bluuebunny/crossref_metadata_2025 huggingface dataset:
Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley–powdery mildew interaction
Authors: Zhou Fasong , Andersen Claus H. , Burhenne Kim , Fischer Pernille Hertz , Collinge David B. , Thordal‐Christensen Hans
Published: March 2003

We propose a model for activation of the epidermal cell hypersensitive response (HR) in the barley/powdery mildew interaction. The model suggests that the plasma membrane proton pump (H+‐ATPase) of epidermal cells is activated following penetration by an avirulent powdery mildew fungus. This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR‐related H2O2 is found in the attachment zones between the epidermal cell and underlying mesophyll cells; (2) scavenger treatment reduces HR; (3) treatment of leaves with low‐pH (3.5) citrate and succinate buffers causes more cells to undergo HR in the compatible interaction, while treatment with the same buffers at pH 5.5 reduces the number of HR‐cells in the incompatible interaction; (4) race‐specific proton extrusion is observed underneath epidermal tissue detached from leaves inoculated 15 h earlier; and (5) treatment of leaves with fusicoccin, an activator of the plasma membrane H+‐ATPase, increases the number of HR‐cells in the compatible interaction.
Over‐expression of a single Ca2+‐dependent protein kinase confers both cold and salt/drought tolerance on rice plants
Authors: Saijo Yusuke , Hata Shingo , Kyozuka Junko , Shimamoto Ko , Izui Katsura
Published: March 2003

A rice gene encoding a calcium‐dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over‐expression of OsCDPK7 enhanced induction of some stress‐responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post‐translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.
Developmental and light‐dependent regulation of a phloem‐localised K+ channel of Arabidopsis thaliana
Authors: Deeken Rosalia , Sanders Christoph , Ache Peter , Hedrich Rainer
Published: March 2003

K+ channels in plants can currently be classified into six families with individual members being involved in nutrient uptake, loading of the xylem and the physiology of stomatal movement. In this study we have focused on akt2/3. This K+ channel, as shown by GUS‐expression analysis, is expressed in the phloem and xylem of the aerial parts of Arabidopsis thaliana. Northern blot analyses revealed the highest akt2/3‐concentrations in the flower stalk, followed by the leaf, flower and stem. During the light period (8 am to 4 pm), transcripts reached a peak around noon (11 am), decayed to almost 50% in the afternoon and reached a low background level in the following dark period. In continuous darkness, however, the K+ channel mRNA content had already decreased beyond the background level by noon. In leaves and flower stalk, the light‐induced transcription of akt2/3 was suppressed by CO2‐free air, indicating that gene activity is under the control of photosynthates. Additionally, when rosette leaves were illuminated and flower stalks shaded, akt2/3‐mRNA transcription was still inhibited in the shaded region. This indicates that channel gene activation is sensitive to photosynthesis‐derived factors from neighboring cells rather than factors mobile in the phloem. We propose that the coupling between sugar production and allocation involves the photosynthate‐ and light‐dependent phloem K+ channel AKT2/3.
Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR‐5 protein
Authors: Ibeas José I. , Lee Hyeseung , Damsz Barbara , Prasad Doddananjappa T. , Pardo José M. , Hasegawa Paul M. , Bressan Ray A. , Narasimhan Meena L.
Published: March 2003

Osmotin is a plant PR‐5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR‐5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an α‐1,2‐mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6‐mannose backbone of the outer chain of cell wall N‐linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an α‐1,3‐mannosyltransferase that adds the terminal mannose to the outer chain branches of N‐linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR‐5 proteins.
Functional identification of an Arabidopsis Snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast
Authors: Kleinow Tatjana , Bhalerao Rishikesh , Breuer Frank , Umeda Masaaki , Salchert Klaus , Koncz Csaba
Published: March 2003

Yeast Snf4 is a prototype of activating γ‐subunits of conserved Snf1/AMPK‐related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast. By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non‐fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT‐binding factor Hap3b, casein kinase I, zinc‐finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP‐hexose transporters, calmodulin, SMC1‐cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C‐terminal regulatory domain of Arabidopsis SnRKs AKIN10 and AKIN11.
A gain‐of‐function phenotype conferred by over‐expression of functional subunits of the COP9 signalosome in Arabidopsis
Authors: Kang Dingming , Wang Xiping , Cao Kaiming , Sun Chongrong , Deng Xing‐Wang , Wei Ning
Published: March 2003

The COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild‐type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6‐1 mutant phenotype. Moreover, light‐grown rFUS6 over‐expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild‐type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long‐hypocotyl phenotype was also observed in transgenic seedlings over‐expressing Arabidopsis COP9. This finding indicates that over‐expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain‐of‐function phenotype relative to the complex. Human GPS1, when expressed in the fus6‐1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low‐abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.
Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co‐expressed in insect cells
Authors: Urbach Serge , Chérel Isabelle , Sentenac Hervé , Gaymard Frédéric
Published: March 2003

KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker‐like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one,
l‐α‐lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion‐exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co‐expressed in insect cells. The use of the two‐hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+‐channel subunits are able to co‐assemble without any discrimination, previously put forward based on co‐expression in Xenopus oocytes of various K+‐channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co‐localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.
In vivo imaging of an elicitor‐induced nitric oxide burst in tobacco
Authors: Foissner Ilse , Wendehenne David , Langebartels Christian , Durner Jörg
Published: March 2003

A growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti‐microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF‐2 DA) has been developed which allows bio‐imaging of NO in vivo. Here we use the cell‐permeable DAF‐2 DA, in conjunction with confocal laser scanning microscopy, for real‐time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO‐induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.
Characterization of cis‐elements required for vascular expression of the Cinnamoyl CoA Reductase gene and for protein–DNA complex formation
Authors: Lacombe Eric , Van Doorsselaere Jan , Boerjan Wout , Boudet Alain M. , Grima‐Pettenati Jacqueline
Published: March 2003

Cinnamoyl‐CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR–GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain‐of‐function experiments identified the sequences between positions −119 and −77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility‐shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel‐shift assays, two DNA–protein interaction sites were mapped. Finally, the role of protein–protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.
Identification of plant actin‐binding proteins by F‐actin affinity chromatography
Authors: Hu Shiquan , Brady Shari R. , Kovar David R. , Staiger Christopher J. , Clark Greg B. , Roux Stanley J. , Muday Gloria K.
Published: March 2003

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin‐binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin‐binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G‐actin) or actin filaments (filamentous actin, F‐actin) have been used to identify actin‐binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G‐actin, F‐actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane‐associated actin‐binding proteins. Extracts of soluble actin‐binding proteins yield distinct patterns when eluted from the G‐actin and F‐actin columns, respectively, leading to the identification of a putative F‐actin‐binding protein of approximately 40 kDa. When plasma membrane‐associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F‐actin column and cross‐reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F‐actin column. These experiments provide a new approach that may help to identify novel actin‐binding proteins from plants.
Plastidic metabolite transporters and their physiological functions in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum
Authors: Häusler Rainer E. , Baur Bernhard , Scharte Judith , Teichmann Thomas , Eicks Michael , Fischer Katrin L. , Flügge Ulf‐Ingo , Schubert Sabine , Weber Andreas , Fischer Karsten
Published: March 2003

The inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose‐6‐phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT‐ and GPT‐specific transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These findings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3‐phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM‐induced), similar to non‐green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic fluxes during the diurnal CAM cycle.
Technical Advance: Cytometric analysis of an epitope‐tagged transit peptide bound to the chloroplast translocation apparatus
Authors: Subramanian Chitra , Ivey Robert , Bruce Barry D.
Published: March 2003

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope‐tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His‐S‐SStp, contains a removable dual‐epitope tag, His‐S, at its N‐terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser‐scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C‐terminal to the transit peptide, His‐S‐SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non‐radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre‐ and post‐import thermolysin treatment, photochemical cross‐linking and organelle fractionation. The interaction was specific for the transit peptide since His‐S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP‐dependence. The addition of GTP showed inhibition of the binding of His‐S‐SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His‐S‐SStp and Toc75 by confocal microscopy revealed a patch‐like labeling, suggesting a co‐ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast‐binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.
Living under a ‘dormant’ canopy: a molecular acclimation mechanism of the desert plant Retama raetam
Authors: Mittler Ron , Merquiol Emmanuelle , Hallak‐Herr Elza , Rachmilevitch Shimon , Kaplan Aaron , Cohen Mira
Published: March 2003

