Plant Physiology

Differential oxidative metabolism and 5-ketoclomazone accumulation are involved in Echinochloa phyllopogon resistance to clomazone.

Publication Date: 2010 Mar 5 PMID: 20207709
Authors: Yasuor, H. - Zou, W. - Tolstikov, V. V. - Tjeerdema, R. S. - Fischer, A. J.
Journal: Plant Physiol

Echinochloa phyllopogon (late watergrass) is a major weed of California rice that has evolved cytochrome P450-mediated metabolic resistance to different herbicides with multiple modes of action. E. phyllopogon populations from Sacramento Valley rice fields have also recently shown resistance to the herbicide clomazone. Clomazone is a proherbicide that must be metabolized to 5-ketoclomazone, which is the active compound that inhibits deoxyxylulose 5-phosphate synthase, a key enzyme of the non-mevalonate isoprenoid pathway. This study evaluated the differential clomazone metabolism within strains of the same species to investigate whether enhanced oxidative metabolism also confers clomazone resistance in E. phyllopogon. Using reverse-phase liquid chromatography-tandem mass spectrometry techniques (RP-LC-MS/MS) in the multi-reaction monitoring mode (MRM) we elucidated that oxidative biotransformations are involved as a mechanism of clomazone resistance in this species. E. phyllopogon plants hydroxylated mostly the isoxazolidinone ring of clomazone, and clomazone hydroxylation activity was greater in resistant than in susceptible plants. The major clomazone metabolites resulted from mono- and di-hydroxylation of the isoxazolidinone ring. Resistant plants accumulated 6- to 12-fold more of the monohydroxylated metabolite than susceptible plants, while susceptible plants accumulated 2.5-fold more of the phytotoxic metabolite of clomazone, 5-ketoclomazone. Our results demonstrate that oxidative metabolism, endows multiple-herbicide-resistant E. phyllopogon with cross-resistance to clomazone through enhanced herbicide degradation and lower accumulation of the toxic metabolite in resistant vs. susceptible plants.

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Mathematical modelling of the central carbohydrate metabolism in Arabidopsis thaliana reveals a substantial regulatory influence of vacuolar invertase on whole plant carbon metabolism.

Publication Date: 2010 Mar 5 PMID: 20207708
Authors: Naegele, T. - Henkel, S. - Hoermiller, I. - Sauter, T. - Sawodny, O. - Ederer, M. - Heyer, A. G.
Journal: Plant Physiol

A mathematical model representing metabolite interconversions in the central carbohydrate metabolism of Arabidopsis thaliana was developed to simulate diurnal dynamics of primary carbon metabolism in a photosynthetically active plant leaf. The model groups enzymatic steps of central carbohydrate metabolism into blocks of interconverting reactions that link easily measurable quantities like CO2 exchange and quasi steady state levels of soluble sugars and starch. When metabolite levels that fluctuate over diurnal cycles are used as a basic condition for simulation, turnover rates for the interconverting reactions can be calculated that approximate measured metabolite dynamics and yield kinetic parameters of interconverting reactions. We used experimental data for Arabidopsis wild type plants, accession Col-0, and a mutant defective in vacuolar invertase AtbetaFruct4 as input data. Reducing invertase activity to mutant levels in the wild type model led to a correct prediction of increased sucrose levels. However, additional changes were needed to correctly simulate levels of hexoses and sugar phosphates, indicating that invertase knock-out causes subsequent changes in other enzymatic parameters. Reduction of invertase activity caused a decline in photosynthesis and export of reduced carbon to associated metabolic pathways and sink organs, e.g. roots, which is in agreement with the reported contribution of vacuolar invertase to sink strength. According to model parameters, there is a role for invertase in leaves, where futile cycling of sucrose appears to have a buffering effect on the pools of sucrose, hexoses and sugar phosphates. Our data demonstrate that modelling complex metabolic pathways is a useful tool to study the significance of single enzyme activities in complex, non-intuitive networks.

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The {alpha} subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency.

Publication Date: 2010 Mar 3 PMID: 20200073
Authors: Nilson, S. E. - Assmann, S. M.
Journal: Plant Physiol

Land plants must balance CO2 assimilation with transpiration in order to minimize drought stress and to maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the alpha subunit of the heterotrimeric G protein in Arabidopsis thaliana, GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells which are hyposensitive to ABA-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous ABA. Leaf level gas exchange analysis shows that gpa1 mutants have wild type assimilation vs. internal CO2 concentration responses but exhibit reduced stomatal conductance compared to Col at ambient and below-ambient internal CO2 concentrations. The increased TE and reduced whole-leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density which is reduced in gpa1 mutants. GPA1 regulates stomatal density via control of epidermal cell size and stomata formation. GPA1 promoter::GUS lines indicate that the GPA1 promoter is active in the stomatal cell lineage further supporting a function for GPA1 in stomatal development in true leaves.

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Probing the reproducibility of leaf growth and molecular phenotypes: a comparison of three Arabidopsis accessions cultivated in ten laboratories.

Publication Date: 2010 Mar 3 PMID: 20200072
Authors: Massonnet, C. - Vile, D. - Fabre, J. - Hannah, M. A. - Caldana, C. - Lisec, J. - Beemster, G. T. - Meyer, R. C. - Messerli, G. - Gronlund, J. T. - Perkovic, J. - Wigmore, E. - May, S. - Bevan, M. W. - Meyer, C. - Rubio-Diaz, S. - Weigel, D. - Micol, J. L. - Buchanan-Wollaston, V. - Fiorani, F. - Walsh, S. - Rinn, B. - Gruissem, W. - Hilson, P. - Hennig, L. - Willmitzer, L. - Granier, C.
Journal: Plant Physiol

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables, metabolite and transcriptome profiles of three Arabidopsis genotypes grown in ten laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented also significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation but the standardization ensured that inter-laboratory variation was not considerably larger than intra-laboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

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AUXIN METABOLISM AND FUNCTION IN THE MULTICELLULAR BROWN ALGA ECTOCARPUS SILICULOSUS.

Publication Date: 2010 Mar 3 PMID: 20200071
Authors: Le Bail, A. - Billoud, B. - Kowalczyk, N. - Kowalczyk, M. - Gicquel, M. - Le Panse, S. - Stewart, S. - Scornet, D. - Cock, J. M. - Ljung, K. - Charrier, B.
Journal: Plant Physiol

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organised as a main primary filament composed of elongated (E) cells and round (R) cells, from which branches differentiate. Modelling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organisation of the branching pattern. In this paper, we show that auxin IAA is detectable in mature E. siliculosus organisms, and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response and transport genes showed that mainly IAA biosynthesis genes from land plants have homologues in the E. siliculosus genome. In addition, application of exogenous auxins and TIBA had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of the progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment, and showed that its expression in a series of morphogenetic mutants was positively correlated with both their E-to-R cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information, and induces a signalling pathway different from that known in land plants.

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The Protein Kinase SnRK2.6 Mediates the Regulation of Sucrose Metabolism and Plant Growth in Arabidopsis.

Publication Date: 2010 Mar 3 PMID: 20200070
Authors: Zheng, Z. - Xu, X. - Crosley, R. A. - Greenwalt, S. A. - Sun, Y. - Blakeslee, B. - Wang, L. - Ni, W. - Sopko, M. S. - Yao, C. - Yau, K. - Burton, S. - Zhuang, M. - McCaskill, D. G. - Gachotte, D. - Thompson, M. - Greene, T. W.
Journal: Plant Physiol

In higher plants three subfamilies of Snf1-related protein kinases have evolved. While the SnRK1 subfamily has been shown to share pivotal roles with the orthologous yeast Snf1 and mammalian AMPK in modulating energy and metabolic homeostasis, functional significance of the two plant-specific subfamilies SnRK2 and SnRK3 in these critical processes is poorly understood. We show here that SnRK2.6, previously identified as crucial in the control of stomatal aperture by abscisic acid (ABA), has a broad expression pattern and participates in the regulation of plant primary metabolism. Inactivation of this gene reduced oil synthesis in Arabidopsis seeds, whereas its overexpression increased sucrose synthesis and fatty acid desaturation in the leaves. Notably, the metabolic alterations in the SnRK2.6 overexpressors were accompanied by amelioration of those physiological processes that require high levels of carbon and energy input, such as plant growth and seed production. However, the mechanisms underlying these functionalities could not be solely attributed to the role of SnRK2.6 as a positive regulator of ABA signaling although we demonstrate that this kinase confers ABA hypersensitivity during seedling growth. Collectively, our results suggest that SnRK2.6 mediates hormonal and metabolic regulation of plant growth and development and that besides the SnRK1 kinases, SnRK2.6 is also implicated in the regulation of metabolic homeostasis in plants.

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The development of an efficient multi-purpose BPMV viral vector set for foreign gene expression and RNA silencing.

Publication Date: 2010 Mar 3 PMID: 20200069
Authors: Zhang, C. - Bradshaw, J. D. - Whitham, S. A. - Hill, J. H.
Journal: Plant Physiol

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants, such as soybean, that are recalcitrant to transformation. Previously, two generations of Bean pod mottle virus (BPMV, genus Comovirus) vectors have been developed for over-expressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean. These vectors are designed for simultaneous expression of multiple foreign genes, insertion of non-coding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of GFP and silencing of phytoene desaturase shows that marker gene assisted silencing is feasible. The results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

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MISSA Is a Highly Efficient in vivo DNA Assembly Method for Plant Multiple-gene Transformation.

