bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium
Authors: Allen Gethyn J. , Sanders Dale 
Published: March 2003

SummaryActivity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole‐vacuole mode of patch‐clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At ‘zero’ to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage‐independent (non‐rectifying) and were typical of Vacuolar K,+‐selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time‐dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl− permeability was assumed then the apparent PCa was lower. However, substitution of Cl− by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl− permeability of the SV channel is invalid. Single‐channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi‐ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg2+‐activated and were demonstrated to be Mg2+‐permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions.The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.
Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins
Authors: Fan Hua‐Ying , Hu Yi , Tudor Matthew , Ma Hong 
Published: March 2003

SummaryMADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled‐coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C‐terminal region of AG in a yeast two‐hybrid selection, 156 clones that encode potential AG‐interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.
Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl
Authors: Gendreau Emmanuel , Höfte Herman , Grandjean Olivier , Brown Spencer , Traas Jan 
Published: March 2003

SummaryA majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light‐grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light‐grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light‐ or dark‐grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark‐grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild‐type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light‐grown hypocotyls is not the consequence of a simple feed‐back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome‐controlled photomorphogenic program.
Upregulation of two ripening‐related genes from a non‐climacteric plant (pepper) in a transgenic climacteric plant (tomato)
Authors: Kuntz  , Chen  , Simkin  , Römer  , Shipton  , Drake  , Schuch  , Bramley  
Published: March 2003

SummaryActivities of promoters from the capsanthin/capsorubin synthase and fibrillin genes, which are molecular markers for ripening in the non‐climacteric pepper fruits, have been studied in transgenic tomato plants that produce fruits of the climacteric type (characterized by an increase in respiration and ethylene production). The promoters of both genes were strongly upregulated during tomato fruit ripening in a manner similar to the induction of these genes in pepper fruits. Induction occurred at the mature green stage preceding ripening (a stage when ethylene production and respiration are known to rise in tomato fruits). Ethylene positively influenced the expression of both genes in tomato. Other plant growth regulators, namely abscisic acid, auxin and polyamines, did not alter gene expression. In contrast, water loss strongly induced both promoters. This dehydration‐mediated gene induction was inhibited by mitochondrial respiration inhibitors (mainly of the alternative oxidase). A slight positive effect with light, apparently not linked to normal photosynthesis but rather to photooxidative stress, was also observed. Taken together, the data indicate that activation of oxidase systems, leading to changes in the cellular redox balance, mediates the induction of both genes in tomato. Various cellular compartments are likely to be contributors to this process, which leads to the developmental regulation of nuclear genes encoding plastid‐located proteins.
Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells
Authors: Kunik Talya , Palanichelvam Karuppaiah , Czosnek Henryk , Citovsky Vitaly , Gafni Yedidya 
Published: March 2003

SummaryThe tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly‐transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem‐associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the β‐glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro‐injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant‐and insect‐cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)‐specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino‐terminus of the protein.
Phosphorylation mediates the nuclear targeting of the maize Rab17 protein
Authors: Jensen Anders B. , Goday Adela , Figueras Mercè , Jessop Alison C. , Pagès Montserrat 
Published: March 2003

SummaryThe maize abscisic acid‐responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In‐frame fusion of Rab17 to the reporter protein β‐glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen‐type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.
BAC representation of two low‐copy regions of the genome of Arabidopsis thaliana
Authors: Bent Elisabeth , Johnson Samantha , Bancroft Ian 
Published: March 2003

SummaryTwo regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel‐purified DNA samples of YACs selected from the YAC‐based physical map of chromosome 4 were used to probe high‐density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty‐four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4‐fold redundant coverage, with a range of 2‐ to 22‐fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.
Ser6 in the maize actin‐depolymerizing factor, ZmADF3, is phosphorylated by a calcium‐stimulated protein kinase and is essential for the control of functional activity
Authors: Smertenko Andrei P. , Jiang Chang‐Jie , Simmons Nicola J. , Weeds Alan G. , Davies Dewi R. , Hussey Patrick J. 
Published: March 2003

SummaryMaize actin‐depolymerizing factor, ZmADF, binds both G‐ and F‐actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal‐responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium‐stimulated protein kinase present in a 40–70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3–4) does not bind G‐ or F‐actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.
Acetyl‐CoA:benzylalcohol acetyltransferase – an enzyme involved in floral scent production in Clarkia breweri
Authors: Dudareva Natalia , D’Auria John C. , Nam Kyoung Hee , Raguso Robert A. , Pichersky Eran 
Published: March 2003

SummaryVolatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl‐CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433‐residue BEAT protein does not show high similarity to any previously characterized protein, but a 35‐residue region from position 135–163 has significant similarity (42–56% identity) to several proteins known or suspected to use an acyl‐CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.
The terminal O‐acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A‐dependent acyl transfer
Authors: St‐Pierre Benoit , Laflamme Pierre , Alarco Anne‐Marie , D Vincenzo , Luca E 
Published: March 2003

