Plant Physiology

Loss of Halophytism by Interference with SOS1 Expression.

Publication Date: 2009 Jul 1 PMID: 19571313
Authors: Oh, D. H. - Leidi, E. - Zhang, Q. - Hwang, S. M. - Li, Y. - Quintero, F. J. - Jiang, X. - D'Urzo, M. P. - Lee, S. Y. - Zhao, Y. - Bahk, J. D. - Bressan, R. A. - Yun, D. J. - Pardo, J. M. - Bohnert, H. J.
Journal: Plant Physiol

The contribution of SOS1, encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild type and RNAi (RNA-interference) lines of the halophytic Arabidopsis-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking Na(+) efflux transporters and increased salt tolerance of wild-type Arabidopsis. Thsos1-RNAi lines of Thellungiella were highly salt-sensitive. A representative line, thsos1-4, showed faster Na(+) accumulation, more severe water loss in shoots under salt stress, and slower removal of Na(+) ion from the root after removal of stress, compared to wild type. Thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than wild type and inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, sodium ions accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella salsuginea that normally can grow in sea water-strength NaCl solutions into a plant as sensitive to sodium ions as Arabidopsis thaliana.

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Abscisic acid has a key role in modulating diverse plant-pathogen interactions.

Publication Date: 2009 Jul 1 PMID: 19571312
Authors: Fan, J. - Hill, L. - Crooks, C. - Doerner, P. - Lamb, C.
Journal: Plant Physiol

We have isolated an activation-tagged Arabidopsis thaliana line, constitutive disease susceptibility2-1D (cds2-1D), that showed enhanced bacterial growth when challenged with various Pseudomonas syringae strains. Systemic acquired resistance and systemic PR1 gene induction were also compromised in cds2-1D. The T-DNA insertion adjacent to NCED5, one of six genes encoding the abscisic acid (ABA) biosynthetic enzyme 9-cis-epoxydioxygenase, caused a massive increase in transcript level and enhanced ABA levels >2-fold. Over expression of NCED genes recreated the enhanced disease susceptibility phenotype. NCED2, 3 and 5 were induced, and ABA accumulated strongly following compatible P. syringae infection. The ABA biosynthetic mutant aba3-1 showed reduced susceptibility to virulent P. syringae, and ABA, whether through exogenous application or endogenous accumulation in response to mild water stress resulted in increased bacterial growth following challenge with virulent P. syringae indicating that ABA suppresses resistance to P. syringae. Likewise ABA accumulation also compromised resistance to the biotrophic oomycete Hyaloperonospora arabidopsis, whereas resistance to the fungus Alternaria brassicicola was enhanced in cds2-1D plants and compromised in aba3-1 plants, indicating ABA promotes resistance to this necrotroph. Comparison of the accumulation of salicylic acid and jasmonic acid in wild type, cds2-1D and aba3-1 plants challenged with P. syringae showed that ABA promotes jasmonic acid accumulation and exhibits a complex antagonistic relationship with salicylic acid. Our findings provide genetic evidence that the abiotic stress signal ABA also has profound roles in modulating diverse plant-pathogen interactions mediated at least in part by cross-talk with the jasmonic acid and salicylic acid biotic stress signal pathways.

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Strain mechanosensing quantitatively controls diameter growth and PtaZFP2 gene expression in poplar.

Publication Date: 2009 Jul 1 PMID: 19571311
Authors: Coutand, C. - Martin, L. - Leblanc-Fournier, N. - Decourteix, M. - Julien, J. L. - Moulia, B.
Journal: Plant Physiol

Mechanical signals are important factors that control plant growth and development. External mechanical loadings lead to a decrease in elongation and a stimulation of diameter growth, a syndrome known as thigmomorphogenesis. A previous study has demonstrated that plants perceive the strains they are subjected to and not forces or stresses. On this basis, an integrative bio-mechanical model of mechanosensing was established ("sum-of-strains model"), and tested on tomato elongation but not for local responses such as diameter growth or gene expression. The first aim of this interdisciplinary work was to provide a quantitative study of the effect of a single transitory bending on poplar diameter growth and on the expression level of a primary mechanosensitive transcription factor gene, PtaZFP2. The second aim of this work was to assess the sum-of-strains model of mechanosensing on these local responses. An original bending device was built to study stem responses according to a controlled range of strains. A single bending modified plant diameter growth and increased the relative abundance of PtaZFP2 transcripts. Integrals of longitudinal strains induced by bending on the responding tissues were highly correlated to local plant responses. The "sum-of-strains" model of mechanosensing established for stem elongation was thus applicable for local responses at two scales: diameter growth and gene expression. These novel results open avenue for the ordering of gene expression profiles as a function of the intensity of mechanical stimulation and provide a generic bio-mechanical core for an integrative model of thigmomorphogenesis linking gene expression with growth responses.

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Arabidopsis thaliana methionine gamma-lyase is regulated according to isoleucine biosynthesis needs, but plays a subordinate role to threonine deaminase.