Publication Date: 2010 Mar 3 PMID: 20200068
Authors: Chen, Q. J. - Xie, M. - Ma, X. X. - Dong, L. - Chen, J. - Wang, X. C.
Journal: Plant Physiol

We describe a highly efficient in vivo DNA assembly method, multiple-round in vivo site-specific assembly (MISSA), which facilitates plant multiple-gene transformation. MISSA is based on conjugational transfer, which is driven by donor strains, and two in vivo site-specific recombination events that are mediated by inducible Cre recombinase and phage lambda site-specific recombination proteins in recipient strains, to enable in vivo transfer and in vivo assembly of multiple transgenic DNA. The assembly reactions can be performed circularly and iteratively through alternate use of the two specially designed donor vectors. As proof-of-principle experiments, we constructed a few plant multigene binary vectors. One of these vectors was generated by 15 rounds of MISSA reactions and was confirmed in transgenic Arabidopsis. As MISSA simplifies the tedious and time-consuming in vitro manipulations to a simple mixing of bacterial strains, it will greatly save time, effort and expense associated with the assembly of multiple transgenic or synthetic DNA. The principle that underlies MISSA is applicable to engineering polygenic traits, biosynthetic pathways or protein complexes in all organisms, such as E.coli, yeast, plants and animals. MISSA also has potential applications in synthetic biology, whether for basic theory or for applied biotechnology aiming at the assembly of genetic pathways for the production of biofuels, pharmaceuticals and industrial compounds from natural or synthetic DNA.

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Concerted modification of flowering time and inflorescence architecture by ectopic expression of TFL1-like genes in Zea mays.

Publication Date: 2010 Mar 3 PMID: 20200067
Authors: Danilveskaya, O. N. - Meng, X. - Ananiev, E. V.
Journal: Plant Physiol

TFL1-like genes are highly conserved in plants and are thought to function in the maintenance of meristem indeterminacy. Recently we described six maize TFL1-related genes, named Zea mays CENTRORADIALIS (ZCN1 - 6). To gain insight into their functions, we generated transgenic maize plants over-expressing their respective cDNAs driven by a constitutive promoter. Overall, ectopic expression of the maize TFL1-like genes produced similar phenotypes, including delayed flowering and altered inflorescence architecture. We observed an apparent relationship between the magnitude of the transgenic phenotypes with the degree of homology between the ZCN proteins. ZCN2, 4, and 5 form a monophylogenetic clade and their over-expression produced the strongest phenotypes. Along with very late flowering, these transgenic plants produced a "bushy" tassel with increased lateral branching and spikelet density compared to non-transgenic siblings. On the other hand, ZCN1, 3, and 6 produced milder effects. Among them, ZCN1 showed moderate effects on flowering time and tassel morphology whereas ZCN3 and ZCN6 did not change flowering time but still showed effects on tassel morphology. In situ hybridizations of tissue from non-transgenic plants revealed that expression of all ZCN genes was associated with vascular bundles but each gene had a specific spatial and temporal pattern. Expression of four ZCN genes localized to the proto-xylem, whereas ZCN5 was expressed in the proto-phloem. Collectively, our findings suggest that ectopic expression of the TFL1-like genes in maize may modify flowering time and inflorescence architecture through maintenance of the indeterminacy of the vegetative and inflorescence meristems.

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Trafficking of the plant cellulose synthase complex.

Publication Date: 2010 Mar 3 PMID: 20200066
Authors: Wightman, R. - Turner, S.
Journal: Plant Physiol

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The role of phloem loading reconsidered.

Publication Date: 2010 Mar 3 PMID: 20200065
Authors: Turgeon, R.
Journal: Plant Physiol

It is generally assumed that the primary role of phloem loading is to drive long-distance transport by elevating hydrostatic pressure in the sieve elements. This concept is consistent with the fact that, in many plants, energy is used to increase the concentrations of photoassimilates in the leaf phloem to levels well above those in mesophyll cells. However, on the basis of recent data, these fundamental assumptions need reevaluation. The data indicate that a large number of woody species - plants with the longest transport distances - load solute passively by maintaining high concentrations of sucrose, and in some cases sugar alcohol, in mesophyll cells. Here I suggest that the adaptive advantage of active phloem loading, in the evolutionary sense, is to permit plants to maintain low foliar concentrations of non-structural carbohydrates (NSC), i.e. sugars, sugar alcohols and starch. Economic considerations of inventory costs indicate that maintaining low NSC levels in leaves improves return on investment in carbohydrate synthesis, leading to a substantial increase in growth potential.

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Combined transcript and metabolite profiling of Arabidopsis thaliana grown under widely variant growth conditions facilitates the identification of novel metabolite mediated regulation of gene expression.

Publication Date: 2010 Feb 26 PMID: 20190096
Authors: Hannah, M. A. - Caldana, C. - Steinhauser, D. - Balbo, I. - Fernie, A. R. - Willmitzer, L.
Journal: Plant Physiol

Regulation of metabolism at the level of transcription and its corollary metabolite-mediated regulation of transcription are well documented mechanisms by which plants adapt to circumstance. That said the function of only a minority of transcription factor networks are fully understood and it seems likely that we have only identified a subset of the metabolites which play a mediator function in the regulation of transcription. Here we describe an integrated genomics approach in which we perform combined transcript and metabolite profiling on Arabidopsis plants challenged by various environmental extremes. We chose this approach to generate a large variance in the levels of all parameters recorded. The data was then statistically evaluated in order to identify metabolites whose level robustly correlated with those of a particularly large number of transcripts. Since correlation alone provides no proof of causality we subsequently attempted to validate these putative mediators of gene expression via a combination of statistical analysis of data available in publicly available databases and iterative experimental evaluation. Data presented here suggest that, on adoption of appropriate caution, the approach can be used for the identification of metabolite mediators of gene expression. As an exemplary case study we document that in plants, as in yeast and mammals, leucine plays an important role as a regulator of gene expression and provide a leucine response gene regulatory network.

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Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMyb12, leads to insect resistance.

Publication Date: 2010 Feb 26 PMID: 20190095
Authors: Misra, P. - Pandey, A. - Tiwari, M. - Chandrashekar, K. - Sidhu, O. P. - Asif, M. H. - Chakrabarty, D. - Singh, P. K. - Trivedi, P. K. - Nath, P. - Tuli, R.
Journal: Plant Physiol

Flavonoids synthesised by phenylpropanoid pathway participate in a myriad of physiological and biochemical processes in plants. Due to diversity of secondary transformations and complexity of regulation of branched pathways, single gene strategies have not been much successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis transcription factor, AtMyb12, in tobacco which resulted enhanced expression of genes involved in the phenylpropanoid pathway, leading to several-fold higher accumulation of flavonols. Global-gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMyb12 regulated a number of pathways, leading to flux availability for phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests, Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial miRNA reversed insect resistance of the AtMyb12-expressing tobacco plants. Our study suggests that AtMyb12 can be strategically used for developing safer insect pest resistant transgenic plants.

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Silencing NaTPI expression increases nectar germin, nectarins and H2O2 levels and inhibits nectar removal from plants in nature.

Publication Date: 2010 Feb 26 PMID: 20190094
Authors: Bezzi, S. - Kessler, D. - Diezel, C. - Muck, A. - Anssour, S. - Baldwin, I. T.
Journal: Plant Physiol

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild type (WT) plants in 4 field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of floral attractants benzylacetone and benzaldehyde, or in the concentrations of nectar sugar and nicotine between WT and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all WT flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of WT flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins (GLPs), and about 1.5-fold more H2O2 compared to WT nectar. Field experiments with WT flowers supplemented with a solution containing sugar and glucose oxidase (GOX) demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

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Differential impact of lipoxygenase 2 and jasmonates on natural and stress-induced senescence in Arabidopsis thaliana.

Publication Date: 2010 Feb 26 PMID: 20190093
Authors: Seltmann, M. A. - Stingl, N. E. - Lautenschlaeger, J. K. - Krischke, M. - Mueller, M. J. - Berger, S.
Journal: Plant Physiol

Jasmonic acid and related oxylipins are controversely discussed to be involved in regulating the initiation and progression of leaf senescence. To this end we analysed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis thaliana. Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes with the strongest increase of jasmonic acid after dark treatment. Arabidopside content did only increase considerably in response to sorbitol treatment. Mono- and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNAi plants were generated which produce constitutively jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase2-RNAi lines and the allene oxid synthase deficient mutant dde2 were less sensitive to sorbitol than the wild type indicating that oxylipins contribute to the response to sorbitol stress.

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Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes.

Publication Date: 2010 Feb 26 PMID: 20181757
Authors: Zhang, Y. - He, J. - Lee, D. - McCormick, S.
Journal: Plant Physiol

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross-talk in the model plant Arabidopsis thaliana, we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both Brefeldin A (BFA) and Wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or Wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/Wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug Latrunculin B (LatB) and the actin stabilization drug Jasplakinolide (Jas) rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross-talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.

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POLYPHOSPHOINOSITIDES ARE ENRICHED IN PLANT MEMBRANE RAFTS AND FORM MICRODOMAINS IN THE PLASMA MEMBRANE.

Publication Date: 2010 Feb 24 PMID: 20181756
Authors: Furt, F. - Konig, S. - Bessoule, J. J. - Sargueil, F. - Zallot, R. - Stanislas, T. - Noirot, E. - Lherminier, J. - Simon-Plas, F. - Heilmann, I. - Mongrand, S.
Journal: Plant Physiol

In this paper, we analyzed the lipid composition of Detergent-Insoluble Membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared to whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the plane of the PM with 60% of the epitope found in clusters of ca. 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the PtdIns(4,5)P2 cluster formation was not significantly sensitive to sterol depletion induced by methyl-beta-cyclodextrin (mbCD). Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction, but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane docking platforms is discussed.

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Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency.