Desert plants are exposed to a combination of environmental stress conditions, including low water availability, extreme temperature fluctuations, high irradiance and nutrient deprivation. Studying desert plants within their natural habitat may therefore reveal novel mechanisms and strategies that enable plants to resist stressful conditions. We studied the acclimation of Retama raetam, an evergreen stem‐assimilating desert plant, to growth within an arid dune ecosystem. Retama raetam contained two different populations of stems: those of the upper canopy, exposed to direct sunlight, and those of the lower canopy, protected from direct sunlight. During the dry season, stems of the upper canopy contained a very low level of a number of essential proteins, including the large and small subunits of rubisco, ascorbate peroxidase and the D1 subunit of the reaction centre of photosystem II. However, RNA encoding these proteins was present; cytosolic transcripts were associated with polysomes, while chloroplastic transcripts were not. Upon water application, as well as following the first rainfall of the season, these ‘photosynthetically suppressed’ stems recovered and accumulated essential proteins within 6–24 h. In contrast, stems of the lower canopy contained the essential proteins throughout the dry season. We suggest that R. raetam uses an acclimation strategy of ‘partial plant dormancy’ in order to survive the dry season. ‘Dormancy’, as evident by the post‐transcriptional suppression of gene expression, as well as the suppression of photosynthesis, was induced specifically in stems of the upper canopy which protect the lower canopy by shading.
Expression of MsLEC1‐ and MsLEC2‐antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities
Authors: Brill Laurence M. , Evans Cory J. , Hirsch Ann M.
Published: March 2003

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1‐ and MsLEC2‐antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector‐control and sense‐transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense‐transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1‐antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.
CKS1At overexpression in Arabidopsis thaliana inhibits growth by reducing meristem size and inhibiting cell‐cycle progression
Authors: De Veylder Lieven , Beemster Gerrit T.S. , Beeckman Tom , Inzé Dirk
Published: March 2003

The SUC1/CKS1 proteins associate with cyclin‐dependent kinases (CDKs) and play an essential role in the regulation of the cell cycle. Recently, an Arabidopsis thaliana SUC1/CKS1 homologous gene, designated CKS1At, has been cloned. Here, overexpression of CKS1At in Arabidopsis is shown to reduce leaf size and root growth rates. Reduced root growth resulted primarily from an increase of the cell‐cycle duration and a shortening of the meristem. Endoreduplication was unaffected. The increased cell‐cycle duration was associated with an equal extension of both the G1 and G2 phases. This inhibition was due to the binding of CDK subunits with CDKs. The reduced growth rates in response to altered cell‐cycle gene expression demonstrates a direct dependence of plant growth rates on cell‐cycle regulation.
Cold‐activation of Brassica napus BN115 promoter is mediated by structural changes in membranes and cytoskeleton, and requires Ca2+ influx
Authors: Sangwan Veena , Foulds Inge , Singh Jas , Dhindsa Rajinder S.
Published: March 2003

Previous studies on cold‐triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold‐induced genes as end‐point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold‐inducible BN115 gene, the β‐glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold‐activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25°C by the membrane rigidifier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25°C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B. Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP‐ribose activated it at 25°C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25°C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25°C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold‐activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases.
Partial complementation of embryo defective mutations: a general strategy to elucidate gene function
Authors: Despres Barbara , Delseny Michel , Devic Martine
Published: March 2003

The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP‐protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.
Localization and targeting of the VP14 epoxy‐carotenoid dioxygenase to chloroplast membranes
Authors: Tan Bao‐Cai , Cline Kenneth , McCarty Donald R.
Published: March 2003

Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9‐cis epoxy‐carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy‐carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy‐carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma‐targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N‐terminal sequence. A putative amphipathic α‐helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S‐transferase failed to associate with the thylakoid membrane. The membrane‐bound VP14 was partially resistant to chaotropic washes such as 0.1 m Na2CO3 (pH 11.5) and 6 m urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.
The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD‐finger family of transcription factors
Authors: Wilson Zoe A. , Morroll Shaun M. , Dawson Janet , Swarup Ranjan , Tighe Paddy J.
Published: March 2003

We report here the molecular characterisation of the Arabidopsis MALE STERILITY1 gene, which is a critical sporophytic controlling factor for anther and pollen development. Homozygous ms1 mutants do not produce viable pollen, but are otherwise phenotypically normal. Degeneration of pollen occurs soon after microspore release from the tetrads, at which time the tapetum also appears abnormally vacuolated. The MS1 gene is expressed at low levels in anthers from closed buds, with expression in the tapetum at the stage of microspore release. No expression is seen in open flowers. The deduced MS1 protein sequence shows strong homology to the PHD‐finger motif found in known transcription factors from humans, yeast and higher plants. Six alleles of ms1 have been identified; all result in premature termination of the MS1 protein and loss of the PHD‐finger motif. MS1 is likely to play a key role in regulating transcription during specific stages of male gametogenesis and anther development. As such, MS1 provides a valuable tool for the manipulation of male sterility in higher plants.
Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth
Authors: Ljung Karin , Bhalerao Rishikesh P. , Sandberg Göran
Published: March 2003