Publication Date: 2009 Jul 1 PMID: 19571310
Authors: Joshi, V. - Jander, G.
Journal: Plant Physiol

The canonical pathway for isoleucine biosynthesis in plants begins with the conversion of threonine to 2-ketobutyrate by threonine deaminase (OMR1). However, demonstration of methionine gamma-lyase (MGL) activity in Arabidopsis thaliana suggested that production of 2-ketobutyrate from methionine can also lead to isoleucine biosynthesis. Rescue of the isoleucine deficit in a threonine deaminase mutant by MGL overexpression, as well as decreased transcription of endogenous A. thaliana MGL in a feedback-insensitive threonine deaminase mutant background, shows that these two enzymes have overlapping functions in amino acid biosynthesis. In mgl mutant flowers and seeds, methionine levels are significantly increased, incorporation of [(13)C]methionine into isoleucine is decreased, but isoleucine levels are unaffected. Accumulation of free isoleucine and other branched-chain amino acids is greatly elevated in response to drought stress in A. thaliana. Gene expression analyses, amino acid phenotypes, and labeled precursor feeding experiments demonstrate that MGL activity is up-regulated by osmotic stress, but likely plays a less prominent role in isoleucine biosynthesis than threonine deaminase. The observation that MGL makes a significant contribution to methionine degradation, particularly in reproductive tissue, suggests practical applications for silencing the expression of MGL in crop plants and thereby increasing the abundance of methionine, a limiting essential amino acid.

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RNR regulation in response to genotoxic stress in Arabidopsis.

Publication Date: 2009 Jul 1 PMID: 19571309
Authors: Roa, H. - Lang, J. - Culligan, K. M. - Keller, M. - Holec, S. - Cognat, V. - Montane, M. H. - Houlne, G. - Chaboute, M. E.
Journal: Plant Physiol

Ribonucleotide Reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis thaliana encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus WT, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in WT in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

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Comparative large-scale analysis of interactions between several crop species and the effector repertoires from multiple pathovars of Pseudomonas and Ralstonia.

Publication Date: 2009 Jul 1 PMID: 19571308
Authors: Wroblewski, T. - Caldwell, K. S. - Piskurewicz, U. - Cavanaugh, K. A. - Xu, H. - Kozik, A. - Ochoa, O. - McHale, L. K. - Lahre, K. - Jelenska, J. - Castillo, J. A. - Blumenthal, D. - Vinatzer, B. A. - Greenberg, J. T. - Michelmore, R. W.
Journal: Plant Physiol

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via Type III Secretion Systems (TTSS). Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response (HR) manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered two extensive co-evolving groups rather than as individual host species co-evolving with single pathovars. To dissect the complexity of this co-evolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce and tomato. Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Non-host species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.

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Multiple sequence motifs in the RbcS transit peptide independently contribute to Toc159-dependent import of proteins into chloroplasts.

Publication Date: 2009 Jul 1 PMID: 19571307
Authors: Lee, D. W. - Lee, S. - Oh, Y. J. - Hwang, I.
Journal: Plant Physiol

A large number of plastid proteins encoded by the nuclear genome are posttranslationally imported into plastids by at least two distinct mechanisms: the Toc159-dependent and Toc132/Toc120-dependent pathways. Light-induced photosynthetic proteins are imported through the Toc159-dependent pathway, whereas constitutive housekeeping plastid proteins are imported into plastids through the Toc132/Toc120 pathway. However, it remains unknown which features of the plastid protein transit peptide (TP) determine the import pathway. We have discovered sequence elements of the Rubisco small subunit TP (RbcS-tp) that play a role in determining import through the Toc159-dependent pathway in vivo. We generated multiple hybrid mutants using the RbcS-tp and the E1alpha subunit of pyruvate dehydrogenase transit peptide (E1alpha-tp) as representative peptides mediating import through the Toc159-dependent and Toc159-independent pathways, respectively. Import experiments using these hybrid mutants in wild-type and ppi2 mutant protoplasts revealed that multiple sequence motifs in the RbcS-tp independently contribute to Toc159-dependent protein import into chloroplasts. One of these motifs is the group of serine residues located in the N-terminal 12-amino acid (aa) segment and the other is the C-terminal T5 region of the RbcS-tp ranging from aa positions 41 to 49. Based on these findings, we propose that multiple sequence elements in the RbcS-tp contribute independently to Toc159-dependent import of proteins into chloroplasts.

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Integrated metabolite and transcript profiling identify a biosynthetic mechanism for hispidol in Medicago truncatula cell cultures.

Publication Date: 2009 Jul 1 PMID: 19571306
Authors: Farag, M. A. - Deavours, B. E. - de Fatima, A. - Naoumkina, M. - Dixon, R. A. - Sumner, L. W.
Journal: Plant Physiol

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection (HPLC-PDA-MS) revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, Mt PRX1, Mt PRX2 and Mt PRX3, were coordinately induced with the accumulation of hispidol. Mt PRX1 and Mt PRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested, but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spill-over when the pool of its precursor, isoliquiritgenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically re-programmed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation.

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Defining core metabolic and transcriptomic responses to oxygen availability in rice embryos and young seedlings.