Publication Date: 2010 Feb 24 PMID: 20181755
Authors: Lanquar, V. - Ramos, M. S. - Lelievre, F. - Barbier-Brygoo, H. - Krieger-Liszkay, A. - Kramer, U. - Thomine, S.
Journal: Plant Physiol

Manganese is an essential element, acting as cofactor in numerous enzymes. In particular, a Mn cluster is indispensable for the function of the oxygen evolving complex of photosystem II. Metal transporters of the natural resistance associated macrophage protein (NRAMP) family have the ability to transport both iron and manganese. AtNRAMP3 and AtNRAMP4 are required for iron mobilization in germinating seeds. The results reported here show that, in adult Arabidopsis thaliana plants, AtNRAMP3 and AtNRAMP4 have an important role in Mn homeostasis. Vacuolar Mn accumulation in mesophyll cells of rosette leaves of adult nramp3nramp4 double mutant plants was dramatically increased when compared to wild type. This suggests that a considerable proportion of the cellular Mn pool passes through the vacuole and is retrieved in an AtNRAMP3/AtNRAMP4-dependent manner. The impaired Mn release from mesophyll vacuoles of nramp3nramp4 double mutant plants is associated with reduced growth under Mn deficiency. However, leaf AtNRAMP3 and AtNRAMP4 protein levels are unaffected by Mn supply. Under Mn deficiency, namp3nramp4 plants contain less functional photosystem II than the wild type. These data are consistent with a shortage of Mn to produce functional photosystem II, whereas mitochondrial Mn dependent superoxide dismutase (SOD) activity is maintained under Mn deficiency in both genotypes. The results presented here suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.

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Regulated Ethylene Insensitivity Through the Inducible Expression of the Arabidopsis etr1-1 Mutant Ethylene Receptor in Tomato.

Publication Date: 2010 Feb 24 PMID: 20181754
Authors: Gallie, D. R.
Journal: Plant Physiol

Ethylene serves as an important hormone controlling several aspects of plant growth and development including fruit ripening and leaf and petal senescence. Ethylene is perceived following its binding to membrane-localized receptors, resulting in their inactivation and the induction of ethylene responses. Five distinct types of receptors are expressed in Arabidopsis and mutant receptors have been described which repress ethylene signaling in a dominant negative manner. One such mutant, i.e., etr1-1, results in a strong ethylene insensitive phenotype in Arabidopsis. In this study, regulated expression of the Arabidopsis etr1-1 in tomato (Solanum lycopersicum) was achieved using an inducible promoter. In the absence of the inducer, transgenic seedlings remained sensitive to ethylene but in its presence, a state of ethylene insensitivity was induced, resulting in the elongation of the hypocotyl and root in dark-grown seedlings in the presence of ethylene, a reduction or absence of an apical hook, and repression of ethylene-inducible E4 expression. The level of ethylene sensitivity could be controlled by the amount of inducer used, demonstrating a linear relationship between the degree of insensitivity and etr1-1 expression. Induction of etr1-1 expression also repressed the epinastic response to ethylene as well as delayed fruit ripening. Restoration of ethylene sensitivity was achieved following the cessation of the induction. These results demonstrate the ability to control ethylene responses temporally and in amount through the control of mutant receptor expression.

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Cloning of papaya chromoplast specific lycopene {beta}-cyclase, CpCYC-b, controlling fruit flesh color reveals conserved microsynteny and a recombination hotspot.

Publication Date: 2010 Feb 24 PMID: 20181753
Authors: Blas, A. L. - Ming, R. - Liu, Z. - Veatch, O. J. - Paull, R. E. - Moore, P. H. - Yu, Q.
Journal: Plant Physiol

Carotenoid pigments in fruits are indicative of the ripening process and potential nutritional value. Papaya fruit flesh color is caused by the accumulation of lycopene or beta-carotene in chromoplasts. It is a distinct feature affecting nutritional composition, fruit quality, shelf life, and consumer preference. To uncover the molecular basis of papaya flesh color, we took map-based cloning and candidate gene approaches using integrated genetic and physical maps. A DNA marker tightly linked to flesh color co-localized on a contig of the physical map with a cDNA probe of the tomato chromoplast-specific lycopene beta-cyclase, CYC-b. Candidate gene sequences were obtained from amplified fragments and verified by sequencing two bacteria artificial chromosomes (BACs) containing the two alleles. Sequence comparison revealed a 2 bp insertion in the coding region of the recessive red flesh allele resulting in a frame-shift mutation and a premature stop codon. A color complementation test in bacteria confirmed that the papaya chromoplast-specific lycopene beta-cyclase, CpCYC-b, is the gene controlling fruit flesh color. Sequence analysis of wild and cultivated papaya accessions showed the presence of this frame-shift mutation in all red flesh accessions examined. Evaluation of DNA markers near CpCYC-b revealed a recombination hotspot, showing that CpCYC-b is located in a gene rich region with a recombination rate at 3.7kb/cM, more than 100-fold higher than the genome average at 400kb/cM. Conserved microsynteny of the CpCYC-b region is indicated by colinearity of two to four genes between papaya, Arabidopsis, grape, and tomato. Our results enhanced our understanding of papaya flesh color inheritance and generated new tools for papaya improvement.

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Transcriptional Profiling of the Arabidopsis Iron Deficiency Response Reveals Conserved Transition Metal Homeostasis Networks.

Publication Date: 2010 Feb 24 PMID: 20181752
Authors: Yang, T. J. - Lin, W. D. - Schmidt, W.
Journal: Plant Physiol

Iron (Fe) deficiency is counteracted by a suite of responses to ensure maintenance of vital processes for which Fe is essential. Here we report on transcriptional changes upon Fe deficiency, investigated in two Arabidopsis (Arabidopsis thaliana) accessions, Col-0 and C24. Functional modules of the Arabidopsis Fe deficiency syndrome were inferred from clustering of Fe-responsive genes according to their co-expression. It was found that the redistribution of transition metals is an integral part of the reduction-based response to Fe starvation. The differential expression of metal transporters under the control of the transcription factor FIT appeared to reflect an anticipated reaction rather than a response to the actual change in metal distribution. In contrast, the regulation of the Zn transporters ZIP2, ZIP3, ZIP4 and ZIP9 was dependent on the cellular Zn level and their regulation by Fe was a secondary effect. Cellular Fe homeostasis was found to be closely coupled to Fe-related processes in the plastids. Using clustered genes as bait in gene fishing experiments, we were able to attribute potentially important roles for gene candidates that have no previously described function in the Fe deficiency response. The results demonstrate a conceptually novel and integrative view into the regulation and interactions that allows Arabidopsis to adapt to suboptimal Fe availability.

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Phosphate Regulation of Lipid Biosynthesis in Arabidopsis is Independent of the Mitochondrial Outer Membrane DGS1 Complex.

Publication Date: 2010 Feb 24 PMID: 20181751
Authors: Moellering, E. R. - Benning, C.
Journal: Plant Physiol

Galactoglycerolipids are major constituents of photosynthetic membranes in chloroplasts. At least three parallel sets of enzymes are involved in their biosynthesis that must be coordinated in response to changing growth conditions. A potential candidate for a protein affecting the activity of different galactoglycerolipid pathways is the recently described DGS1 (dgd1 SUPPRESSOR1) protein of Arabidopsis thaliana localized in the mitochondrial outer membrane. It was discovered based on a specific gain-of-function point mutation allele, dgs1-1, that causes a partial restoration of chloroplast galactoglycerolipid deficiency in the digalactosyldiacylglycerol1 (dgd1) mutant, which is defective in the lipid galactosyltransferase, DGD1. The dgs1-1 allele causes the accumulation of hydrogen peroxide which leads to an activation of an alternative, DGD1-independent galactoglycerolipid biosynthesis pathway in chloroplasts. Analysis presented here shows that the DGS1 protein is a component of a large protein complex, which explains the previously observed dominant negative phenotype following the expression of the dgs1-1 allele. The dgs1-1 allele causes the loss of mitochondrial alternative oxidase protein which might be related to the accumulation of hydrogen peroxide in the dgs1-1 mutant background. This effect was posttranscriptional because mRNA levels for the major form of alternative oxidase were not affected in dgs1-1 mutant seedlings. Unlike dgs1-1, a loss-of-function allele, dgs1-2, had no effect on plant growth, alternative oxidase, and lipid composition to the extent tested, leaving the quest for a possible molecular function of DGS1 open. Apparently, the DGS1 wild-type protein does not directly affect lipid metabolism in mitochondria or chloroplasts.

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AtNUDX6, an ADP-ribose/NADH pyrophosphohydrolase in Arabidopsis, positively regulates NPR1-dependent salicylic acid signaling.

Publication Date: 2010 Feb 24 PMID: 20181750
Authors: Ishikawa, K. - Yoshimura, K. - Harada, K. - Fukusaki, E. - Ogawa, T. - Tamoi, M. - Shigeoka, S.
Journal: Plant Physiol

Here, we investigated the physiological role of AtNUDX6, the gene encoding ADP-ribose/NADH pyrophosphohydrolase, using its overexpressor (Pro35S:AtNUDX6) or disruptant (KO-nudx6). The level of NADH in Pro35S:AtNUDX6 and KO-nudx6 plants was decreased and increased, respectively, compared with that of the control plants, while the level of ADP-ribose was not changed in either plant. The activity of pyrophosphohydrolase toward NADH was enhanced and reduced in the Pro35S:AtNUDX6 and KO-nudx6 plants, respectively. The decrease in the activity of NADH pyrophosphohydrolase and the increase in the level of NADH were observed in the rosette and cauline leaves, but not in the roots, of the KO-nudx6 plants. Notably, the expression level of AtNUDX6 and the activity of NADH pyrophosphohydrolase in the control plants, but not in the KO-nudx6 plants, were increased by the treatment with salicylic acid (SA). The expression of SA-induced genes (PR1, WRKY70, NIMIN1, and NIMIN2) depending on Nonexpresser of Pathogenesis-Related genes 1 (NPR1), a key component required for pathogen resistance, was significantly suppressed and enhanced in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively, under the treatment with SA. Induction of thioredoxin 5 (TRX-h5) expression, which catalyzes an SA-induced NPR1 activation, was suppressed and accelerated in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively. The expression of isochorismate synthase 1 required for regulation of SA synthesis through the NPR1-mediated feedback loop was decreased and increased in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively. Judging from seed germination rates, the KO-nudx6 plants had enhanced sensitivity to the toxicity of high level SA. The present results indicated that AtNUDX6 is a modulator of NADH rather than ADP-ribose metabolism, and that, through induction of TRX-h5 expression, AtNUDX6 significantly impacts the plant immune response as a positive regulator of NPR1-dependent SA signaling pathways.