The distribution and biosynthesis of indole‐3‐acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg−1 of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred‐fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten‐fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue‐dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue‐specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.
Cytochrome b6f mutation specifically affects thermal dissipation of absorbed light energy in Arabidopsis
Authors: Munekage Yuri , Takeda Satomi , Endo Tsuyoshi , Jahns Peter , Hashimoto Takashi , Shikanai Toshiharu
Published: March 2003

Light‐induced lumenal acidification controls the efficiency of light harvesting by inducing thermal dissipation of excess absorbed light energy in photosystem II. We isolated an Arabidopsis mutant, pgr1 (proton gradient regulation), entirely lacking thermal dissipation, which was observed as little non‐photochemical quenching of chlorophyll fluorescence. Map‐based cloning showed that pgr1 had a point mutation in petC encoding the Rieske subunit of the cytochrome b6f complex. Although the electron transport rate was not affected at low light intensity, it was significantly restricted at high light intensity in pgr1, indicating that the lumenal acidification was not sufficient to induce thermal dissipation. This view was supported by (i) slow de‐epoxidation of violaXanthin, which is closely related to lumenal acidification, and (ii) reduced 9‐aminoacridine fluorescence quenching. Although lumenal acidification was insufficient to induce thermal dissipation, growth rate was not affected under low light growth conditions in pgr1. These results suggest that thermal dissipation is precisely regulated by lumenal pH to maintain maximum photosynthetic activity. We showed that pgr1 was sensitive to changes in light conditions, demonstrating that maximum activity of the cytochrome b6f complex is indispensable for short‐term acclimation.
Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana
Authors: Tanaka Ryouichi , Koshino Yoshihiro , Sawa Shinichiro , Ishiguro Sumie , Okada Kiyotaka , Tanaka Ayumi
Published: March 2003

The light‐harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de‐etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a : b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full‐green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light‐harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green‐gel system increased by 20%. (3) The ratio of the light‐harvesting complex II apoproteins (LHCP) to 47‐kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10–20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.
Brassinosteroid functions to protect the translational machinery and heat‐shock protein synthesis following thermal stress
Authors: Dhaubhadel Sangeeta , Browning Karen S. , Gallie Daniel R. , Krishna Priti
Published: March 2003

In addition to their essential role in plant development, brassinosteroids have the ability to protect plants from various environmental stresses. Currently it is not understood how brassinosteroids control plant stress responses at the molecular level. We have begun an investigation into the molecular mechanisms underlying 24‐epibrassinolide (EBR)‐mediated stress resistance. Earlier we found that treatment of Brassica napus seedlings with EBR leads to a significant increase in their basic thermotolerance, and results in higher accumulation of four major classes of heat‐shock proteins (hsps) as compared to untreated seedlings. Surprisingly, previous studies have shown that while hsp levels were significantly higher in treated seedlings during the recovery period, transcripts corresponding to these hsps were present at higher levels in untreated seedlings. To understand mechanisms controlling hsp synthesis in EBR‐treated and untreated seedlings, we studied protein synthesis in vivo as well as in vitro, and assessed the levels of components of the translational machinery in these seedlings. We report here that increased accumulation of hsps in EBR‐treated seedlings results from higher hsp synthesis, even when the mRNA levels are lower than in untreated seedlings, and that several translation initiation and elongation factors are present at significantly higher levels in EBR‐treated seedlings as compared to untreated seedlings. These results suggest that EBR treatment limits the loss of some of the components of the translational apparatus during prolonged heat stress, and increases the level of expression of some of the components of the translational machinery during recovery, which correlates with a more rapid resumption of cellular protein synthesis following heat stress and a higher survival rate.
A role for nitrilase 3 in the regulation of root morphology in sulphur‐starving Arabidopsis thaliana
Authors: Kutz Alexandra , Müller Axel , Hennig Peter , Kaiser Werner M. , Piotrowski Markus , Weiler Elmar W.
Published: March 2003

Arabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole‐3‐acetonitrile to indole‐3‐acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a β‐cyano‐l‐alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in bolting and flowering plants, but its level increases strongly when plants experience sulphur deprivation. No other nitrilase genes respond to sulphur supply/deficiency. Neither N‐ nor P‐deprivation cause detectable changes in NIT3 promoter activity. In transgenic plants expressing uidA under the control of the NIT3 promoter (NIT3p::uidA), sulphate deprivation leads to the appearance of β‐glucuronidase activity in shoots and particularly in roots, most strongly in the conductive tissues and lateral root primordia. Deletion analysis allowed localization of the sulphur‐responsive element to a 317 bp segment of the NIT3 promoter encompassing nt −2151 to −1834 upstream of the transcriptional start point. Both nitrilase polypeptide and nitrilase activity were also induced by sulphur starvation. NIT3 promoter activity was strongly induced by O‐acetylserine, suggesting that, as is the case with enzymes of sulphate assimilation, sulphate deficiency may be communicated to NIT3 via an increase in the level of the cysteine precursor, O‐acetylserine. During sulphur deprivation, a preferential depletion of the pool of the indole‐3‐acetonitrile precursor glucobrassicin compared with that of total glucosinolates was noticed. In the absence of an external sulphate supply, plants developed longer roots with a higher number of lateral roots. The increased growth of the root system occurred at the expense of shoot growth which was retarded under conditions of sulphur starvation. Taken together, these results suggest that a regulatory loop appears to exist by which sulphate deficiency, through an increase in glucobrassicin turnover and nitrilase 3 accumulation, initiates the production of extra auxin leading to increased root growth and branching, thus allowing the root system to penetrate new areas of soil effectively to gain access to fresh supplies of sulphur.
The IRE gene encodes a protein kinase homologue and modulates root hair growth in Arabidopsis
Authors: Oyama Tokitaka , Shimura Yoshiro , Okada Kiyotaka
Published: March 2003

We identified an Arabidopsis mutant, incomplete root hair elongation (ire), whose root hairs are about 40% shorter than the wild type owing to the early cessation of their growth. In contrast, the hy5 mutant has root hairs about twice as long as the wild type, possibly because of delayed cessation of growth. Thus IRE and HY5 are likely to modify the duration of growth of root hairs. We cloned the IRE locus using the T‐DNA tagged line. The IRE locus encodes a protein that includes a serine/threonine protein kinase domain. The primary structure of this kinase domain shows significant similarity to a group of protein kinases among various eukaryotes. In the Arabidopsis genome there are at least three genes that are closely related to IRE. The IRE transcript was detected in every organ examined. However, the IRE promoter–GUS fusion gene was strongly expressed in the elongating root hair cells, suggesting the cell‐autonomous function of IRE in root hairs. GUS activity was also detected in pollen grains, which develop by tip growth, suggesting that IRE has a common role in the tip growth of plant cells.
Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N‐mediated resistance to tobacco mosaic virus
Authors: Liu Yule , Schiff Michael , Marathe Rajendra , Dinesh‐Kumar S. P.
Published: March 2003

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll‐interleukin‐1 receptor/nucleotide binding site/leucine‐rich repeat (TIR‐NBS‐LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N‐mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1‐, EDS1‐, and NPR1/NIM1‐ like genes in N‐mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1‐like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR‐NBS‐LRR class of resistance proteins. Requirement of Rar1‐ like gene for N‐mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR‐NBS‐LRR and CC‐NBS‐LRR proteins. The TRV based VIGS approach as described here to study N‐mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.
On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum
Authors: Winter Kai‐Uwe , Saedler Heinz , Theißen Günter
Published: March 2003

Class B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2‐like and DAL12‐like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild‐type plants and in different class B mutants. In wild‐type plants and in a conditional mutant grown at a permissive temperature, gain‐of‐function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss‐of‐function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non‐permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain‐of‐function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.
Light regulated modulation of Z‐box containing promoters by photoreceptors and downstream regulatory components, COP1 and HY5, in Arabidopsis
Authors: Yadav Vandana , Kundu Snehangshu , Chattopadhyay Debasis , Negi Prem , Wei Ning , Deng Xing‐Wang , Chattopadhyay Sudip
Published: March 2003

The Z‐box is one of the light‐responsive elements (LREs) found in the promoters of light inducible genes. We have studied the light responsive characteristics of Z‐box containing synthetic as well as native promoters. We show that promoters with Z‐box as a single LRE or paired with another LRE can respond to a broad spectrum of light. The response is primarily mediated by phyA, phyB and CRY1 photoreceptors at their respective wavelengths of light. We have demonstrated that CAB1 and Z‐GATA containing promoters are down‐regulated in hy5 mutants in the light. On the other hand, a promoter with Z‐box alone is down‐regulated in hy5 mutants both in dark and in light conditions, suggesting involvement of a similar regulatory system in the regulation of the promoter in two distinct developmental pathways: skotomorphogenesis and photomorphogenesis. Furthermore, similar to the CAB1 promoter, a Z‐GATA containing promoter is derepressed in cop1 mutants in the dark. DNA–protein interaction studies reveal the presence of a DNA‐binding activity that is specific to Z‐box. These results provide insights into the regulation of the Z‐box LRE mediated by various light signaling components.