Publication Date: 2009 Jul 1 PMID: 19571305
Authors: Narsai, R. - Howell, K. A. - Carroll, A. - Ivanova, A. - Millar, A. H. - Whelan, J.
Journal: Plant Physiol

Analysis reveals that there is limited overlap in the sets of transcripts that show significant changes in abundance during anaerobiosis in different plant species. This may be due to the fact that a combination of primary effects, changes due to the presence or absence of oxygen, and secondary effects, responses to primary changes or tissue and developmental responses, are measured together and not differentiated from each other. In order to dissect out these responses, the effect of the presence or absence of oxygen was investigated using three different experimental designs using rice as a model system. A total of 110 metabolites and 9,596 transcripts were found to change significantly in response to oxygen availability in at least one experiment. However, only one-quarter of these showed complementary responses to oxygen in all three experiments, allowing the core response to oxygen availability to be defined. A total of 10 metabolites and 1,136 genes could be defined as aerobic responders (up-regulated in the presence of oxygen and down-regulated in its absence) and 13 metabolites and 730 genes as anaerobic responders (up-regulated in the absence of oxygen and down-regulated in its presence). Defining core sets of transcripts that were sensitive to oxygen provided insights into alterations in metabolism, specifically carbohydrate and lipid metabolism and the putative regulatory mechanisms that allow rice to grow under anaerobic conditions. Transcript abundance of a specific set of transcription factors were sensitive to oxygen availability during all the different experiments conducted, putatively identifying primary regulators of gene expression under anaerobic conditions. Combined with the possibility of selective transcript degradation, these transcriptional processes are involved in the core response of rice to anaerobiosis.

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BOBBER1 is a non-canonical Arabidopsis small heat shock protein required for both development and thermotolerance.

Publication Date: 2009 Jul 1 PMID: 19571304
Authors: Perez, D. E. - Hoyer, J. S. - Johnson, A. I. - Moody, Z. R. - Lopez, J. - Kaplinsky, N. J.
Journal: Plant Physiol

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis thaliana small heat shock protein. BOBBER1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss of function mutants are embryo lethal we used a partial loss of function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and post-embryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants BOB1 is cytoplasmic at basal temperatures but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.

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Recent advances in PAMP-triggered immunity against bacteria: pattern recognition receptors watch over and raise the alarm.

Publication Date: 2009 Jun 26 PMID: 19561123
Authors: Nicaise, V. - Roux, M. - Zipfel, C.
Journal: Plant Physiol

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Under-Explored Niches in Research on Plant Pathogenic Bacteria.

Publication Date: 2009 Jun 26 PMID: 19561122
Authors: Allen, C. - Bent, A. - Charkowski, A.
Journal: Plant Physiol

Despite rapid advances on certain aspects of plant pathogenic bacteria, many economically important pathosystems are largely unexplored and biologically relevant life stages of even familiar systems remain poorly understood. We know remarkably little about end-stage disease, latent infections, survival away from the host, interactions among multiple microbes in a plant, and the effects of quantitative virulence factors. While no thoughtful researcher would dispute the effectiveness of reductionist experiments, we propose that this approach be combined with a broader perspective that includes the ecology, histopathology, and community population biology of phytopathogenic bacteria. We offer examples of exciting recent discoveries resulting from this natural history-based approach. In particular, in situ studies using biologically realistic inoculation followed by analyses with microscopy, gene expression profiling, community analyses, or application of key computational tools can offer new insights into old questions. Research that combines cutting-edge tools with a biological perspective is especially lacking on high-impact diseases of subsistence crops. Understanding the biology underlying important practical issues such as copper resistance, eradication from seed and cuttings, and rapid, sensitive detection could be of significant utility. Overall, we endorse a broader biological approach to research on plant pathogenic bacteria.

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Turnover of fatty acids during natural senescence of Arabidopsis, Brachypodium and Switchgrass and in Arabidopsis beta-oxidation mutants.

Publication Date: 2009 Jun 26 PMID: 19561121
Authors: Yang, Z. - Ohlrogge, J. B.
Journal: Plant Physiol

During leaf senescence macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds or other storage organs. In this study, we determined the fatty acid (FA) levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium (Brachypodium distachyon) and switchgrass leaves during natural senescence. In young leaves, fatty acids represent 4-5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 microg per leaf (0.6 microg.mg(-1) dry weight). Triacylglycerol (TAG) levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7 and kat2 which are blocked in enzyme activities of beta-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and wild type, indicating that several mutations in beta-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.

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The mechanism of iron homeostasis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 and its relationship to oxidative stress.

Publication Date: 2009 Jun 26 PMID: 19561120
Authors: Shcolnick, S. - Summerfield, T. C. - Reytman, L. - Sherman, L. A. - Keren, N.
Journal: Plant Physiol

In this paper, we demonstrate the connection between intracellular iron storage and oxidative stress response in cyanobacteria. Iron is essential for the survival of all organisms. However, the redox properties that makes iron a valuable cofactor also lead to oxidative interactions, resulting in the formation of harmful radicals. Therefore, iron accumulation in cells should be tightly regulated, a process in which Ferritin family proteins play an important role. Synechocystis sp. PCC 6803 contains two ferritin type storage complexes, bacterioferritin (BFR) and MrgA. Previous studies demonstrated the role of BFR and MrgA in iron storage. In addition, MrgA was found to play a key role in oxidative stress response. Here we examined the dual role of the ferritin family proteins using physiological and transcriptomic approaches. Microarray analysis of iron-limited wild-type and DeltamrgA cultures revealed a substantial up-regulation of oxidative stress related genes in mutant cells. The PerR regulator was found to play an important role in that process. Furthermore, we were able to demonstrate the connection between internal iron quota, the presence of the two storage complexes and the sensitivity to externally applied oxidative stress. These data suggest a pivotal role for the ferritin-type proteins of Synechocystis sp. PCC 6803 in coordinating iron homeostasis and in oxidative stress response. The combined action of the two complexes allows for the safe accumulation and release of iron from storage by minimizing damage resulting from interactions between reduced iron and the oxygen radicals that are produced in abundance by the photosynthetic apparatus.