SummaryThe gene encoding acetyl CoA:deacetylvindoline 4‐O‐acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross‐reacting with anti‐DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light‐treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of ≈ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit‐ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O‐acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.
A mutant inLycopersicon esculentumMill. with highly reduced VA mycorrhizal colonization: isolation and preliminary characterisation
Authors: Barker S. J. , Stummer B. , Gao L. , Dispain I. , O’Connor P. J. , Smith S. E. 
Published: March 2003

Summary This paper reports the successful isolation and preliminary characterisation of a mutant of

Lycopersicon esculentum

Mill. with highly reduced vesicular‐arbuscular (VA) mycorrhizal colonization. The mutation is recessive and has been designated

rmc

. Colonization by

G. mosseae

is characterised by poor development of external mycelium and a few abnormal appressoria. Vesicles were never formed by this fungus in association with the mutant.

Gi. margarita

formed large amounts of external mycelium, complex branched structures and occasional auxiliary cells. Small amounts of internal colonization also occurred. Laser scanning confocal microscopy (LSCM) gave a clear picture of the differences in development of

G. intraradices

and

Gi. margarita

in mutant and wild‐type roots and confirmed that the fungus is restricted to the root surface of the mutants. The amenability of tomato for molecular genetic characterisation should enable us to map and clone the mutated gene, and thus identify one of the biochemical bases for inability to establish a normal mycorrhizal symbiosis. The mutant represents a key advance in molecular research on VA mycorrhizal symbiosis.
Cytosolic acidification but not auxin at physiological concentration is an activator of MAP kinases in tobacco cells
Authors: Tena Guillaume , Renaudin Jean‐Pierre 
Published: March 2003

Summary In higher plants, MAP kinase cascades are involved in the transduction of numerous stress‐related signals but much less is known about the effect of mitogenic signals. We have analysed MAP kinase activation in tobacco cells after treatment by auxin, a growth factor required at physiological concentrations for mitosis in plant cell cultures. From in‐gel assay of myelin basic protein kinase and from immunochemical detection of ERK related kinases, we show that the mitogenic effect of auxin, which was confirmed by the specific increase of several mRNAs species, did not rely on MAP kinase activation within the first 2 hours. These data contest previous results which could be due to the activation of MAP kinase by a signal other than auxin. In the second part of this study, we show that the treatment of the cells with high concentrations of various weak lipophilic acids such as auxin, in a non‐physiological concentration range, butyric or acetic acid is sufficient to activate transiently a MAP kinase. The data show that MAP kinase activation is the consequence of cytosolic acidification. Moreover, it is not sensitive to the protein kinase inhibitor staurosporine. These results suggest a functional role for cytosolic acidification as a second messenger mediating MAP kinase activation in the response of plant cells to various stresses.
BRANCHED SILKLESSmediates the transition from spikelet to floral meristem duringZea maysear development
Authors: Colombo Lucia , Marziani Giovanna , Masiero Simona , Wittich Peter E. , Schmidt Robert J. , Gorla Mirella Sari , Pè M. Enrico 
Published: March 2003

Summary The molecular and genetic control of inflorescence and flower development has been studied in great detail in model dicotyledonous plants such as

Arabidopsis

and

Antirrhinum

. In contrast, little is known about these important developmental steps in monocotyledonous species. Here we report the analysis of the

Zea mays

mutant

branched silkless1–2 (bd1–2)

, allelic to

bd1

, which we have used as a tool to study the transition from spikelet to floret development in maize. Floret development is blocked in the female inflorescence (the ear) of

bd1–2

plants, whereas florets develop almost normally in the male inflorescence (the tassel). Detailed phenotypic analyses indicate that in

bd1–2

mutants ear inflorescence formation initiates normally, however, the spikelet meristems do not proceed to form floret meristems. The ear spikelets, at anthesis, contain various numbers of spikelet‐like meristems and glume‐like structures. Furthermore, growth of branches from the base of the ear is often observed. Expression analyses show that the floral‐specific MADS box genes

Zea mays AGAMOUS1

(

ZAG1

), ZAG2 and

Zea mays MADS 2

(

ZMM2

) are not expressed in ear florets in

bd1–2

mutants, whereas their expression in tassel florets is similar to that of wild type. Taken together, these data indicate that the development from spikelet to floret meristem is differentially controlled in the ear and tassel in the monoecious grass species

Zea mays

, and that

BRANCHED SILKLESS

plays an important role in regulating the transition from spikelet meristem to floral meristem during the development of the female inflorescence of maize.
The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase
Authors: Nuccio Michael L. , Russell Brenda L. , Nolte Kurt D. , Rathinasabapathi Bala , Gage Douglas A. , Hanson Andrew D. 
Published: March 2003

Summary Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two‐step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well‐established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline monooxygenase (CMO), a stromal enzyme with a Rieske‐type [2Fe‐2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet‐deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe‐2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post‐transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02– 0.05 μmol g–1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30‐fold. The choline precursors mono‐ and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet‐deficient plants engineered with choline‐oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.