bluuebunny/crossref_metadata_2025 huggingface dataset:
Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato
Authors: Bendahmane Abdelhafid , Querci Maddalena , Kanyuka Konstantin , Baulcombe David C.
Published: March 2003

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium‐mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2‐mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper‐nucleotide binding site‐leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.
Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity
Authors: Becker Jürgen , Kempf Ralf , Jeblick Wolfgang , Kauss Heinrich
Published: March 2003

The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response. However, elicitor competence developed while conditioning the surface‐abraded seedlings by rotating them in buffer for 4 h. Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clasto‐lactacystin β‐lactone (LAC) and carboxybenzoyl‐
l‐leucyl‐
l‐leucyl‐
l‐leucinal (LLL). In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition. Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA. LAC and LLL caused an accumulation of ubiquitin‐conjugated proteins and enhanced the expression of a proteasome α‐subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general. Together, our results suggest that the ubiquitin–proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast.
Arabidopsis thaliana SHAGGY‐related protein kinases (AtSK11 and 12) function in perianth and gynoecium development
Authors: Dornelas Marcelo C. , Van Lammeren Andie A. M. , Kreis Martin
Published: March 2003

In higher plants, the correct patterning of the floral meristem in terms of organ type, number and form is the result of a concerted expression of a network of genes. We describe phenotypes of flower patterning, resulting from a reduction of transcript levels of the Arabidopsis SHAGGY‐related protein kinase genes AtSK11(ASKα) and AtSK12(ASKγ). The AtSK genes are plant homologues of the Drosophila shaggy (SGG) gene and the mammalian Glycogen‐Synthase Kinase‐3 (GSK‐3). The SGG protein kinase is a key component of the wingless signalling pathway and is required for the establishment of tissue patterning and cell fate determination. The expression patterns of the AtSK11(ASKα) and AtSK12(ASKγ) genes during wild‐type Arabidopsis inflorescence development, detected by in situ hybridisation, have been shown to be consistent with a possible role in floral meristem patterning. AtSK11(ASKα) and AtSK12(ASKγ) transcripts were detected at the periphery of the inflorescence meristem and in the floral meristem. At later stages the expression of the AtSK genes became localised in specific regions of developing flower organ primordia. Furthermore, we have obtained and analysed transgenic plants containing AtSK11(ASKα) and AtSK12(ASKγ) gene specific antisense constructs. These plants developed flowers showing a higher number of perianth organs and an alteration of the apical‐basal patterning of the gynoecium.
Arbuscular mycorrhizal fungi induce the non‐mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the ‘yellow pigment’ and other apocarotenoids
Authors: Walter Michael H. , Fester Thomas , Strack Dieter
Published: March 2003

Plants and certain bacteria use a non‐mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS) and 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long‐known ‘yellow pigment’. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an ‘apocarotenoid complex’ of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.
Phytochrome A resets the circadian clock and delays tuber formation under long days in potato
Authors: Yanovsky Marcelo J. , Izaguirre Miriam , Wagmaister Javier A. , Gatz Christiane , Jackson Stephen D. , Thomas Brian , Casal Jorge J.
Published: March 2003

Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far‐red or far‐red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild‐type plants and phyA over‐expressors, but had little effect in phyA under‐expressors. Far‐red light was also able to reset the rhythm of leaf movement in wild‐type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA‐deficient potato plants. Resetting of the rhythm by far‐red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far‐red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light‐grown potato plants.
Soluble phospholipase A2 activity is induced before oxylipin accumulation in tobacco mosaic virus‐infected tobacco leaves and is contributed by patatin‐like enzymes
Authors: Dhondt Sandrine , Geoffroy Pierrette , Stelmach Boguslawa A. , Legrand Michel , Heitz Thierry
Published: March 2003