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A dynamic interface for capsaicinoid systems biology.

Publication Date: 2009 Jun 24 PMID: 19553373
Authors: Mazourek, M. - Pujar, A. - Borovsky, Y. - Paran, I. - Mueller, L. - Jahn, M. M.
Journal: Plant Physiol

Capsaicinoids are the pungent alkaloids that give hot peppers their spiciness. While capsaicinoids are relatively simple molecules, much is unknown about their biosynthesis which spans diverse metabolisms of essential amino acids, phenylpropanoids, benzenoids, and fatty acids. Pepper (Capsicum sp.) is not a model organism, but has access to the resources developed in model plants through comparative approaches. To aid research in this system, we have implemented a comprehensive model of capsaicinoid biosynthesis and made it publicly available within the SolCyc database at the SOL Genomics Network (http://www.sgn.cornell.edu). As a preliminary test of this model, and to build its value as a resource, targeted transcripts were cloned as candidates for nearly all of the structural genes for capsaicinoid biosynthesis. In support of the role of these transcripts in capsaicinoid biosynthesis beyond correct spatial and temporal expression, their predicted subcellular localizations were compared against the biosynthetic model and experimentally determined compartmentalization in Arabidopsis. To enable their use in a positional candidate gene approach in the Solanaceae, these genes were genetically mapped in pepper. These data were integrated into SGN, a clade oriented database that incorporates community annotation of genes, enzymes, phenotypes, mutants and genomic loci. Here we describe the creation and integration of these resources as a holistic and dynamic model of the characteristic specialized metabolism of pepper.

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Modeling the Hydraulics of Root Growth in Three Dimensions with Phloem Water Sources.

Publication Date: 2009 Jun 19 PMID: 19542299
Authors: Wiegers, B. S. - Cheer, A. Y. - Silk, W. K.
Journal: Plant Physiol

Primary growth is characterized by cell expansion facilitated by water uptake generating hydrostatic (turgor) pressure to inflate the cell, stretching the rigid cell walls. The multiple source theory of root growth hypothesizes that root growth involves transport of water from both the soil surrounding the growth zone and from the mature tissue higher in the root via phloem and protophloem. Here, protophloem water sources are used as boundary conditions in a classical, three-dimensional model of growth sustaining water potentials in primary roots. The model predicts small radial gradients in water potential, with a significant longitudinal gradient. The results improve the agreement of theory with empirical studies for water potential in the primary growth zone of roots of Zea mays. A sensitivity analysis quantifies the functional importance of apical phloem differentiation in permitting growth and reveals that the presence of phloem water sources makes the growth-sustaining water relations of the root relatively insensitive to changes in root radius and hydraulic conductivity. Adaptation to drought and other environmental stress is predicted to involve more apical differentiation of phloem and/or higher phloem delivery rates to the growth zone.

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The rice aquaporin Lsi1 mediates uptake of methylated arsenic species.

Publication Date: 2009 Jun 19 PMID: 19542298
Authors: Li, R. Y. - Ago, Y. - Liu, W. J. - Mitani, N. - Feldmann, J. - McGrath, S. P. - Ma, J. F. - Zhao, F. J.
Journal: Plant Physiol

Pentavalent methylated arsenic species such as monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two Si transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild-type having 3.5-fold higher V max than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild-type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.

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AINTEGUMENTA and AINTEGUMENTA-like 6 act redundantly to regulate Arabidopsis floral growth and patterning.

Publication Date: 2009 Jun 19 PMID: 19542297
Authors: Krizek, B. A.
Journal: Plant Physiol

An Arabidopsis thaliana flower consists of four types of organs arranged in a stereotypical pattern. This complex floral structure is elaborated from a small number of floral meristem cells partitioned from the shoot apical meristem during reproductive development. The positioning of floral primordia within the periphery of the shoot apical meristem depends on transport of the phytohormone auxin with floral anlagen arising at sites of auxin maxima. An early marker of lateral organ fate is the AP2/ERF-type transcription factor AINTEGUMENTA (ANT), which has been proposed to act downstream of auxin in organogenic growth. Here, I show that the related, AINTEGUMENTA-like 6 (AIL6)/PLETHORA3 (PLT3) gene acts redundantly with ANT during flower development. ant ail6 double mutants show defects in floral organ positioning, identity and growth. These floral defects are correlated with changes in the expression levels and patterns of two floral organ identity genes, APETALA3 (AP3) and AGAMOUS (AG). ant ail6 flowers also display altered expression of an auxin responsive reporter suggesting that auxin accumulation and/or responses are not normal. Furthermore, I show that ANT expression in incipient and young floral primordia depends on auxin transport within the inflorescence meristem. These results show that ANT and AIL6 are important regulators of floral growth and patterning and that they may act downstream of auxin in these processes.

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Real-time Detection of Caspase-3-like Proteases Activation in Vivo Using Fluorescence Resonance Energy Transfer during Plant Programmed Cell Death Induced by Ultraviolet-C Overexposure.

Publication Date: 2009 Jun 17 PMID: 19535476
Authors: Zhang, L. - Xu, Q. - Xing, D. - Gao, C. - Xiong, H.
Journal: Plant Physiol

Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the timescale of caspase-3-like proteases activation was investigated in single living plant cells undergoing PCD induced by ultraviolet-C (UV-C) overexposure. The real-time detection of caspase-3-like proteases activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis protoplasts. At 30 min after exposure to UV-C, caspase-3-like proteases activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like proteases activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western blotting analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with mutant DEVD tag. In summary, the results represent direct evidence for the activation of caspase-3-like proteases in UV-C-induced PCD, and FRET technique is a powerful tool for monitoring key events of PCD in living cells in real-time.

