The Plant Cell current issue

Oxylipin Signaling in Plant Stress Responses

Eckardt, N. A. — 2008-04-28

The IMEter Predicts an Intron's Ability to Boost Gene Expression

Farquharson, K. L. — 2008-04-28

Aquaporins and Chloroplast Membrane Permeability

Eckardt, N. A. — 2008-04-28

Cellulose Synthesis in Phytophthora infestans Pathogenesis

Mach, J. — 2008-04-28

Heritability of the Tomato Fruit Metabolome

Eckardt, N. A. — 2008-04-28

Pond Scum Genomics: The Genomes of Chlamydomonas and Ostreococcus

Peers, G., Niyogi, K. K. — 2008-04-28

Mode of Inheritance of Primary Metabolic Traits in Tomato

2008-04-28

To evaluate components of fruit metabolic composition, we have previously metabolically phenotyped tomato (Solanum lycopersicum) introgression lines containing segmental substitutions of wild species chromosome in the genetic background of a cultivated variety. Here, we studied the hereditability of the fruit metabolome by analyzing an additional year's harvest and evaluating the metabolite profiles of lines heterozygous for the introgression (ILHs), allowing the evaluation of putative quantitative trait locus (QTL) mode of inheritance. These studies revealed that most of the metabolic QTL (174 of 332) were dominantly inherited, with relatively high proportions of additively (61 of 332) or recessively (80 of 332) inherited QTL and a negligible number displaying the characteristics of overdominant inheritance. Comparison of the mode of inheritance of QTL revealed that several metabolite pairs displayed a similar mode of inheritance of QTL at the same chromosomal loci. Evaluation of the association between morphological and metabolic traits in the ILHs revealed that this correlation was far less prominent, due to a reduced variance in the harvest index within this population. These data are discussed in the context of genomics-assisted breeding for crop improvement, with particular focus on the exploitation of wide biodiversity.

The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization

Murata, J., Roepke, J., Gordon, H., De Luca, V. — 2008-04-28

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Promoter-Proximal Introns in Arabidopsis thaliana Are Enriched in Dispersed Signals that Elevate Gene Expression

Rose, A. B., Elfersi, T., Parra, G., Korf, I. — 2008-04-28

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression. We found that the sequences responsible for elevating expression are dispersed throughout an enhancing intron, as is a candidate motif that is overrepresented in first introns and whose occurrence in tested introns is proportional to its effect on expression. The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.

Heme, a Plastid-Derived Regulator of Nuclear Gene Expression in Chlamydomonas

von Gromoff, E. D., Alawady, A., Meinecke, L., Grimm, B., Beck, C. F. — 2008-04-28

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.

The Highly Similar Arabidopsis Homologs of Trithorax ATX1 and ATX2 Encode Proteins with Divergent Biochemical Functions

2008-04-28

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.

ARABIDOPSIS TRITHORAX1 Dynamically Regulates FLOWERING LOCUS C Activation via Histone 3 Lysine 4 Trimethylation

2008-04-28

Trithorax function is essential for epigenetic maintenance of gene expression in animals, but little is known about trithorax homologs in plants. ARABIDOPSIS TRITHORAX1 (ATX1) was shown to be required for the expression of homeotic genes involved in flower organogenesis. Here, we report a novel function of ATX1, namely, the epigenetic regulation of the floral repressor FLOWERING LOCUS C (FLC). Downregulation of FLC accelerates the transition from vegetative to reproductive development in Arabidopsis thaliana. In the atx1 mutant, FLC levels are reduced and the FLC chromatin is depleted of trimethylated, but not dimethylated, histone 3 lysine 4, suggesting a specific trimethylation function of ATX1. In addition, we found that ATX1 directly binds the active FLC locus before flowering and that this interaction is released upon the transition to flowering. This dynamic process stands in contrast with the stable maintenance of homeotic gene expression mediated by trithorax group proteins in animals but resembles the dynamics of plant Polycomb group function.