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Tracing cadmium from culture to spikelet: non-invasive imaging and quantitative characterization of absorption, transport and accumulation of cadmium in an intact rice plant.

Publication Date: 2010 Feb 19 PMID: 20172965
Authors: Fujimaki, S. - Suzui, N. - Ishioka, N. S. - Kawachi, N. - Ito, S. - Chino, M. - Nakamura, S. I.
Journal: Plant Physiol

We characterized the absorption and short-term translocation of cadmium (Cd) in rice (Oryza sativa L. cv. Nipponbare) quantitatively using serial images observed with a positron-emitting tracer imaging system (PETIS). We fed a positron-emitting (107)Cd (half-life: 6.5 h) tracer to the hydroponic culture solution and non-invasively obtained serial images of Cd distribution in intact rice plants at a vegetative stage and at the grain-filling stage every four minutes for 36 h. The rates of absorption of Cd by the root were proportional to Cd concentrations in the culture solution within the tested range of 0.05 to 100 nM. It was estimated that the radial transport from the culture to the xylem in the root tissue was completed in less than 10 min. Cd moved up through the shoot organs with velocities of a few centimeters per hour at the both stages, which was obviously slower than the bulk flow in the xylem. Finally, Cd arrived at the panicles 7 h after feeding and accumulated there constantly, although no Cd was observed in the leaf blades within the initial 36 h. The nodes exhibited the most intensive Cd accumulation in the shoot at the both stages and Cd transport from the basal nodes to crown root tips was observed at the vegetative stage. We concluded that the nodes are the central organ where xylem-to-phloem transfer takes place and play a pivotal role in the half-day travel of Cd from the soil to the grains at the grain-filling stage.

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Nonhost resistance of barley to different fungal pathogens is associated with largely distinct, quantitative transcriptional responses.

Publication Date: 2010 Feb 19 PMID: 20172964
Authors: Zellerhoff, N. - Himmelbach, A. - Dong, W. - Bieri, S. - Schaffrath, U. - Schweizer, P.
Journal: Plant Physiol

Nonhost resistance protects plants against attack by the vast majority of potential pathogens including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare ssp. vulgare, cv "Ingrid") to three different pairs of adapted (host) and non-adapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust) and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost-interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wildtype- or the mutated mlo5-allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens similar patterns of over- and under-represented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely non-overlapping sets of pathogen-responsive genes involved in similar metabolic or signalling pathways.

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Mutations in the hyperosmotic stress responsive mitochondrial BASIC AMINO ACID CARRIER 2 enhance proline accumulation in Arabidopsis.

Publication Date: 2010 Feb 19 PMID: 20172963
Authors: Toka, I. - Planchais, S. - Cabassa, C. - Justin, A. M. - De Vos, D. - Richard, L. - Savoure, A. - Carol, P.
Journal: Plant Physiol

Mitochondrial carrier family proteins are diverse in their substrate specificity, organellar location and gene expression. In Arabidopsis thaliana, 58 genes encode these six-transmembrane-domain proteins. We investigated the biological role of the basic amino acid carrier BAC2 from Arabidopsis that is structurally and functionally similar to ARG11, a yeast ornithine and arginine carrier, and to Arabidopsis BAC1. By studying the expression of BAC2 and the consequences of its mutation in Arabidopsis, we showed that BAC2 is a genuine mitochondrial protein and that Arabidopsis requires expression of the BAC2 gene in order to use arginine. The BAC2 gene is induced by hyperosmotic stress (with either 0.2 M NaCl or 0.4 M mannitol) and dark-induced senescence. The BAC2 promoter contains numerous stress-related cis regulatory elements and the transcriptional activity of BAC2:GUS is upregulated by stress and senescence. Under hyperosmotic stress bac2 mutants express the P5CS1 proline biosynthetic gene more strongly than wild type and this correlates with a greater accumulation of proline. Our data suggest that BAC2 is a hyperosmotic-stress inducible transporter of basic amino acids that contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis.

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Identification of miRNAs involved in Pathogen-Associated Molecular Pattern-triggered plant innate immunity.

Publication Date: 2010 Feb 17 PMID: 20164210
Authors: Li, Y. - Zhang, Q. - Zhang, J. - Wu, L. - Qi, Y. - Zhou, J. M.
Journal: Plant Physiol

Pathogen-associated molecular patterns (PAMPs) trigger plant defenses when perceived by surface-localized immune receptors. PAMP-triggered immunity (PTI) plays a vital role in the resistance of plants to numerous potential pathogens. microRNA(miRNA) biogenesis is known to be important for PTI, but miRNA species involved in this process have not been fully explored. Here we show that the miRNA effector protein, Argonuate1 (AGO1), is required for a number of PTI responses including PAMP-induced callose deposition, gene expression and seedling growth inhibition. Deep-sequencing of AGO1-bound small RNAs led to the identification of a number of miRNAs that are up- or down-regulated by flg22, a well-studied PAMP. Overexpression of selected miRNAs in stable transgenic plants demonstrated that miR160a positively regulate PAMP-induced callose deposition, whereas miR398b and miR773 negatively regulate PAMP-induced callose deposition and disease resistance to bacteria, suggesting a complexity of the miRNA regulation in plant innate immunity.

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The lesion mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner.

Publication Date: 2010 Feb 17 PMID: 20164209
Authors: Mosher, S. - Moeder, W. - Nishimura, N. - Jikumaru, Y. - Joo, S. H. - Urquhart, W. - Klessig, D. F. - Kim, S. K. - Nambara, E. - Yoshioka, K.
Journal: Plant Physiol

A number of Arabidopsis thaliana lesion mimic mutants exhibits alterations in both abiotic stress responses as well as pathogen resistance. One of these mutants, cpr22, which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent (Yoshioka et al., 2001., Yoshioka et al., 2006). These defense responses in cpr22 are suppressed under high humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not wild type, showed a rapid increase in SA levels, followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were up-regulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, were down-regulated, but others, like ABI1 and HAB1 were up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA, not only in abiotic stress responses, but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.

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Annexins - Components of the calcium and reactive oxygen signaling network.

Publication Date: 2010 Feb 12 PMID: 20154100
Authors: Laohavisit, A. - Brown, A. T. - Cicuta, P. - Davies, J. M.
Journal: Plant Physiol

Despite the importance of reactive oxygen species (ROS) in plant immunity, stress signaling and development (Mori and Schroeder, 2004; Gadjev et al., 2006) the molecular identities of Ca(2+)-permeable channels responding to ROS have not been established. Here we propose annexins as candidate channel proteins. Elevation of cytoplasmic free calcium ([Ca(2+)]cyt) as a regulatory step in plant immunity, stress adaptation and development will rely on spatio-temporal control of Ca(2+) -permeable channel activity at endomembranes and plasma membrane (PM) (McAinsh and Pitman, 2009). Higher plant genomes encode multiple potential Ca(2+)-permeable channel sub-units that could contribute to cell- and stimulus-specific patterns of [Ca(2+)]cyt elevation (McAinsh and Pitttman, 2009; Ward et al., 2009). Numerous factors are involved in modulating channel events including voltage, membrane stretch, pH, phosphorylation, G proteins, amino acids, cyclic nucleotides and ROS (Ward et al., 2009). Identifying ROS-responsive Ca(2+)-permeable channels and their encoding genes will help elucidate ROS-Ca(2+) signaling networks. Annexins could be able to form an ROS-stimulated passive Ca(2+) transport pathway. Here we examine the evidence leading to this proposal and show that plant annexins are capable of forming a Ca(2+)-permeable conductance in an oxidised membrane simulating ROS signaling conditions.

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The catalytic and protein-protein interaction domains are required for APM1 function.

Publication Date: 2010 Feb 12 PMID: 20154099
Authors: Hosein, F. N. - Bandyopadhyay, A. - Ann Peer, W. - Murphy, A. S.
Journal: Plant Physiol

The M1 metallopeptidase APM1 is essential for embryonic, vegetative, and reproductive development in Arabidopsis. Here we show that, like mammalian M1 proteases, APM1 appears to have distinct enzymatic and protein-protein interaction domains and functions as a homodimer. Arabidopsis seedlings treated with ezetimibe, an inhibitor of M1 protein-protein interactions, mimicked a subset of apm1 phenotypes distinct from those resulting from treatment with PAQ-22, an inhibitor of M1 catalytic activity, suggesting that the APM1 catalytic and interaction domains can function independently. apm1-1 knockdown mutants transformed with catalytically inactive APM1 did not prevent seedling lethality. However, apm1-2 has a functional enzymatic domain but lacks the C-terminus, and transformation with catalytically inactive APM1 rescued the mutant. Overexpression of human IRAP rescued all apm1 phenotypes, suggesting that the catalytic activity was sufficient to compensate for loss of APM1 function, while overexpression of catalytically inactive IRAP only rescued apm1-2. Increased catalytic activity alone is not sufficient to compensate for loss-of-APM1-function, as overexpression of another Arabidopsis M1 family member lacking an extended C-terminus did not rescue apm1-1. The protein interactions facilitating enzymatic activity appear to be dependent on the C-terminus of APM1, as transformation with an open reading frame containing an internal deletion of a portion of the C-terminus or a point mutation in a dileucine motif did not rescue the mutant. These results suggest that both the catalytic and interaction domains are necessary for APM1 function, but that APM1 function and dimerization does not require these domains to be present in the same linear molecule.

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Phytochrome regulation of branching in Arabidopsis.