Recent evidence suggests that oxidized lipid‐derived molecules play significant roles in inducible plant defence responses against microbial pathogens, either by directly deterring parasite multiplication, or as signals involved in the induction of sets of defence genes. The synthesis of these oxylipins was hypothesized to be initiated by the phospholipase A2‐mediated release of unsaturated fatty acids from membrane lipids. Here, we demonstrate that, in tobacco leaves reacting hypersensitively to tobacco mosaic virus, a strong increase in soluble phospholipase A2 (PLA2) activity occurs at the onset of necrotic lesion appearance. This rapid PLA2 activation occurred before the accumulation of 12‐oxophytodienoic and jasmonic acids, two fatty acid‐derived defence signals. Three PLA2 isoforms were separated and the most active enzyme was partially purified, its N‐terminal sequence displaying similarity with patatin, the major storage protein in potato tubers. Three related tobacco patatin‐like cDNAs, called NtPat1, NtPat2 and NtPat3, were cloned, with NtPat2 encoding the PLA2 isolated from infected leaves. RT–PCR experiments showed a rapid transcriptional activation of the three NtPat genes in virus‐infected leaves, preceding the increase in PLA2 activity. Recombinant NtPat1 and NtPat3 enzymes were active in an assay using labelled bacterial membranes, and also displayed high bona fide PLA2 activity on phosphatidylcholine substrate. These results point to a possible new role of patatin‐like phospholipases in inducible plant defence responses. The induction kinetics together with the enzymatic activity data indicate that the NtPat proteins may provide precursors for oxylipin synthesis during the hypersensitive response to pathogens.
The Arabidopsis SUCROSE UNCOUPLED‐6 gene is identical to ABSCISIC ACID INSENSITIVE‐4: involvement of abscisic acid in sugar responses
Authors: Huijser Casper , Kortstee Anne , Pego Jónatas , Weisbeek Peter , Wisman Ellen , Smeekens Sjef
Published: March 2003

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled‐6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase‐dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE‐4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase‐dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid‐ and sugar‐dependent way.
The Arabidopsis sugar‐insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response
Authors: Laby Ron J. , Kincaid M. Sean , Kim Donggiun , Gibson Susan I.
Published: March 2003

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar‐insensitive (sis) mutants of Arabidopsis that, unlike wild‐type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA‐biosynthesis mutant aba2 and the ABA‐insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose‐ and mannose‐insensitive phenotypes. Wild‐type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.
A zinc finger protein RHL41 mediates the light acclimatization response in Arabidopsis
Authors:   Asako Iida, Kazuoka Toru , Torikai Satomi , Kikuchi Hiromi , Oeda Kenji
Published: March 2003

Arabidopsis thaliana plants showed an increased tolerance to high‐intensity light when pre‐exposed to medium‐intensity light. This response, known as light acclimatization, depended on the quantity of light, the period of irradiation, and the quality of light. Among characterized acclimatization‐induced cDNA clones, we identified a zinc finger protein rhl41 (responsive to high light) gene, that was rapidly up‐regulated in proportion to the time of irradiation and the light intensity. Transgenic Arabidopsis plants over‐expressing the rhl41 gene showed an increased tolerance to high‐intensity light, and also morphological changes of thicker and dark green leaves. Interestingly, the palisade parenchyma was highly developed in the leaves of the transgenic plants, which is one of the long‐term acclimatization responses in Arabidopsis plants. The anthocyanin content (a light protectant) as well as the chlorophyll content also increased. Antisense transgenic plants exhibited decreased tolerance to high irradiation. We propose that the RHL41 zinc finger protein has a key role in the acclimatization response to changes in light intensity.
A potato α‐glucosidase gene encodes a glycoprotein‐processing α‐glucosidase II‐like activity. Demonstration of enzyme activity and effects of down‐regulation in transgenic plants
Authors: Taylor* Mark A. , Ross Heather A. , McRae Diane , Stewart Derek , Roberts Ian , Duncan George , Wright Frank , Millam Steve , Davies Howard V.
Published: March 2003

In order to elucidate more fully the function of a potato gene (MAL1) encoding α‐glucosidase activity, transgenic plants in which MAL1 expression was down‐regulated were generated using antisense technology. In transgenic lines severely down‐regulated in the expression of MAL1, total α‐glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein‐processing α‐glucosidase II of mammalian and yeast origin than to other plant α‐glucosidases. Using [14C‐Glc]‐labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down‐regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down‐regulation produced an extremely stunted phenotype – the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down‐regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down‐regulation. In leaves from four antisense lines, the steady‐state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.
Metabolic engineering of beta‐carotene and lycopene content in tomato fruit
Authors: Rosati Carlo , Aquilani Riccardo , Dharmapuri Sridhar , Pallara Patrizia , Marusic Carla , Tavazza Raffaela , Bouvier Florence , Camara Bilal , Giuliano Giovanni
Published: March 2003

Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta‐carotene (pro‐vitamin A). Lycopene is transformed into beta‐carotene by the action of lycopene beta‐cyclase (β‐Lcy). We introduced, via Agrobacterium‐mediated transformation, DNA constructs aimed at up‐regulating (OE construct) or down‐regulating (AS construct) the expression of the β‐Lcy gene in a fruit‐specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta‐carotene content. The fruits from these plants display different colour phenotypes, from orange to orange‐red, depending on the lycopene/beta‐carotene ratio. Fruits from AS transformants show up to 50% inhibition of β‐Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.
Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene
Authors: Sarrobert Catherine , Thibaud Marie‐Christine , Contard‐David Pascale , Gineste Sandrine , Bechtold Nicole , Robaglia Christophe , Nussaume Laurent
Published: March 2003