EMB2473/MIRO1, an Arabidopsis Miro GTPase, Is Required for Embryogenesis and Influences Mitochondrial Morphology in Pollen

Yamaoka, S., Leaver, C. J. — 2008-04-28

The regulation of mitochondrial biogenesis, subcellular distribution, morphology, and metabolism are essential for all aspects of plant growth and development. However, the molecular mechanisms involved are still unclear. Here, we describe an analysis of the three Arabidopsis thaliana orthologs of the evolutionarily conserved Miro GTPases. Two of the genes, MIRO1 and MIRO2, are transcribed ubiquitously throughout the plant tissues, and their gene products localize to mitochondria via their C-terminal transmembrane domains. While insertional mutations in the MIRO2 gene do not have any visible impact on plant development, an insertional mutation in the MIRO1 gene is lethal during embryogenesis at the zygote to four-terminal-cell embryo stage. It also substantially impairs pollen germination and tube growth. Laser confocal and transmission electron microscopy revealed that the miro1 mutant pollen exhibits abnormally enlarged or tube-like mitochondrial morphology, leading to the disruption of continuous streaming of mitochondria in the growing pollen tube. Our findings suggest that mitochondrial morphology is influenced by MIRO1 and plays a vital role during embryogenesis and pollen tube growth.

Haplo-Insufficiency of MPK3 in MPK6 Mutant Background Uncovers a Novel Function of These Two MAPKs in Arabidopsis Ovule Development

Wang, H., Liu, Y., Bruffett, K., Lee, J., Hause, G., Walker, J. C., Zhang, S. — 2008-04-28

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage–dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3^–/– mpk6^+/– plants are fertile. Genetic analysis revealed that the female sterility of mpk3^+/– mpk6^–/– plants is a sporophytic effect. In mpk3^+/– mpk6^–/– mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3^+/– mpk6^–/– ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development.

Exclusion of a Proton ATPase from the Apical Membrane Is Associated with Cell Polarity and Tip Growth in Nicotiana tabacum Pollen Tubes

2008-04-28

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca²⁺, H⁺, K⁺, and Cl^– -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H⁺-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H⁺ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H⁺ flux profiles. Furthermore, there is no net flux of H⁺ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.

The AGL62 MADS Domain Protein Regulates Cellularization during Endosperm Development in Arabidopsis

Kang, I.-H., Steffen, J. G., Portereiko, M. F., Lloyd, A., Drews, G. N. — 2008-04-28

Endosperm, a storage tissue in the angiosperm seed, provides nutrients to the embryo during seed development and/or to the developing seedling during germination. A major event in endosperm development is the transition between the syncytial phase, during which the endosperm nuclei undergo many rounds of mitosis without cytokinesis, and the cellularized phase, during which cell walls form around the endosperm nuclei. The molecular processes controlling this phase transition are not understood. In agl62 seeds, the endosperm cellularizes prematurely, indicating that AGL62 is required for suppression of cellularization during the syncytial phase. AGL62 encodes a Type I MADS domain protein that likely functions as a transcription factor. During seed development, AGL62 is expressed exclusively in the endosperm. During wild-type endosperm development, AGL62 expression is strong during the syncytial phase and then declines abruptly just before cellularization. By contrast, in mutant seeds containing defects in some FERTILIZATION-INDEPENDENT SEED (FIS) class Polycomb group genes, the endosperm fails to cellularize and AGL62 expression fails to decline. Together, these data suggest that AGL62 suppresses cellularization during the syncytial phase of endosperm development and that endosperm cellularization is triggered via direct or indirect AGL62 inactivation by the FIS polycomb complex.

Function of Nicotiana tabacum Aquaporins as Chloroplast Gas Pores Challenges the Concept of Membrane CO2 Permeability

Uehlein, N., Otto, B., Hanson, D. T., Fischer, M., McDowell, N., Kaldenhoff, R. — 2008-04-28

Photosynthesis is often limited by the rate of CO₂ diffusion from the atmosphere to the chloroplast. The primary resistances for CO₂ diffusion are thought to be at the stomata and at photosynthesizing cells via a combination resulting from resistances of aqueous solution as well as the plasma membrane and both outer and inner chloroplast membranes. In contrast with stomatal resistance, the resistance of biological membranes to gas transport is not widely recognized as a limiting factor for metabolic function. We show that the tobacco (Nicotiana tabacum) plasma membrane and inner chloroplast membranes contain the aquaporin Nt AQP1. RNA interference–mediated decreases in Nt AQP1 expression lowered the CO₂ permeability of the inner chloroplast membrane. In vivo data show that the reduced amount of Nt AQP1 caused a 20% change in CO₂ conductance within leaves. Our discovery of CO₂ aquaporin function in the chloroplast membrane opens new opportunities for mechanistic examination of leaf internal CO₂ conductance regulation.