Publication Date: 2010 Feb 12 PMID: 20154098
Authors: Finlayson, S. A. - Krishnareddy, S. R. - Kebrom, T. H. - Casal, J. J.
Journal: Plant Physiol

The red to far-red ratio (R:FR) perceived by phytochromes controls plastic traits of plant architecture, including branching. Despite the significance of branching for plant fitness and productivity, there is little quantitative and mechanistic information concerning phytochrome control of branching responses in Arabidopsis thaliana. Here we show that in Arabidopsis, the negative effects of the phytochrome B (phyB) mutation and of low R:FR on branching were largely due to reduced bud outgrowth capacity and an increased degree of correlative inhibition acting on the buds rather than due to a reduced number of leaves and buds available for branching. Phytochrome effects on the degree of correlative inhibition required functional BRC1, BRC2, AXR1, MAX2 and MAX4. The analysis of gene expression in selected buds indicated that BRC1 and BRC2 are part of different gene networks. The BRC1 network is linked to the growth capacity of specific buds while the BRC2 network is associated with coordination of growth among branches. We conclude that the branching integrators BRC1 and BRC2 are necessary for responses to phytochrome, but they contribute differentially to these responses, likely acting through divergent pathways.

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Multiple Roles and Interaction Factors of an E-Box Element in Chlamydomonas reinhardtii.

Publication Date: 2010 Feb 12 PMID: 20154097
Authors: Seitz, S. B. - Weisheit, W. - Mittag, M.
Journal: Plant Physiol

The two subunits of the circadian RNA-binding protein CHLAMY1 from Chlamydomonas reinhardtii are involved in maintaining period (C1 subunit) and phase (C3 subunit) of the circadian clock. C1 co-regulates the level of C3. Overexpression of C1 causes a parallel increase in C3. Both subunits can also integrate temperature information, resulting in hyper-phosphorylation of C1 and up-regulation of C3 at low temperature. Temperature-dependent up-regulation of C3 is mediated predominantly by an E-box element and only partially by two DREB1A-boxes that are situated within the C3 promoter. The E-box element is also involved in circadian C3 expression. Here, we show that the C3 promoter region drives C3 co-regulation by C1. We also found that replacement of the E-box prevents the co-regulation of C3 in strains overexpressing C1. In contrast, replacement of any of the two DREB1A-boxes does not influence either the co-regulation of C3 by increased levels of C1 or circadian C3 expression. Thus, the E-box has multiple key roles including temperature-dependent up-regulation of C3, its circadian expression and its co-regulation by C1. Using mobility shift assays and DNA-affinity chromatography along with mass spectrometry, we characterized proteins binding specifically to the E-box region and identified five of them. By immunoblotting, we could further show that C3 itself that was detected in nuclear extracts can be found in the E-box binding protein complex. Our data indicate a complex transcriptional mechanism of C3 up-regulation and a positive feedback of C3 on its own promoter region.

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Metabolome and photochemical analysis of rice plants over-expressing Arabidopsis NAD kinase gene.

Publication Date: 2010 Feb 12 PMID: 20154096
Authors: Takahara, K. - Kasajima, I. - Takahashi, H. - Hashida, S. N. - Itami, T. - Onodera, H. - Toki, S. - Yanagisawa, S. - Kawai-Yamada, M. - Uchimiya, H.
Journal: Plant Physiol

The chloroplastic nicotinamide adenine dinucleotide kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis thaliana, which is vulnerable to high light. Since, rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we compared effects of the NADK2 expression in rice to that in Arabidopsis by developing transgenic rice plants that constitutively expressed Arabidopsis chloroplastic NAD kinase gene (NK2 lines). NK2 lines showed enhanced activity of NAD kinase and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO2 assimilation rates in NK2 line, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.

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Irritable walls: The plant extracellular matrix and signaling.

Publication Date: 2010 Feb 12 PMID: 20154095
Authors: Seifert, G. J. - Blaukopf, C.
Journal: Plant Physiol

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Depletion of the Membrane-Associated Acyl-Coenzyme A-Binding Protein ACBP1 Enhances the Ability of Cold Acclimation in Arabidopsis.

Publication Date: 2010 Mar PMID: 20107029
Authors: Du, Z. Y. - Xiao, S. - Chen, Q. F. - Chye, M. L.
Journal: Plant Physiol

In Arabidopsis (Arabidopsis thaliana), a family of six genes encodes acyl-coenzyme A-binding proteins (ACBPs). A member of this family, ACBP1, contains an amino-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum. To investigate ACBP1 function, ACBP1-overexpressing transgenic Arabidopsis plants were characterized using lipid analysis. ACBP1 overexpressors showed reduction in several species of diunsaturated phosphatidylcholine (PC), prompting us to investigate if they were altered in response to freezing stress. ACBP1 overexpressors demonstrated increased freezing sensitivity accompanied by a decrease in PC and an increase in phosphatidic acid (PA), while acbp1 mutant plants showed enhanced freezing tolerance associated with PC accumulation and PA reduction. We also showed binding of a recombinant eukaryotic ACBP (ACBP1) to PA, indicative of the possibility of enhanced PA interaction in ACBP1 overexpressors. Since phospholipase Dalpha1 (PLDalpha1) is a major enzyme promoting the hydrolysis of PC to PA, PLDalpha1 expression was examined and was observed to be higher in ACBP1 overexpressors than in acbp1 mutant plants. In contrast, the expression of PLDdelta, which plays a positive role in freezing tolerance, declined in the ACBP1 overexpressors but increased in acbp1 mutant plants. Given that ACBP1 is localized to the endoplasmic reticulum and plasma membrane, it may regulate the expression of PLDalpha1 and PLDdelta by maintaining a membrane-associated PA pool through its ability to bind PA. Moreover, both genotypes showed no alterations in proline and soluble sugar content or in cold-regulated (COR6.6 and COR47) gene expression, suggesting that the ACBP1-mediated response is PLD associated and is independent of osmolyte accumulation.

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Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy and Xylella fastidiosa-Infected Grapevines.

Publication Date: 2010 Mar PMID: 20107028
Authors: Perez-Donoso, A. G. - Sun, Q. - Roper, M. C. - Greve, L. C. - Kirkpatrick, B. - Labavitch, J. M.
Journal: Plant Physiol

The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- beta -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease.

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Actin reorganization underlies phototropin-dependent positioning of nuclei in Arabidopsis leaf cells.

Publication Date: 2010 Mar PMID: 20107027
Authors: Iwabuchi, K. - Minamino, R. - Takagi, S.
Journal: Plant Physiol

In epidermal and mesophyll cells of Arabidopsis (Arabidopsis thaliana) leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue light receptor phototropin2 in mesophyll cells. Here, we investigate the involvement of phototropin and the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness, whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in Arabidopsis, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.

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Characterization of drr1, an Alkamide-Resistant Mutant of Arabidopsis, Reveals an Important Role for Small Lipid Amides in Lateral Root Development and Plant Senescence.

Publication Date: 2010 Mar PMID: 20107026
Authors: Morquecho-Contreras, A. - Mendez-Bravo, A. - Pelagio-Flores, R. - Raya-Gonzalez, J. - Ortiz-Castro, R. - Lopez-Bucio, J.
Journal: Plant Physiol

Alkamides belong to a class of small lipid signals of wide distribution in plants, which are structurally related to the bacterial quorum-sensing signals N-acyl-l-homoserine lactones. Arabidopsis (Arabidopsis thaliana) seedlings display a number of root developmental responses to alkamides, including primary root growth inhibition and greater formation of lateral roots. To gain insight into the regulatory mechanisms by which these compounds alter plant development, we performed a mutant screen for identifying Arabidopsis mutants that fail to inhibit primary root growth when grown under a high concentration of N-isobutyl decanamide. A recessive N-isobutyl decanamide-resistant mutant (decanamide resistant root [drr1]) was isolated because of its continued primary root growth and reduced lateral root formation in response to this alkamide. Detailed characterization of lateral root primordia development in the wild type and drr1 mutants revealed that DRR1 is required at an early stage of pericycle cell activation to form lateral root primordia in response to both N-isobutyl decanamide and N-decanoyl-l-homoserine lactone, a highly active bacterial quorum-sensing signal. Exogenously supplied auxin similarly inhibited primary root growth and promoted lateral root formation in wild-type and drr1 seedlings, suggesting that alkamides and auxin act by different mechanisms to alter root system architecture. When grown both in vitro and in soil, drr1 mutants showed dramatically increased longevity and reduced hormone- and age-dependent senescence, which were related to reduced lateral root formation when exposed to stimulatory concentrations of jasmonic acid. Taken together, our results provide genetic evidence indicating that alkamides and N-acyl-l-homoserine lactones can be perceived by plants to modulate root architecture and senescence-related processes possibly by interacting with jasmonic acid signaling.

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Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems.

Publication Date: 2010 Mar PMID: 20107024
Authors: Iyer-Pascuzzi, A. S. - Symonova, O. - Mileyko, Y. - Hao, Y. - Belcher, H. - Harer, J. - Weitz, J. S. - Benfey, P. N.
Journal: Plant Physiol

The ability to nondestructively image and automatically phenotype complex root systems, like those of rice (Oryza sativa), is fundamental to identifying genes underlying root system architecture (RSA). Although root systems are central to plant fitness, identifying genes responsible for RSA remains an underexplored opportunity for crop improvement. Here we describe a nondestructive imaging and analysis system for automated phenotyping and trait ranking of RSA. Using this system, we image rice roots from 12 genotypes. We automatically estimate RSA traits previously identified as important to plant function. In addition, we expand the suite of features examined for RSA to include traits that more comprehensively describe monocot RSA but that are difficult to measure with traditional methods. Using 16 automatically acquired phenotypic traits for 2,297 images from 118 individuals, we observe (1) wide variation in phenotypes among the genotypes surveyed; and (2) greater intergenotype variance of RSA features than variance within a genotype. RSA trait values are integrated into a computational pipeline that utilizes supervised learning methods to determine which traits best separate two genotypes, and then ranks the traits according to their contribution to each pairwise comparison. This trait-ranking step identifies candidate traits for subsequent quantitative trait loci analysis and demonstrates that depth and average radius are key contributors to differences in rice RSA within our set of genotypes. Our results suggest a strong genetic component underlying rice RSA. This work enables the automatic phenotyping of RSA of individuals within mapping populations, providing an integrative framework for quantitative trait loci analysis of RSA.