A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.
Localization and control of expression of Nt‐Syr1, a tobacco snare protein
Authors: Leyman Barbara , Geelen Danny , Blatt Michael R.
Published: March 2003

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin‐related protein Nt‐Syr1 from Nicotiana in a screen for ABA‐related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus‐dependent expression of Nt‐Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt‐Syr1. The Nt‐Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well‐watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt‐Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt‐Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt‐Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA‐evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt‐Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress‐related signalling pathways, both dependent on and independent of ABA. Nt‐Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.
Promotive effect of brassinosteroids on cell division involves a distinct CycD3‐induction pathway in Arabidopsis
Authors: Hu Yuxin , Bao Fang , Li Jiayang
Published: March 2003

Brassinosteroids (BRs) are steroid hormones that play an essential role in plant growth and development. However, the contradictory results of previous studies make their role in cell division unclear. Using a cDNA array, we identified genes that respond to BR in the det2 suspension culture of Arabidopsis, and found that epi‐brassinolide upregulated transcription of the CycD3, a D‐type plant cyclin gene through which cytokinin activates cell division. RNA gel‐blot analysis and cell culturing showed that epi‐brassinolide may promote cell division through CycD3, and can substitute cytokinin in culturing of Arabidopsis callus and suspension cells. The CycD3 induction by epi‐brassinolide was further shown to involve de novo protein synthesis, but no protein phosphorylation or dephosphorylation. Induction was also found to occur in cells of a BR‐insensitive mutant, bri1, suggesting that BR induces CycD3 transcription through a previously unknown signal pathway in plants.
The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway
Authors: Merlot Sylvain , Gosti Françoise , Guerrier Danièle , Vavasseur Alain , Giraudat Jérôme
Published: March 2003

The ArabidopsisABI1andABI2genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutationsabi1–1andabi2–1,which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants ofABI1were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction‐of‐function allele ofABI2,abi2–1R1. Thein vitrophosphatase activity of the abi2–1R1 protein is approximately 100‐fold lower than that of the wild‐type ABI2 protein.Abi2–1R1plants displayed a wild‐type ABA sensitivity. However, doubly mutant plants combining theabi2–1R1allele and a loss‐of‐function allele at theABI1locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild‐type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.
Ethylene‐dependent salicylic acid regulates an expanded cell death response to a plant pathogen
Authors: O'Donnell Philip J. , Jones Jeffrey B. , Antoine Francis R. , Ciardi Joseph , Klee Harry J.
Published: March 2003

The molecular events associated with susceptible plant responses to disease‐causing organisms are not well understood. We have previously shown that ethylene‐insensitive tomato plants infected with Xanthomonas campestris pv. vesicatoria have greatly reduced disease symptoms relative to wild‐type cultivars. Here we show that salicylic acid (SA) is also an important component of the susceptible disease response. SA accumulates in infected wild‐type tissues and is correlated with necrosis but does not accumulate in ethylene‐insensitive plants. Exogenous feeding of SA to ethylene‐deficient plants restores necrosis, indicating that reduced disease symptoms are associated with failure to accumulate SA. These results indicate a mechanism for co‐ordination of phytohormone signals that together constitute a susceptible response to pathogens.
The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2;1
Authors: Schaeffer Aurélie , Bronner Roberte , Benveniste Pierre , Schaller Hubert
Published: March 2003

The Arabidopsis genome contains three distinct genes encoding sterol‐C24‐methyltransferases (SMTs) involved in sterol biosynthesis. The expression of one of them, STEROL METHYLTRANSFERASE 2;1, was modulated in 35S::SMT2;1 Arabidopsis in order to study its physiological function. Plants overexpressing the transgene accumulate sitosterol, a 24‐ethylsterol which is thought to be the typical plant membrane reinforcer, at the expense of campesterol. These plants displayed a reduced stature and growth that could be restored by brassinosteroid treatment. Plants showing co‐suppression of SMT2;1 were characterized by a predominant 24‐methylsterol biosynthetic pathway leading to a high campesterol content and a depletion in sitosterol. Pleiotropic effects on development such as reduced growth, increased branching, and low fertility of high‐campesterol plants were not modified by exogenous brassinosteroids, indicating specific sterol requirements to promote normal development. Thus SMT2;1 has a crucial role in balancing the ratio of campesterol to sitosterol in order to fit both growth requirements and membrane integrity.
Expression of a plant gene with sequence similarity to animal TGF‐β receptor interacting protein is regulated by brassinosteroids and required for normal plant development
Authors: Jiang Jingrui , Clouse Steven D.
Published: March 2003