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HORMONOMETER: A tool for discerning transcript signatures of hormone action in the Arabidopsis transcriptome.

Publication Date: 2009 Jun 17 PMID: 19535475
Authors: Volodarsky, D. - Leviatan, N. - Otcheretianski, A. - Fluhr, R.
Journal: Plant Physiol

Plant hormones regulate growth and response to environmental change. Hormone action ultimately modifies cellular physiological processes and gene activity. To facilitate transcriptome evaluation of novel mutants and environmental responses there is a need to rapidly assess the possible contribution of hormone action to changes in the levels of gene transcripts. We developed a vector-based algorithm that rapidly compares lists of transcripts yielding correlation values. The application as described here, called "HORMONOMETER", was used to analyze hormone related activity in a transcriptome. The veracity of the resultant analysis was established by comparison to cognate and non-cognate hormone transcriptomes as well as to mutants and to selected plant-environmental interactions. The HORMONOMETER accurately predicted correlations between hormone-action and biosynthetic mutants for which transcriptome data are available. A high degree of correlation was detected between many hormones, particularly at early time points of hormone action. Unforeseen complexity was detected in the analysis of mutants and in plant-herbivore interactions. The "HORMONOMETER" provides a diagnostic tool for evaluating the physiological state-of-being of the plant from the point of view of transcripts regulated by hormones and yields biological insight into the multiple response components that enable plant adaptation to the environment. A Web-based interface has been developed to facilitate external interfacing with this platform.

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More productive than maize in the Midwest. How does Miscanthus do it?

Publication Date: 2009 Jun 17 PMID: 19535474
Authors: Dohleman, F. G. - Long, S. P.
Journal: Plant Physiol

In the first side-by-side large scale trials of these two C4 crops in the US Corn-Belt, Miscanthus (Miscanthus x giganteus) was 59% more productive than grain maize (Zea mays). Total productivity is the product of the total solar radiation incident per unit land area (Qtot) and the efficiencies of light interception (epsiloni) and its conversion into above-ground biomass (epsilonca). Averaged over two growing seasons, epsilonca did not differ, but epsiloni was 61% higher for Miscanthus, which developed a leaf canopy earlier and maintained it later. The diurnal course of photosynthesis was measured on sunlit and shaded leaves of each species on 26 dates. The daily integral of leaf-level photosynthetic CO2 uptake (A') differed slightly when integrated across two growing seasons, but was up to 60% higher in maize in mid-summer. The average leaf area of Miscanthus was double that of maize, with the result that calculated canopy photosynthesis was 44% higher in Miscanthus, corresponding closely to the biomass differences. To determine the basis of differences in mid-season leaf photosynthesis, light and CO2 responses were analyzed to determine in vivo biochemical limitations. Maize had a higher maximum velocity of PEP carboxylation (Vpmax), velocity of PEP regeneration (Vpr), light saturated rate of photosynthesis (Asat) and higher maximum quantum efficiency of CO-2 assimilation (PhiCO2 max). These biochemical differences, however, were more than offset by the larger leaf area and its longer duration in Miscanthus. The results indicate that the full potential of C4 photosynthetic productivity is not achieved by modern temperate maize cultivars.

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Expansion Mechanisms and Functional Annotations of Hypothetical Genes in Rice Genome.

Publication Date: 2009 Jun 17 PMID: 19535473
Authors: Jiang, S. Y. - Christoffels, A. - Ramamoorthy, R. - Ramachandran, S.
Journal: Plant Physiol

In each completely sequenced genome, 30-50% of genes are annotated as uncharacterized hypothetical genes. In the rice genome, 10918 hypothetical genes were annotated in the latest version (release 6) of MSU rice genome annotation. We have implemented an integrative approach to analyze their duplication/expansion and function. The analyses show that tandem/segmental duplication and transposition/ retrotransposition have significantly contributed to the expansion of hypothetical genes despite their different contribution rates. A total of 3769 hypothetical genes have been detected from retrogene, tandem, segmental, Pack-MULE or LTR-related duplication/expansion. The nonsynonymous substitutions per site (Ka) and synonymous substitutions per site (Ks) analyses showed that 21.65% of them were still functional, accounting for 7.47% of total hypothetical genes. Global expression analyses have identified 1672 expressed hypothetical genes. Among them, 415 genes might function in a developmental stage-specific manner. Anti-sense strand expression and small RNA analyses have demonstrated that high percentage of these hypothetical genes might play important roles in negatively regulating gene expression. Homologous searches against Arabidopsis, maize, sorghum and Indica rice genomes suggest that most of the hypothetical genes could be annotated from recently evolved genomic sequences. These data advance the understanding of rice hypothetical genes as being involved in lineage-specific expansion, and that they function in a specific developmental stage. Our analyses also provide a valuable means to facilitate the characterization and functional annotation of hypothetical genes in other organisms.

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The Mg-Chelatase H Subunit Binds Abscisic Acid and Functions in Abscisic Acid Signaling: New Evidence in Arabidopsis.