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Analysis of Arabidopsis with highly reduced levels of malate and fumarate sheds light on the role of these organic acids as storage carbon molecules.

Publication Date: 2010 Mar PMID: 20107023
Authors: Zell, M. B. - Fahnenstich, H. - Maier, A. - Saigo, M. - Voznesenskaya, E. V. - Edwards, G. E. - Andreo, C. - Schleifenbaum, F. - Zell, C. - Drincovich, M. F. - Maurino, V. G.
Journal: Plant Physiol

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C(3) plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C(3) plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C(4) acids, which may be a constitutive function in plants.

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The Submergence Tolerance Regulator Sub1A Mediates Stress-Responsive Expression of AP2/ERF Transcription Factors.

Publication Date: 2010 Mar PMID: 20107022
Authors: Jung, K. H. - Seo, Y. S. - Walia, H. - Cao, P. - Fukao, T. - Canlas, P. E. - Amonpant, F. - Bailey-Serres, J. - Ronald, P. C.
Journal: Plant Physiol

We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

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MCA1 and MCA2 That Mediate Ca2+ Uptake Have Distinct and Overlapping Roles in Arabidopsis.

Publication Date: 2010 Mar PMID: 20097794
Authors: Yamanaka, T. - Nakagawa, Y. - Mori, K. - Nakano, M. - Imamura, T. - Kataoka, H. - Terashima, A. - Iida, K. - Kojima, I. - Katagiri, T. - Shinozaki, K. - Iida, H.
Journal: Plant Physiol

Ca(2+) is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca(2+)-permeable channels or transporters involved in Ca(2+) uptake in roots are largely unknown. We recently identified a candidate for the Ca(2+)-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1. Here, we investigated the only paralog of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca(2+) uptake deficiency in yeast cells lacking a Ca(2+) channel composed of Mid1 and Cch1. Reverse transcription polymerase chain reaction analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques, and stems, and histochemical observation showed that an MCA2 promoter::GUS fusion reporter gene was universally expressed in 10-d-old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase agar method. Ca(2+) uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg(2+) added to medium compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca(2+) uptake in roots and an overlapping role with MCA1 in plant growth.

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Photosynthetic Performance and Fertility Are Repressed in GmAOX2b Antisense Soybean.

Publication Date: 2010 Mar PMID: 20097793
Authors: Chai, T. T. - Simmonds, D. - Day, D. A. - Colmer, T. D. - Finnegan, P. M.
Journal: Plant Physiol

The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function.

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Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis.

Publication Date: 2010 Mar PMID: 20097792
Authors: Kagale, S. - Links, M. G. - Rozwadowski, K.
Journal: Plant Physiol

The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

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Cross-kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses.

Publication Date: 2010 Mar PMID: 20097791
Authors: Mustroph, A. - Lee, S. C. - Oosumi, T. - Zanetti, M. E. - Yang, H. - Ma, K. - Yaghoubi-Masihi, A. - Fukao, T. - Bailey-Serres, J.
Journal: Plant Physiol

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.

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The significance of protein maturation by plastidic type I signal peptidase 1 for thylakoid development in Arabidopsis chloroplasts.

Publication Date: 2010 Mar PMID: 20097790
Authors: Shipman-Roston, R. L. - Ruppel, N. J. - Damoc, C. - Phinney, B. S. - Inoue, K.
Journal: Plant Physiol

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids.

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Evolutionarily Conserved Regulatory Mechanisms of Abscisic Acid Signaling in Land Plants: Characterization of ABSCISIC ACID INSENSITIVE1-Like Type 2C Protein Phosphatase in the Liverwort Marchantia polymorpha.

Publication Date: 2010 Mar PMID: 20097789
Authors: Tougane, K. - Komatsu, K. - Bhyan, S. B. - Sakata, Y. - Ishizaki, K. - Yamato, K. T. - Kohchi, T. - Takezawa, D.
Journal: Plant Physiol

Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the amino-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the beta -glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

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The Heat-Inducible Transcription Factor HsfA2 Enhances Anoxia Tolerance in Arabidopsis.

Publication Date: 2010 Mar PMID: 20089772
Authors: Banti, V. - Mafessoni, F. - Loreti, E. - Alpi, A. - Perata, P.
Journal: Plant Physiol

Anoxia induces several heat shock proteins, and a mild heat pretreatment can acclimatize Arabidopsis (Arabidopsis thaliana) seedlings to subsequent anoxic treatment. In this study, we analyzed the response of Arabidopsis seedlings to anoxia, heat, and combined heat + anoxia stress. A significant overlap between the anoxic and the heat responses was observed by whole-genome microarray analysis. Among the transcription factors induced by both heat and anoxia, the heat shock factor A2 (HsfA2), known to be involved in Arabidopsis acclimation to heat and to other abiotic stresses, was strongly induced by anoxia. Heat-dependent acclimation to anoxia is lost in an HsfA2 knockout mutant (hsfa2) as well as in a double mutant for the constitutively expressed HsfA1a/HsfA1b (hsfA1a/1b), indicating that these three heat shock factors cooperate to confer anoxia tolerance. Arabidopsis seedlings that overexpress HsfA2 showed an increased expression of several known targets of this transcription factor and were markedly more tolerant to anoxia as well as to submergence. Anoxia failed to induce HsfA2 target proteins in wild-type seedlings, while overexpression of HsfA2 resulted in the production of HsfA2 targets under anoxia, correlating well with the low anoxia tolerance experiments. These results indicate that there is a considerable overlap between the molecular mechanisms of heat and anoxia tolerance and that HsfA2 is a player in these mechanisms.

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The Stromal Chloroplast Deg7 Protease Participates in the Repair of Photosystem II after Photoinhibition in Arabidopsis.

Publication Date: 2010 Mar PMID: 20089771
Authors: Sun, X. - Fu, T. - Chen, N. - Guo, J. - Ma, J. - Zou, M. - Lu, C. - Zhang, L.
Journal: Plant Physiol

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).

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R2R3-NaMYB8 Regulates the Accumulation of Phenylpropanoid-Polyamine Conjugates, Which Are Essential for Local and Systemic Defense against Insect Herbivores in Nicotiana attenuata.

Publication Date: 2010 Mar PMID: 20089770
Authors: Kaur, H. - Heinzel, N. - Schottner, M. - Baldwin, I. T. - Galis, I.
Journal: Plant Physiol

Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine, increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY-2 cells regulates PPC biosynthesis, and silenced its expression by RNA interference in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPC biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and dicaffeoylspermidine showed no changes in reproductive parameters of the plants. In contrast, a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically preinduced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared with wild-type plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatiotemporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the optimal defense theory: plants preferentially protect their most fitness-enhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness.

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Glycolysis and the Tricarboxylic Acid Cycle Are Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of Lotus japonicus.

Publication Date: 2010 Mar PMID: 20089769
Authors: Rocha, M. - Licausi, F. - Araujo, W. L. - Nunes-Nesi, A. - Sodek, L. - Fernie, A. R. - van Dongen, J. T.
Journal: Plant Physiol

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.

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Down-Regulation of the CSLF6 Gene Results in Decreased (1,3;1,4)-{beta}-D-Glucan in Endosperm of Wheat.

Publication Date: 2010 Mar PMID: 20089768
Authors: Nemeth, C. - Freeman, J. - Jones, H. D. - Sparks, C. - Pellny, T. K. - Wilkinson, M. D. - Dunwell, J. - Andersson, A. A. - Aman, P. - Guillon, F. - Saulnier, L. - Mitchell, R. A. - Shewry, P. R.
Journal: Plant Physiol

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

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Regulation of plant glycine decarboxylase by s-nitrosylation and glutathionylation.

Publication Date: 2010 Mar PMID: 20089767
Authors: Palmieri, M. C. - Lindermayr, C. - Bauwe, H. - Steinhauser, C. - Durner, J.
Journal: Plant Physiol

Mitochondria play an essential role in nitric oxide (NO) signal transduction in plants. Using the biotin-switch method in conjunction with nano-liquid chromatography and mass spectrometry, we identified 11 candidate proteins that were S-nitrosylated and/or glutathionylated in mitochondria of Arabidopsis (Arabidopsis thaliana) leaves. These included glycine decarboxylase complex (GDC), a key enzyme of the photorespiratory C(2) cycle in C3 plants. GDC activity was inhibited by S-nitrosoglutathione due to S-nitrosylation/S-glutathionylation of several cysteine residues. Gas-exchange measurements demonstrated that the bacterial elicitor harpin, a strong inducer of reactive oxygen species and NO, inhibits GDC activity. Furthermore, an inhibitor of GDC, aminoacetonitrile, was able to mimic mitochondrial depolarization, hydrogen peroxide production, and cell death in response to stress or harpin treatment of cultured Arabidopsis cells. These findings indicate that the mitochondrial photorespiratory system is involved in the regulation of NO signal transduction in Arabidopsis.

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Reconstruction of Metabolic Pathways, Protein Expression, and Homeostasis Machineries across Maize Bundle Sheath and Mesophyll Chloroplasts: Large-Scale Quantitative Proteomics Using the First Maize Genome Assembly.

Publication Date: 2010 Mar PMID: 20089766
Authors: Friso, G. - Majeran, W. - Huang, M. - Sun, Q. - van Wijk, K. J.
Journal: Plant Physiol

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C(4) photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.

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Characterization of the Rice PHO1 Gene Family Reveals a Key Role for OsPHO1;2 in Phosphate Homeostasis and the Evolution of a Distinct Clade in Dicotyledons.

Publication Date: 2010 Mar PMID: 20081045
Authors: Secco, D. - Baumann, A. - Poirier, Y.
Journal: Plant Physiol

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

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Comparison of Developmental and Stress-Induced Nodule Senescence in Medicago truncatula.

