bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryTelomeres 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

SummaryThe 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

SummaryTo 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

SummaryThe 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

SummaryStomatal 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

SummaryThe 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

SummaryFlowers 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

SummaryGenetic 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

Summary 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

SummaryA 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

SummaryWe 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

SummaryUnlike 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

SummaryExpression 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

SummarySPY 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.