Brassinosteroids (BRs) regulate the expression of numerous genes associated with plant development, and require the activity of a Ser/Thr receptor kinase to realize their effects. In animals, the transforming growth factor‐β (TGF‐β) family of peptides acts via Ser/Thr receptor kinases to have a major impact on several pathways involved in animal development and adult homeostasis. TGF‐β receptor‐interacting protein (TRIP‐1) was previously shown by others to be an intracellular substrate of the TGF‐β type II receptor kinase which plays an important role in TGF‐β signaling. TRIP‐1 is a WD‐repeat protein that also has a dual role as an essential subunit of the eukaryotic translation initiation factor eIF3 in animals, yeast and plants, thereby revealing a putative link between a developmental signaling pathway and the control of protein translation. In yeast, expression of a TRIP‐1 homolog has also been closely associated with cell proliferation and progression through the cell cycle. We report here the novel observation that transcript levels of TRIP‐1 homologs in plants are regulated by BR treatment under a variety of conditions, and that transgenic plants expressing antisense TRIP‐1 RNA exhibit a broad range of developmental defects, including some that resemble the phenotype of BR‐deficient and ‐insensitive mutants. This correlative evidence suggests that a WD‐domain protein with reported dual functions in vertebrates and fungi might mediate some of the molecular mechanisms underlying the regulation of plant growth and development by BRs.
Interaction of phytochromes A and B in the control of de‐etiolation and flowering in pea
Authors: Weller James L. , Beauchamp Nicola , Kerckhoffs L. Huub J. , Platten J. Damien , Reid James B.
Published: March 2003

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White‐light‐grown phyA phyB double mutant plants show severely impaired de‐etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de‐etiolation under continuous, high‐irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de‐etiolation or shade‐avoidance, but undergoes partial de‐etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night‐breaks with white light, whereas phyB appears to play a major role in detection of light quality in end‐of‐day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft‐transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.
Evidence for a disease‐resistance pathway in rice similar to the NPR1‐mediated signaling pathway in Arabidopsis
Authors: Chern Maw‐Sheng , Fitzgerald Heather A. , Yadav Ram C. , Canlas Patrick E. , Dong Xinnian , Ronald Pamela C.
Published: March 2003

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over‐expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over‐expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two‐hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade–sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1‐1 and npr1‐2 mutations, but not by the nim1‐4 mutation. The NPR1–rTGA2.1 interaction was confirmed by an in vitro pull‐down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis‐element required sequence‐specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1‐mediated resistance.
Regulation of drought tolerance by gene manipulation of 9‐cis‐epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis
Authors: Iuchi Satoshi , Kobayashi Masatomo , Taji Teruaki , Naramoto Masaaki , Seki Motoaki , Kato Tomohiko , Tabata Satoshi , Kakubari Yoshitaka , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

Abscisic acid (ABA), a plant hormone, is involved in responses to environmental stresses such as drought and high salinity, and is required for stress tolerance. ABA is synthesized de novo in response to dehydration. 9‐cis‐epoxycarotenoid dioxygenase (NCED) is thought to be a key enzyme in ABA biosynthesis. Here we demonstrate that the expression of an NCED gene of Arabidopsis, AtNCED3, is induced by drought stress and controls the level of endogenous ABA under drought‐stressed conditions. Overexpression of AtNCED3 in transgenic Arabidopsis caused an increase in endogenous ABA level, and promoted transcription of drought‐ and ABA‐inducible genes. Plants overexpressing AtNCED3 showed a reduction in transpiration rate from leaves and an improvement in drought tolerance. By contrast, antisense suppression and disruption of AtNCED3 gave a drought‐sensitive phenotype. These results indicate that the expression of AtNCED3 plays a key role in ABA biosynthesis under drought‐stressed conditions in Arabidopsis. We improved drought tolerance by gene manipulation of AtNCED3 causing the accumulation of endogenous ABA.
A high‐throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana
Authors: Tornero Pablo , Dangl Jeffery L.
Published: March 2003

Measuring the growth of pathogenic bacteria in leaves is a mainstay of plant pathology studies. We have made significant improvements to standard methods that will not only increase the throughput but also reduce the space limitations. Additionally, the method described here is as accurate as the standard method. Briefly, we infected leaves by dipping whole seedlings of Arabidopsis into a bacterial solution containing a surfactant. After harvest, the seedlings were then simply shaken in buffer. The resulting bacterial solutions were diluted in microtitre plates and spotted onto agar plates. Colony‐forming units were then counted 40 h after plating. Therefore, we have eliminated most of the labour‐intensive steps involved in measuring the growth of bacteria in Arabidopsis, and describe a method that could be automated. The assay is sensitive enough to detect small differences between pathogens or ecotypes.
Identification and regulation of high light‐induced genes in Chlamydomonas reinhardtii
Authors: Im Chung Soon , Grossman Arthur R.
Published: March 2003