Publication Date: 2010 Mar PMID: 20081044
Authors: Perez Guerra, J. C. - Coussens, G. - De Keyser, A. - De Rycke, R. - De Bodt, S. - Van De Velde, W. - Goormachtig, S. - Holsters, M.
Journal: Plant Physiol

Mature indeterminate Medicago truncatula nodules are zonated with an apical meristem, an infection zone, a fixation zone with nitrogen-fixing bacteroids, and a "developmental" senescence zone that follows nodule growth with a conical front originating in the center of the fixation zone. In nitrogen-fixing cells, senescence is initiated coincidently with the expression of a family of conserved cysteine proteases that might be involved in the degradation of symbiotic structures. Environmental stress, such as prolonged dark treatment, interferes with nodule functioning and triggers a fast and global nodule senescence. Developmental and dark stress-induced senescence have several different structural and expression features, suggesting at least partly divergent underlying molecular mechanisms.

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Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in papaver.

Publication Date: 2010 Mar PMID: 20081043
Authors: Poulter, N. S. - Staiger, C. J. - Rappoport, J. Z. - Franklin-Tong, V. E.
Journal: Plant Physiol

The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

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Arabidopsis auxin mutants are compromised in systemic acquired resistance and exhibit aberrant accumulation of various indolic compounds.

Publication Date: 2010 Mar PMID: 20081042
Authors: Truman, W. M. - Bennett, M. H. - Turnbull, C. G. - Grant, M. R.
Journal: Plant Physiol

Systemic acquired resistance is a widespread phenomenon in the plant kingdom that confers heightened and often enduring immunity to a range of diverse pathogens. Systemic immunity develops through activation of plant disease resistance protein signaling networks following local infection with an incompatible pathogen. The accumulation of the phytohormone salicylic acid in systemically responding tissues occurs within days after a local immunizing infection and is essential for systemic resistance. However, our knowledge of the signaling components underpinning signal perception and the establishment of systemic immunity are rudimentary. Previously, we showed that an early and transient increase in jasmonic acid in distal responding tissues was central to effective establishment of systemic immunity. Based upon predicted transcriptional networks induced in naive Arabidopsis (Arabidopsis thaliana) leaves following avirulent Pseudomonas syringae challenge, we show that a variety of auxin mutants compromise the establishment of systemic immunity. Linking together transcriptional and targeted metabolite studies, our data provide compelling evidence for a role of indole-derived compounds, but not auxin itself, in the establishment and maintenance of systemic immunity.

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Maturation stress generation in poplar tension wood studied by synchrotron radiation microdiffraction.

Publication Date: 2010 Mar PMID: 20071605
Authors: Clair, B. - Almeras, T. - Pilate, G. - Jullien, D. - Sugiyama, J. - Riekel, C.
Journal: Plant Physiol

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress, called maturation stress. Maturation stress performs essential biomechanical functions such as optimizing the mechanical resistance of the stem, performing adaptive movements, and ensuring long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides x Populus trichocarpa 'I45-51'). Synchrotron radiation microdiffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated with the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 and the outer part of the S2 layer. The microfibril angle in the S2 layer was found to be lower in its inner part than in its outer part, especially in tension wood. In tension wood only, this decrease occurred together with an increase in cellulose lattice spacing, and this happened before the G-layer was visible. The relative increase in lattice spacing was found close to the usual value of maturation strains, strongly suggesting that microfibrils of this layer are put into tension and contribute to the generation of maturation stress.

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Rapid auxin-induced cell expansion and gene expression: a four-decade-old question revisited.

Publication Date: 2010 Mar PMID: 20071604
Authors: Schenck, D. - Christian, M. - Jones, A. - Luthen, H.
Journal: Plant Physiol

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The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning.

Publication Date: 2010 Mar PMID: 20071603
Authors: de Carbonnel, M. - Davis, P. - Roelfsema, M. R. - Inoue, S. - Schepens, I. - Lariguet, P. - Geisler, M. - Shimazaki, K. - Hangarter, R. - Fankhauser, C.
Journal: Plant Physiol

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

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Combined Bimolecular Fluorescence Complementation and Forster Resonance Energy Transfer Reveals Ternary SNARE Complex Formation in Living Plant Cells.

Publication Date: 2010 Mar PMID: 20071602
Authors: Kwaaitaal, M. - Keinath, N. F. - Pajonk, S. - Biskup, C. - Panstruga, R.
Journal: Plant Physiol

Various fluorophore-based microscopic methods, comprising Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.

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Fatty Acid activation in cyanobacteria mediated by acyl-acyl carrier protein synthetase enables Fatty Acid recycling.

Publication Date: 2010 Mar PMID: 20061450
Authors: Kaczmarzyk, D. - Fulda, M.
Journal: Plant Physiol

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.

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Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm.

Publication Date: 2010 Mar PMID: 20061449
Authors: Jerkovic, A. - Kriegel, A. M. - Bradner, J. R. - Atwell, B. J. - Roberts, T. H. - Willows, R. D.
Journal: Plant Physiol

Bran from bread wheat (Triticum aestivum 'Babbler') grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.

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CORNET: A User-Friendly Tool for Data Mining and Integration.

Publication Date: 2010 Mar PMID: 20053712
Authors: De Bodt, S. - Carvajal, D. - Hollunder, J. - Van den Cruyce, J. - Movahedi, S. - Inze, D.
Journal: Plant Physiol

As an overwhelming amount of functional genomics data have been generated, the retrieval, integration, and interpretation of these data need to be facilitated to enable the advance of (systems) biological research. For example, gathering and processing microarray data that are related to a particular biological process is not straightforward, nor is the compilation of protein-protein interactions from numerous partially overlapping databases identified through diverse approaches. However, these tasks are inevitable to address the following questions. Does a group of differentially expressed genes show similar expression in diverse microarray experiments? Was an identified protein-protein interaction previously detected by other approaches? Are the interacting proteins encoded by genes with similar expression profiles and localization? We developed CORNET (for CORrelation NETworks) as an access point to transcriptome, protein interactome, and localization data and functional information on Arabidopsis (Arabidopsis thaliana). It consists of two flexible and versatile tools, namely the coexpression tool and the protein-protein interaction tool. The ability to browse and search microarray experiments using ontology terms and the incorporation of personal microarray data are distinctive features of the microarray repository. The coexpression tool enables either the alternate or simultaneous use of diverse expression compendia, whereas the protein-protein interaction tool searches experimentally and computationally identified protein-protein interactions. Different search options are implemented to enable the construction of coexpression and/or protein-protein interaction networks centered around multiple input genes or proteins. Moreover, networks and associated evidence are visualized in Cytoscape. Localization is visualized in pie charts, thereby allowing multiple localizations per protein. CORNET is available at http://bioinformatics.psb.ugent.be/cornet.

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Fluorescence resonance energy transfer-sensitized emission of yellow cameleon 3.60 reveals root zone-specific calcium signatures in Arabidopsis in response to aluminum and other trivalent cations.

Publication Date: 2010 Mar PMID: 20053711
Authors: Rincon-Zachary, M. - Teaster, N. D. - Sparks, J. A. - Valster, A. H. - Motes, C. M. - Blancaflor, E. B.
Journal: Plant Physiol

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca(2+)](cyt)) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca(2+)](cyt) were observed in response to glutamic acid (Glu), ATP, and aluminum (Al(3+)). Each chemical induced a [Ca(2+)](cyt) signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca(2+)](cyt) increases that were similar among the different root zones, whereas Al(3+) evoked [Ca(2+)](cyt) transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al(3+)-induced [Ca(2+)](cyt) increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca(2+)](cyt) response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca(2+)](cyt) signature resulting from Al(3+) treatment originated mostly from cortical cells located at 300 to 500 mu m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca(2+) channel blockers, elicited [Ca(2+)](cyt) responses similar to those induced by Al(3+). The trivalent ion-induced [Ca(2+)](cyt) signatures in roots of an Al(3+)-resistant and an Al(3+)-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca(2+)](cyt) changes we report here may not be tightly linked to Al(3+) toxicity but rather to a general response to trivalent cations.

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Chloroplastic phosphoadenosine phosphosulfate metabolism regulates Basal levels of the prohormone jasmonic Acid in Arabidopsis leaves.

Publication Date: 2010 Mar PMID: 20053710
Authors: Rodriguez, V. M. - Chetelat, A. - Majcherczyk, P. - Farmer, E. E.
Journal: Plant Physiol

Levels of the enzymes that produce wound response mediators have to be controlled tightly in unwounded tissues. The Arabidopsis (Arabidopsis thaliana) fatty acid oxygenation up-regulated8 (fou8) mutant catalyzes high rates of alpha -linolenic acid oxygenation and has higher than wild-type levels of the alpha -linolenic acid-derived wound response mediator jasmonic acid (JA) in undamaged leaves. fou8 produces a null allele in the gene SAL1 (also known as FIERY1 or FRY1). Overexpression of the wild-type gene product had the opposite effect of the null allele, suggesting a regulatory role of SAL1 acting in JA synthesis. The biochemical phenotypes in fou8 were complemented when the yeast (Saccharomyces cerevisiae) sulfur metabolism 3'(2'), 5'-bisphosphate nucleotidase MET22 was targeted to chloroplasts in fou8. The data are consistent with a role of SAL1 in the chloroplast-localized dephosphorylation of 3'-phospho-5'-adenosine phosphosulfate to 5'-adenosine phosphosulfate or in a closely related reaction (e.g. 3',5'-bisphosphate dephosphorylation). Furthermore, the fou8 phenotype was genetically suppressed in a triple mutant (fou8 apk1 apk2) affecting chloroplastic 3'-phospho-5'-adenosine phosphosulfate synthesis. These results show that a nucleotide component of the sulfur futile cycle regulates early steps of JA production and basal JA levels.

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Channel-Like Characteristics of the Low-Affinity Barley Phosphate Transporter PHT1;6 When Expressed in Xenopus Oocytes.