The Arabidopsis P4-ATPase ALA3 Localizes to the Golgi and Requires a {beta}-Subunit to Function in Lipid Translocation and Secretory Vesicle Formation

2008-04-28

Vesicle budding in eukaryotes depends on the activity of lipid translocases (P₄-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P₄-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a β-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

The Arabidopsis thaliana Type I Isopentenyl Diphosphate Isomerases Are Targeted to Multiple Subcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

Phillips, M. A., D'Auria, J. C., Gershenzon, J., Pichersky, E. — 2008-04-28

To form the building blocks of isoprenoids, isopentenyl diphosphate (IPP) isomerase activity, which converts IPP to dimethylallyl diphosphate (DMAPP), appears to be necessary in cytosol, plastids, and mitochondria. Arabidopsis thaliana contains only two IPP isomerases (Isopentenyl Diphosphate Isomerase1 [IDI1] and IDI2). Both encode proteins with N-terminal extensions similar to transit peptides and are expressed in all organs, with IDI1 less abundant than IDI2. Examination of enhanced green fluorescent protein fusions established that IDI1 is mainly in the plastid, whereas IDI2 is mainly in the mitochondria. Both proteins are also in the cytosol as a result of their translation from naturally occurring shorter transcripts lacking transit peptides, as demonstrated by 5' rapid amplification of cDNA ends cloning. IPP isomerase activity in the cytosol was confirmed by uniform labeling of IPP- and DMAPP-derived units of the cytoplasmic isoprenoid product, sitosterol, when labeled mevalonate was administered. Analysis of mutant lines showed that double mutants were nonviable, while homozygous single mutants had no major morphological or chemical differences from the wild type except for flowers with fused sepals and underdeveloped petals on idi2 mutants. Thus, each of the two Arabidopsis IPP isomerases is found in multiple but partially overlapping subcellular locations, and each can compensate for the loss of the other through partial redundancy in the cytosol.

The F-Box Protein ACRE189/ACIF1 Regulates Cell Death and Defense Responses Activated during Pathogen Recognition in Tobacco and Tomato

2008-04-28

Virus-induced gene silencing identified the Avr9/Cf-9 RAPIDLY ELICITED gene ACRE189 as essential for the Cf-9– and Cf-4–mediated hypersensitive response (HR) in Nicotiana benthamiana. We report a role for ACRE189 in disease resistance in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). ACRE189 (herein renamed Avr9/Cf-9–INDUCED F-BOX1 [ACIF1]) encodes an F-box protein with a Leu-rich-repeat domain. ACIF1 is widely conserved and is closely related to F-box proteins regulating plant hormone signaling. Silencing of tobacco ACIF1 suppressed the HR triggered by various elicitors (Avr9, Avr4, AvrPto, Inf1, and the P50 helicase of Tobacco mosaic virus [TMV]). ACIF1 is recruited to SCF complexes (a class of ubiquitin E3 ligases), and the expression of ACIF1 F-box mutants in tobacco compromises the HR similarly to ACIF1 silencing. ACIF1 affects N gene–mediated responses to TMV infection, including lesion formation and salicylic acid accumulation. Loss of ACIF1 function also reduced confluent cell death induced by Pseudomonas syringae pv tabaci. ACIF1 silencing in Cf9 tomato attenuated the Cf-9–dependent HR but not Cf-9 resistance to Cladosporium fulvum. Resistance conferred by the Cf-9 homolog Cf-9B, however, was compromised in ACIF1-silenced tomato. Analysis of public expression profiling data suggests that Arabidopsis thaliana homologs of ACIF1 (VFBs) regulate defense responses via methyl jasmonate– and abscisic acid–responsive genes. Together, these findings support a role of ACIF1/VFBs in plant defense responses.

Cellulose Synthesis in Phytophthora infestans Is Required for Normal Appressorium Formation and Successful Infection of Potato

2008-04-28

Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.

The Coiled-Coil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen Recognition and Signaling

2008-04-28

Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure–function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase–activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.