Publication Date: 2009 Jun 17 PMID: 19535472
Authors: Wu, F. Q. - Xin, Q. - Cao, Z. - Liu, Z. Q. - Du, S. Y. - Mei, C. - Zhao, C. X. - Wang, X. F. - Shang, Y. - Jiang, T. - Zhang, X. F. - Yan, L. - Zhao, R. - Cui, Z. N. - Liu, R. - Sun, H. L. - Yang, X. L. - Su, Z. - Zhang, D. P.
Journal: Plant Physiol

Using a newly-developed ABA-affinity chromatography technique, we showed that the Mg-chelatase H subunit ABAR/CHLH specifically binds ABA through the C-terminal half but not the N-terminal half. A set of potential agonists/antagonists to ABA including 2-trans, 4-trans-ABA, gibberellin GA3, cytokinin-like regulator 6-BA, auxin IAA and auxin-like substance NAA and jasmonic acid methyl ester MeJA did not bind ABAR/CHLH. A C-terminal C370-truncated ABAR with 369 amino acid residues (631-999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently, expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescence protein (GFP) in wild-type plants conferred ABA-hypersensitivity in all the major ABA responses including seed germination, post-germination growth and stomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cch mutant of ABAR/CHLH gene restored the ABA sensitivity of the mutant in all the ABA responses. However, the effect of expression of the ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effect of the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles of ABAR/CHLH gene from the mutant pool in the Arabidopsis Biological Resource Center (ABRC) via the Arabidopsis TILLING (Targeting Induced Local Lesions in Genomes). The abar-2 mutant has a point mutation resulting in the N-terminal Leu348-->Phe and the abar-3 mutant also in the N-terminal Ser183-->Phe. The two mutants show altered ABA-related phenotypes in seed germination and post-germination growth but not in stomatal movement. These new findings support the idea that ABAR/CHLH is an ABA receptor, and reveal that the C-terminal half of ABAR/CHLH plays a central role in ABA signaling, which is consistent with its ABA-binding ability, but the N-terminal half is also functionally required likely through a regulatory action on the C-terminal half.

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Truffles regulate plant root morphogenesis via the production of auxin and ethylene.

Publication Date: 2009 Jun 17 PMID: 19535471
Authors: Splivallo, R. - Fischer, U. - Gobel, C. - Feussner, I. - Karlovsky, P.
Journal: Plant Physiol

Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction i. e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle T. melanopsorum induce alterations in root morphology of the host Cistus incanus and the non-host Arabidopsis thaliana (i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and IAA fully mimicked the root morphology induced by the mycelium for both host and non-host plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungal induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5::GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffle synthesize ethylene from L-methionine probably through the alpha-keto-gamma-(methylthio) butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions.

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A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid (2-O-[caffeoyl]-L-malate) Biosynthesis.

Publication Date: 2009 Jun 12 PMID: 19525325
Authors: Sullivan, M. L.
Journal: Plant Physiol

Red clover (Trifolium pratense L.) leaves accumulate several micromol g(-1) fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa L.) lack both polyphenol oxidase and o-diphenols and breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in development of forages utilizing this natural system of protein protection. A possible pathway for phaselic acid biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs were identified in red clover. HCT1 shares >75% amino acid identity with a number of well-characterized shikimate O-HCTs implicated in monolignol biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has limited sequence identity to any previously identified HCT. Expression analyses indicate HCT1 mRNA accumulates to four-fold higher levels in stems than leaves, whereas HCT2 mRNA accumulates to ten-fold higher levels in leaves than stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate HCT1 transfers caffeoyl or p-coumaroyl moieties from a CoA-thiolester to shikimate, but not malate whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a CoA-thiolester to malate but not shikimate. Together, these results indicate HCT1 is involved in monolignol biosynthesis and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.

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BT2, a BTB protein, mediates multiple responses to nutrients, stresses, and hormones in Arabidopsis thaliana.

Publication Date: 2009 Jun 12 PMID: 19525324
Authors: Mandadi, K. K. - Misra, A. - Ren, S. - McKnight, T. D.
Journal: Plant Physiol

The Arabidopsis thaliana gene BT2 encodes a 41-kilodalton protein that possesses an N-terminal BTB domain, a central TAZ domain, and a C-terminal calmodulin-binding domain. We previously demonstrated that BT2 could activate telomerase expression in mature Arabidopsis leaves. Here, we report its distinct role in mediating diverse hormone, stress and metabolic responses. We serendipitously discovered that steady-state expression of BT2 mRNA was regulated diurnally and controlled by the circadian clock, with maximum expression in the dark. This pattern of expression suggested that BT2 mRNA could be linked to the availability of photosynthate in the plant. Exogenous sugars decreased BT2 expression, whereas exogenous nitrogen increased expression. bt2 loss-of-function mutants displayed a hypersensitive response to both sugar-mediated inhibition of germination and to abscisic-acid (ABA)-mediated inhibition of germination, thus supporting a role of ABA in sugar signaling in germination and development. Moreover, constitutive expression of BT2 imparted resistance to both sugars and ABA at germination, suggesting that BT2 suppresses sugar and ABA responses. In support of the previously described antagonistic relationship between ABA and auxin, we found that BT2 positively regulated certain auxin responses in plants, as revealed by knocking down BT2 expression in the high-auxin mutant yucca. Accumulation of BT2 mRNA was affected by a variety of hormones, nutrients and stresses, and BT2 was required for response to many of these same factors. Together, these results suggest that BT2 is a central component of an interconnected signaling network that detects and responds to multiple inputs.

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Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1.

