smaller and some of them had irregular shape. Tuberisation of the antisense‐StubGAL83 lines was delayed, the size of the tubers was reduced while the number of tubers per plant was increased. These results together suggest that StubGAL83 affects root and tuber development probably by altering the metabolic status of the leaves.
Reduced amino acid content in transgenic potato tubers due to antisense inhibition of the leaf H+/amino acid symporter StAAP1
Authors: Koch Wolfgang , Kwart Marion , Laubner Marion , Heineke Dieter , Stransky Harald , Frommer Wolf B. , Tegeder Mechthild
Published: March 2003

Transport processes across the plasma membrane of leaf vascular tissue are essential for transport and distribution of assimilates. In potato, leaves are the predominant sites for nitrate reduction and amino acid biosynthesis. From there, assimilated amino acids are exported through the phloem to supply tubers with organic nitrogen. To study the role of amino acid transporters in long‐distance transport and allocation of organic nitrogen in potato plants, a gene encoding a functional, leaf‐expressed amino acid permeaseStAAP1was isolated. Similar to the sucrose transporter SUT1,StAAP1expression was induced during the sink‐to‐source transition, indicating a role in phloem loading. To test the role of StAAP1, expression was inhibited by an antisense approach. Transgenic plants with reducedStAAP1expression were phenotypically indistinguishable from wild type, as were photosynthetic capacity and tuber yield. However, tubers from antisenseStAAP1plants showed up to 50% reduction in free amino acid contents. In comparison, starch content was not affected or tended to increase relative to wild type. The reduction in all amino acids except aspartate in the antisense plants is consistent with the properties of amino acid permeases (AAPs) found in heterologous systems. The results demonstrate an important role forStAAP1in long‐distance transport of amino acids and highlight the importance of plasma membrane transport for nutrient distribution in plants.
The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression
Authors: Lee Gyu In , Howe Gregg A.
Published: March 2003

Wound‐induced systemic expression of defensive proteinase inhibitor (PI) genes in tomato plants requires the action of systemin and its precursor protein prosystemin. Although it is well established that systemin induces PI expression through the octadecanoid pathway for jasmonic acid (JA) biosynthesis, relatively little is known about how systemin and JA interact to promote long‐distance signaling between damaged and undamaged leaves. Here, this question was addressed by characterizing a systemin‐insensitive mutant (spr1) that was previously identified as a suppressor of prosystemin‐mediated responses. In contrast to JA biosynthetic or JA signaling mutants that lack both local and systemic PI expression in response to wounding, spr1 plants were deficient mainly in the systemic response. Consistent with this phenotype, spr1 plants exhibited normal PI induction in response to oligosaccharide signals that are thought to play a role in the local wound response. Moreover, spr1 abolished JA accumulation in response to exogenous systemin, and reduced JA accumulation in wounded leaves to approximately 57% of wild‐type (WT) levels. Analysis of reciprocal grafts between spr1 and WT plants showed that spr1 impedes systemic PI expression by blocking the production of the long‐distance wound signal in damaged leaves, rather than inhibiting the recognition of that signal in systemic undamaged leaves. These experiments suggest that Spr1 is involved in a signaling step that couples systemin perception to activation of the octadecanoid pathway, and that systemin acts at or near the site of wounding (i.e. in rootstock tissues) to increase JA synthesis to a level that is required for the systemic response. It was also demonstrated that spr1 plants are not affected in the local or systemic expression of a subset of rapidly induced wound‐response genes, indicating the existence of a systemin‐independent pathway for wound signaling.
An abscisic acid‐sensitive checkpoint in lateral root development of Arabidopsis
Authors: De Smet Ive , Signora Laurent , Beeckman Tom , Inzé Dirk , Foyer Christine H. , Zhang Hanma
Published: March 2003

Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post‐embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10‐fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA‐induced lateral root inhibition is mediated by an auxin‐independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin‐dependent regulatory pathway for the post‐emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3‐1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA‐sensitive, auxin‐independent checkpoint for lateral root development in Arabidopsis at the post‐emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.