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light‐activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2‐induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low‐CO2 conditions. Genes specifically activated by high light, even under high‐CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.
Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana
Authors: Tähtiharju Sari , Palva Tapio
Published: March 2003

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought‐induced expression of these genes was ABA‐dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue‐specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold‐and ABA‐induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.
Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation
Authors: Kovar Joy L. , Zhang Jun , Funke Roel P. , Weeks Donald P.
Published: March 2003

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85–20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild‐type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild‐type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 µm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild‐type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP‐ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 × 10−4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 × 10−6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP‐ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.
The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH
Authors: Gerbeau Patricia , Amodeo Gabriela , Henzler Tobias , Santoni Véronique , Ripoche Pierre , Maurel Christophe
Published: March 2003

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4‐fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two‐phase partitioning and their water transport properties were characterized by stopped‐flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (Pf) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3‐fold higher Pf and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their Pf and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half‐inhibition of Pf at 50–100 µm free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half‐inhibition of Pf at pH 7.2–7.5. Thus, both Ca2+ and H+ contribute to a membrane‐delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.
A novelin vitrosystem for simultaneous import of precursor proteins into mitochondria and chloroplasts
Authors: Rudhe Charlotta , Chew Orinda , Whelan James , Glaser Elzbieta
Published: March 2003

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novelin vitrosystem for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre‐sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre‐sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre‐sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre‐sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.
A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana
Authors: Swidzinski Jodi A. , Sweetlove Lee J. , Leaver Christopher J.
Published: March 2003

Programmed cell death (PCD) is a form of cellular suicide requiring active gene expression, and occurs in both animals and plants. While the cascade of events and the genes that control PCD have been extensively studied in animals, we remain largely ignorant about the similar process in plant cells. Many of the key proteins of animal cell death such as the Bcl‐2 family and the caspase family of proteases do not appear to be conserved in plants, suggesting that plants may employ unique mechanisms to execute PCD. To identify genetic elements of PCD in plants, we monitored changes in transcript levels of approximately 100 selected genes during cell death in an Arabidopsis cell suspension culture using a cDNA microarray. PCD was induced in the cell cultures by two independent means (heat treatment or by allowing the cultures to senesce) to allow the distinction to be drawn between changes in gene expression that are related to PCD and those that are specific to a particular treatment. We argue that genes whose expression is altered during PCD induced by two different means may be generally involved in all types of PCD. We show that certain oxidative stress‐related genes, including CSD1, CSD3, and GPX, in addition to cysteine proteinases, some transcription factors, and HR‐related genes may serve as markers of a core plant cell death programme. Additionally we observe a down‐regulation of the mitochondrial adenine nucleotide transporter and suggest that this may be an early event in the execution of plant PCD.
A knock‐out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis
Authors: Park Joon‐Hyun , Halitschke Rayko , Kim Ho Bang , Baldwin Ian T. , Feldmann Kenneth A. , Feyereisen René
Published: March 2003

Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential roles in pollen maturation and dehiscence and wound‐induced defence against biotic attacks. To better understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis knock‐out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100‐fold 1 h after wounding in wild‐type plants, do not increase after wounding in the aos mutant. In addition, the mutant showed severe male sterility due to defects in anther and pollen development. The male‐sterile phenotype was completely rescued by exogenous application of methyl jasomonate and by complementation with constitutive expression of the AOS gene. RT–PCR analysis showed that the induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be inducible by wound and jasmonate application in the wild‐type, was absent in the aos mutant. In transgenic plants constitutively expressing AOS, wound‐induced JA levels were 50–100% higher compared to wild‐type plants. Taken together with JA deficiency in the aos mutant, our results show that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in unwounded plants.
The broad‐spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS‐LRR genes with an unusual amino acid repeat in the LRR region
Authors: Ernst Karin , Kumar Amar , Kriseleit Doris , Kloos Dorothee‐U. , Phillips Mark S. , Ganal Martin W.
Published: March 2003

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map‐based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide‐binding site (NBS) and a leucine‐rich‐repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118‐kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.
The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells
Authors: Simon‐Plas Françoise , Elmayan Taline , Blein Jean‐Pierre
Published: March 2003

A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91–phox in AOS production in elicited tobacco cells.