Publication Date: 2009 Jun 12 PMID: 19525323
Authors: Kim, S. H. - Kwon, S. I. - Saha, D. - Anyanwu, N. C. - Gassmann, W.
Journal: Plant Physiol

The Pseudomonas syringae - Arabidopsis thaliana (Arabidopsis) interaction is an extensively studied plant-pathogen system. Arabidopsis possesses approximately 150 putative resistance genes encoding nucleotide binding site (NBS) and leucine-rich repeat (LRR) domain-containing proteins. The majority of these belong to the Toll/Interleukin-1 receptor (TIR)-NBS-LRR class. Comparative studies with the coiled coil (CC)-NBS-LRR genes RPS2, RPM1 and RPS5 and isogenic P. syringae strains expressing single corresponding avirulence genes have been particularly fruitful in dissecting specific and common resistance signaling components. However, the major TIR-NBS-LRR class is represented by a single known P. syringae resistance gene, RPS4. We previously identified hopA1 from P. syringae pv. syringae strain 61 as an avirulence gene that signals through EDS1, indicating that the corresponding resistance gene RPS6 belongs to the TIR-NBS-LRR class. Here we report the identification of RPS6 based on a forward-genetic screen and map-based cloning. Among resistance proteins of known function, the deduced amino acid sequence of RPS6 shows highest similarity to the TIR-NBS-LRR resistance protein RAC1 that determines resistance to the oomycete pathogen Albugo candida. Similar to RPS4 and other TIR-NBS-LRR genes, RPS6 generates alternatively spliced transcripts, although the alternative transcript structures are RPS6-specific. We previously characterized SRFR1 as a negative regulator of avrRps4-triggered immunity. Interestingly, mutations in SRFR1 also enhanced HopA1-triggered immunity in rps6 mutants. In conclusion, the cloning of RPS6 and comparisons with RPS4 will contribute to a closer dissection of the TIR-NBS-LRR resistance pathway in Arabidopsis.

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A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine.

Publication Date: 2009 Jun 12 PMID: 19525322
Authors: Hugueney, P. - Provenzano, S. - Verries, C. - Ferrandino, A. - Meudec, E. - Batelli, G. - Merdinoglu, D. - Cheynier, V. - Schubert, A. - Ageorges, A.
Journal: Plant Physiol

Anthocyanins are major pigments in coloured grape berries and most of them are mono- or di-methoxylated. We report here the functional characterization of an anthocyanin O-methyltransferase (AOMT) from Vitis vinifera L.. The expression pattern in two cultivars with different anthocyanin methylation profiles (Syrah and Nebbiolo) showed a peak at start ripening (veraison), when the concentration of all methylated anthocyanins begins to increase. The purified recombinant AOMT protein was active on both anthocyanins and flavonols in vitro, with Km in the micromolar range, and was dependent on divalent cations for activity. AOMT showed a preference for 3',5' methylation when a 3',4',5' hydroxylated anthocyanin substrate was tested. In order to assess its in planta activity, we performed transient expression of AOMT in tobacco leaves expressing the PAP1 transcription factor from Arabidopsis thaliana. PAP1 expression in leaves induced the accumulation of the non methylated anthocyanin delphinidin 3-rutinoside. The co-expression of PAP1 and AOMT resulted in an accumulation of malvidin 3-rutinoside. We also showed that AOMT localized exclusively in the cytoplasm of tobacco leaf cells. These results demonstrate the ability of this enzyme to methylate anthocyanins both in vitro and in vivo, indicating that AOMT play a major role in anthocyanin biosynthesis in grape berries.

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CYP86B1 is required for very long chain -hydroxyacids and ,-dicarboxylic acids synthesis in root and seed suberin polyester.

Publication Date: 2009 Jun 12 PMID: 19525321
Authors: Compagnon, V. - Diehl, P. - Benveniste, I. - Meyer, D. - Schaller, H. - Schreiber, L. - Franke, R. - Pinot, F.
Journal: Plant Physiol

Suberin composition of various plants including Arabidopsis thaliana has shown the presence of very long chain fatty acid (VLCFA) derivatives C20, in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis this subfamily is represented by CYP86B1 and CYP86B2, that share about 45% identity with CYP86A1, a fatty acid omega-hydroxylase implicated in root suberin monomer synthesis. Here we show that CYP86B1 is located to the ER and is highly expressed in roots. Indeed, CYP86B1 promoter-driven GUS expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knock-outs and two weak ones were RNAi silenced-mediated lines. These ralph (root aliphatic plant hydroxylase) lines had a root and a seed coat aliphatic polyester composition in which C22 and C24 -hydroxyacids and alpha,omega-dicarboxylic acids were strongly reduced. However these changes do not affect seed coat permeability and ions content in leaves. The presumed precursors, C22 and C24 fatty acids accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a VLCFA hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development.

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ROOT UV-B SENSITIVE2 Acts With RUS1 in a Root UV-B Sensing Pathway.