A MYB Transcription Factor Regulates Very-Long-Chain Fatty Acid Biosynthesis for Activation of the Hypersensitive Cell Death Response in Arabidopsis

2008-04-28

Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants.

General Detoxification and Stress Responses Are Mediated by Oxidized Lipids through TGA Transcription Factors in Arabidopsis

2008-04-28

12-oxo-phytodienoic acid and several phytoprostanes are cyclopentenone oxylipins that are formed via the enzymatic jasmonate pathway and a nonenzymatic, free radical–catalyzed pathway, respectively. Both types of cyclopentenone oxylipins induce the expression of genes related to detoxification, stress responses, and secondary metabolism, a profile clearly distinct from that of the cyclopentanone jasmonic acid. Microarray analyses revealed that 60% of the induction by phytoprostanes and 30% of the induction by 12-oxo-phytodienoic acid was dependent on the TGA transcription factors TGA2, TGA5, and TGA6. Moreover, treatment with phytoprostanes and 12-oxo-phytodienoic acid inhibited cell division and root growth, a property also shared by jasmonic acid. Besides being potent signals, cyclopentenones and other lipid peroxidation products are reactive electrophiles that can covalently bind to and damage proteins. To this end, we show that at least two of the induced detoxification enzymes efficiently metabolize cyclopentenones in vitro. Accumulation of two of these metabolites was detectable during Pseudomonas infection. The cyclopentenone oxylipin gene induction profile resembles the defense response induced by a variety of lipophilic xenobiotics. Hence, oxidized lipids may activate chemosensory mechanisms of a general broad-spectrum detoxification network involving TGA transcription factors.

Modulation of Nitrosative Stress by S-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth in Arabidopsis

Lee, U., Wie, C., Fernandez, B. O., Feelisch, M., Vierling, E. — 2008-04-28

Nitric oxide (NO) is a key signaling molecule in plants. This analysis of Arabidopsis thaliana HOT5 (sensitive to hot temperatures), which is required for thermotolerance, uncovers a role of NO in thermotolerance and plant development. HOT5 encodes S-nitrosoglutathione reductase (GSNOR), which metabolizes the NO adduct S-nitrosoglutathione. Two hot5 missense alleles and two T-DNA insertion, protein null alleles were characterized. The missense alleles cannot acclimate to heat as dark-grown seedlings but grow normally and can heat-acclimate in the light. The null alleles cannot heat-acclimate as light-grown plants and have other phenotypes, including failure to grow on nutrient plates, increased reproductive shoots, and reduced fertility. The fertility defect of hot5 is due to both reduced stamen elongation and male and female fertilization defects. The hot5 null alleles show increased nitrate and nitroso species levels, and the heat sensitivity of both missense and null alleles is associated with increased NO species. Heat sensitivity is enhanced in wild-type and mutant plants by NO donors, and the heat sensitivity of hot5 mutants can be rescued by an NO scavenger. An NO-overproducing mutant is also defective in thermotolerance. Together, our results expand the importance of GSNOR-regulated NO homeostasis to abiotic stress and plant development.

Phospholipase D{alpha}3 Is Involved in the Hyperosmotic Response in Arabidopsis

Hong, Y., Pan, X., Welti, R., Wang, X. — 2008-04-28

Rapid activation of phospholipase D (PLD), which hydrolyzes membrane lipids to generate phosphatidic acid (PA), occurs under various hyperosmotic conditions, including salinity and water deficiency. The Arabidopsis thaliana PLD family has 12 members, and the function of PLD activation in hyperosmotic stress responses has remained elusive. Here, we show that knockout (KO) and overexpression (OE) of previously uncharacterized PLD3 alter plant response to salinity and water deficit. PLD3 uses multiple phospholipids as substrates with distinguishable preferences, and alterations of PLD3 result in changes in PA level and membrane lipid composition. PLD3-KO plants display increased sensitivities to salinity and water deficiency and also tend to induce abscisic acid–responsive genes more readily than wild-type plants, whereas PLD3-OE plants have decreased sensitivities. In addition, PLD3-KO plants flower later than wild-type plants in slightly dry conditions, whereas PLD3-OE plants flower earlier. These data suggest that PLD3 positively mediates plant responses to hyperosmotic stresses and that increased PLD3 expression and associated lipid changes promote root growth, flowering, and stress avoidance.