Publication Date: 2009 Jun 10 PMID: 19515790
Authors: Leasure, C. D. - Tong, H. - Yuen, G. - Hou, X. - Sun, X. - He, Z. H.
Journal: Plant Physiol

Ultraviolet-B (UVB; 280-320 nm) perception and signaling are well-known phenomena in plants, although no specific UVB photoreceptors have yet been identified. We previously reported on the rus1 mutants in Arabidopsis, which display a block to development under very low-fluence-rate (VLF) UV-B (< 0.1 micromol m(-1) s(-1)) after the seedling emerges from the seed. Here we report the analysis and cloning of the root UVB sensitive2-1 (rus2-1) mutation in Arabidopsis. The phenotype of rus2-1 mutant seedlings is virtually indistinguishable from the phenotype of rus1 seedlings. A map-based approach was used to clone RUS2. RUS2 encodes a DUF647-containing protein that is homologous to the RUS1 protein. rus1-2 rus2-1 double mutant seedlings have the same phenotype as both rus1 and rus2 single mutants, suggesting that the two genes work in the same pathway. RUS2-GFP shows a similar expression pattern as that of RUS1-GFP, and RUS1 and RUS2 proteins interact physically in yeast. This protein-protein interaction depends on the DUF647 domain and site-directed mutagenesis identified specific residues in the DUF647 that are required for both protein-protein interaction and physiological function. Six RUS genes are found in Arabidopsis, rice and moss, and one RUS member, RUS3, is conserved in plants and animals. Our results demonstrate that RUS2 works with RUS1 in a root UV-B sensing pathway that plays a vital role in Arabidopsis early seedling morphogenesis and development.

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Transcriptional gene silencing mediated by a plastid inner envelope phosphoenolpyruvate/phosphate translocator CUE1 in Arabidopsis.

Publication Date: 2009 Jun 10 PMID: 19515789
Authors: Shen, J. - Ren, X. - Cao, R. - Liu, J. - Gong, Z.
Journal: Plant Physiol

Mutations in ROS1 (REPRESSOR OF SILENCING 1) lead to the transcriptional gene silencing (TGS) of ProRD29A:LUC and Pro35S:NPTII reporter genes. We performed a genetic screen to find suppressors of ros1 that identified two mutant alleles in the Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (CUE1) gene, which encodes a plastid inner envelope phosphoenolpyruvate/phosphate translocator. The cue1 mutations released the TGS of Pro35S:NPTII and the transcriptionally silent endogenous locus TSI (TRANSCRIPTIONAL SILENCING INFORMATION) in a manner that was independent on DNA methylation but dependent on chromatin modification. The cue1 mutations did not affect the TGS of ProRD29A:LUC in ros1, which was dependent on RNA-directed DNA methylation. It is possible that signals from chloroplasts help to regulate the epigenetic status of subset of genomic loci in the nucleus.

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Lifestyles of the effector-rich: Genome-enabled characterization of bacterial plant pathogens.

Publication Date: 2009 Jun 10 PMID: 19515788
Authors: Collmer, A. - Schneider, D. J. - Lindeberg, M.
Journal: Plant Physiol

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High throughput quantification of root growth using a novel image analysis tool.

Publication Date: 2009 Jun 10 PMID: 19515787
Authors: French, A. - Ubeda-Tomas, S. - Holman, T. J. - Bennett, M. J. - Pridmore, T.
Journal: Plant Physiol

Measuring the dynamics of plant growth is fundamental to the understanding of plant development processes. This paper describes a high throughput, automatic method to trace Arabidopsis thaliana seedling roots grown on agarose plates. From the trace, additional software can quantify length, curvature and stimulus response parameters such as onset of gravitropism. The method combines a particle filtering algorithm with a graph-based method to trace the centre-line of a root. This top-down approach is robust to a variety of noise effects, and is reasonably flexible across different image sets. The resulting tool requires minimal interaction from the user, and is able to process long timelapse sequences with user interaction only required on the first frame. The tool is described first, followed by its use on two sample datasets, one measuring root lengths and the other additionally analysing the gravitropic response and curvature. The tool, RootTrace is open source; both the program and source code will be available online.

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Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A.

Publication Date: 2009 Jun 11 PMID: 19502356
Authors: Maruyama, K. - Takeda, M. - Kidokoro, S. - Yamada, K. - Sakuma, Y. - Urano, K. - Fujita, M. - Yoshiwara, K. - Matsukura, S. - Morishita, Y. - Sasaki, R. - Suzuki, H. - Saito, K. - Shibata, D. - Shinozaki, K. - Yamaguchi-Shinozaki, K.
Journal: Plant Physiol

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis. Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration, but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically, and consequently many kinds of monosaccharides, disaccharides, trisaccharides and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.

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Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars.

Publication Date: 2009 Jun 5 PMID: 19502355
Authors: Fan, R. C. - Peng, C. C. - Xu, Y. H. - Wang, X. F. - Li, Y. - Shang, Y. - Du, S. Y. - Zhao, R. - Zhang, X. Y. - Zhang, L. Y. - Zhang, D. P.
Journal: Plant Physiol

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remains unclear in plant and animal cells. Here, we report that, a sucrose transporter MdSUT1 and a sorbitol transporter MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two hybrid, immuno-coprecipitation and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown both to interact physically with an apple ER-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis homologue of MdCYB5, AtCYB5-A, interacts also with the two sugar transporters and functions similarly. The point mutations Leu73-->Pro in MdSUT1 and Leu117-->Pro in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.

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Identification of Nutrient-Responsive Arabidopsis and Rapeseed MicroRNAs by Comprehensive Real-Time Polymerase Chain Reaction Profiling and Small RNA Sequencing.

Publication Date: 2009 Jul PMID: 19465578
Authors: Pant, B. D. - Musialak-Lange, M. - Nuc, P. - May, P. - Buhtz, A. - Kehr, J. - Walther, D. - Scheible, W. R.
Journal: Plant Physiol

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

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