Publication Date: 2010 Mar PMID: 20053709
Authors: Preuss, C. P. - Huang, C. Y. - Gilliham, M. - Tyerman, S. D.
Journal: Plant Physiol

Remobilization of inorganic phosphate (P(i)) within a plant is critical for sustaining growth and seed production under external P(i) fluctuation. The barley (Hordeum vulgare) transporter HvPHT1;6 has been implicated in P(i) remobilization. In this report, we expressed HvPHT1;6 in Xenopus laevis oocytes, allowing detailed characterization of voltage-dependent fluxes and currents induced by HvPHT1;6. HvPHT1;6 increased efflux of P(i) near oocyte resting membrane potentials, dependent on external P(i) concentration. Time-dependent inward currents were observed when membrane potentials were more negative than -160 mV, which was consistent with nH(+):HPO(4)(2-) (n > 2) cotransport, based on simultaneous radiotracer and oocyte voltage clamping, dependent upon P(i) concentration gradient and pH. Time- and voltage-dependent inward currents through HvPHT1;6 were also observed for SO(4)(2-)and to a lesser degree for NO(3)(-)Cl(-)but not for malate. Inward and outward currents showed linear dependence on the concentration of external HPO(4)(2-)similar to low-affinity P(i) transport in plant studies. The electrophysiological properties of HvPHT1;6, which locates to the plasma membrane when expressed in onion (Allium cepa) epidermal cells, are consistent with its suggested role in the remobilization of P(i) in barley plants.

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The RON1/FRY1/SAL1 Gene Is Required for Leaf Morphogenesis and Venation Patterning in Arabidopsis.

Publication Date: 2010 Mar PMID: 20044451
Authors: Robles, P. - Fleury, D. - Candela, H. - Cnops, G. - Alonso-Peral, M. M. - Anami, S. - Falcone, A. - Caldana, C. - Willmitzer, L. - Ponce, M. R. - Van Lijsebettens, M. - Micol, J. L.
Journal: Plant Physiol

To identify genes involved in vascular patterning in Arabidopsis (Arabidopsis thaliana), we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The rotunda1-1 (ron1-1) mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3' (2'),5'-bisphosphate nucleotidase activities and has not, to our knowledge, previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in the ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning and found that ron1 synergistically interacts with auxin resistant1 and hemivenata-1 but not with cotyledon vascular pattern1 (cvp1) and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myoinositol and indole-3-acetonitrile, a precursor of auxin.

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WOX4 Promotes Procambial Development.

Publication Date: 2010 Mar PMID: 20044450
Authors: Ji, J. - Strable, J. - Shimizu, R. - Koenig, D. - Sinha, N. - Scanlon, M. J.
Journal: Plant Physiol

Plant shoot organs arise from initial cells that are recruited from meristematic tissues. Previous studies have shown that members of the WUSCHEL-related HOMEOBOX (WOX) gene family function to organize various initial cell populations during plant development. The function of the WOX4 gene is previously undescribed in any plant species. Comparative analyses of WOX4 transcription and function are presented in Arabidopsis (Arabidopsis thaliana), a simple-leafed plant with collateral vasculature, and in tomato (Solanum lycopersicum), a dissected-leafed species with bicollateral venation. WOX4 is transcribed in the developing vascular bundles of root and shoot lateral organs in both Arabidopsis and tomato. RNA interference-induced down-regulation of WOX4 in Arabidopsis generated small plants whose vascular bundles accumulated undifferentiated ground tissue and exhibited severe reductions in differentiated xylem and phloem. In situ hybridization analyses of Atwox4-RNA interference plants revealed delayed and reduced expression of both the phloem developmental marker ALTERED PHLOEM1 and HOMEOBOX GENE8, a marker of the vascular procambium. Overexpression of SlWOX4 correlated with overproliferation of xylem and phloem in transgenic tomato seedlings. The cumulative data suggest that the conserved WOX4 function is to promote differentiation and/or maintenance of the vascular procambium, the initial cells of the developing vasculature.

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ZmPIN1-Mediated Auxin Transport Is Related to Cellular Differentiation during Maize Embryogenesis and Endosperm Development.

Publication Date: 2010 Mar PMID: 20044449
Authors: Forestan, C. - Meda, S. - Varotto, S.
Journal: Plant Physiol

To study the influence of PINFORMED1 (PIN1)-mediated auxin transport during embryogenesis and endosperm development in monocots, the expression pattern of the three identified ZmPIN1 genes was determined at the transcript level. Localization of the corresponding proteins was also analyzed during maize (Zea mays) kernel development. An anti-indole-3-acetic acid (IAA) monoclonal antibody was used to visualize IAA distribution and correlate the direction of auxin active transport, mediated by ZmPIN1 proteins, with the actual amount of auxin present in maize kernels at different developmental stages. ZmPIN1 genes are expressed in the endosperm soon after double fertilization occurs; however, unlike other tissues, the ZmPIN1 proteins were never polarly localized in the plasma membrane of endosperm cells. ZmPIN1 transcripts and proteins also colocalize in developing embryos, and the ZmPIN1 proteins are polarly localized in the embryo cell plasma membrane from the first developmental stages, indicating the existence of ZmPIN1-mediated auxin fluxes. Auxin distribution visualization indicates that the aleurone, the basal endosperm transfer layer, and the embryo-surrounding region accumulate free auxin, which also has a maximum in the kernel maternal chalaza. During embryogenesis, polar auxin transport always correlates with the differentiation of embryo tissues and the definition of the embryo organs. On the basis of these reports and of the observations on tissue differentiation and IAA distribution in defective endosperm-B18 mutant and in N-1-naphthylphthalamic acid-treated kernels, a model for ZmPIN1-mediated transport of auxin and the related auxin fluxes during maize kernel development is proposed. Common features between this model and the model previously proposed for Arabidopsis (Arabidopsis thaliana) are discussed.

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Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms.

Publication Date: 2010 Mar PMID: 20044448
Authors: Keeling, C. I. - Dullat, H. K. - Yuen, M. - Ralph, S. G. - Jancsik, S. - Bohlmann, J.
Journal: Plant Physiol

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

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A PIP1 Aquaporin Contributes to Hydrostatic Pressure-Induced Water Transport in Both the Root and Rosette of Arabidopsis.

Publication Date: 2010 Mar PMID: 20034965
Authors: Postaire, O. - Tournaire-Roux, C. - Grondin, A. - Boursiac, Y. - Morillon, R. - Schaffner, A. R. - Maurel, C.
Journal: Plant Physiol

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (K(ros)) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased K(ros) by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of K(ros) by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11-18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of K(ros) and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

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Low Glucose Uncouples Hexokinase1-Dependent Sugar Signaling from Stress and Defense Hormone Abscisic Acid and C2H4 Responses in Arabidopsis.

Publication Date: 2010 Mar PMID: 20034964
Authors: Cho, Y. H. - Sheen, J. - Yoo, S. D.
Journal: Plant Physiol

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Kinetic and spectral resolution of multiple nonphotochemical quenching components in Arabidopsis leaves.

Publication Date: 2010 Mar PMID: 20032080
Authors: Lambrev, P. H. - Nilkens, M. - Miloslavina, Y. - Jahns, P. - Holzwarth, A. R.
Journal: Plant Physiol

Using novel specially designed instrumentation, fluorescence emission spectra were recorded from Arabidopsis (Arabidopsis thaliana) leaves during the induction period of dark to high-light adaptation in order to follow the spectral changes associated with the formation of nonphotochemical quenching. In addition to an overall decrease of photosystem II fluorescence (quenching) across the entire spectrum, high light induced two specific relative changes in the spectra: (1) a decrease of the main emission band at 682 nm relative to the far-red (750-760 nm) part of the spectrum (Delta F(682)); and (2) an increase at 720 to 730 nm (Delta F(720)) relative to 750 to 760 nm. The kinetics of the two relative spectral changes and their dependence on various mutants revealed that they do not originate from the same process but rather from at least two independent processes. The Delta F(720) change is specifically associated with the rapidly reversible energy-dependent quenching. Comparison of the wild-type Arabidopsis with mutants unable to produce or overexpressing the PsbS subunit of photosystem II showed that PsbS was a necessary component for Delta F(720). The spectral change Delta F(682) is induced both by energy-dependent quenching and by PsbS-independent mechanism(s). A third novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high turnover rate of photosystem II. Its induction and relaxation occur on a time scale of a few minutes. Analysis of the spectral inhomogeneity of nonphotochemical quenching allows extraction of mechanistically valuable information from the fluorescence induction kinetics when registered in a spectrally resolved fashion.

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Expression quantitative trait Loci analysis of two genes encoding rubisco activase in soybean.

Publication Date: 2010 Mar PMID: 20032079
Authors: Yin, Z. - Meng, F. - Song, H. - Wang, X. - Xu, X. - Yu, D.
Journal: Plant Physiol

Rubisco activase (RCA) catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis. However, until now, little was known about the molecular genetics of RCA in soybean (Glycine max), one of the most important legume crops. Here, we cloned and characterized two genes encoding the longer alpha -isoform and the shorter beta -isoform of soybean RCA (GmRCA alpha and GmRCA beta, respectively). The two corresponding cDNAs are divergent in both the translated and 3 ' untranslated regions. Analysis of genomic DNA sequences suggested that the corresponding mRNAs are transcripts of two different genes and not the products of a single alternatively splicing pre-mRNA. Two additional possible alpha -form RCA-encoding genes, GmRCA03 and GmRCA14, and one additional beta -form RCA-encoding gene, GmRCA11, were also isolated. To examine the function and modulation of RCA genes in soybean, we determined the expression levels of GmRCA alpha and GmRCA beta, Rubisco initial activity, photosynthetic rate, and seed yield in 184 soybean recombinant inbred lines. Correlation of gene expression levels with three other traits indicates that RCA genes could play an important role in regulating soybean photosynthetic capacity and seed yield. Expression quantitative trait loci mapping revealed four trans-expression quantitative trait loci for GmRCA alpha and GmRCA beta. These results could provide a new approach for the modulation of RCA genes to improve photosynthetic rate and plant growth in soybean and other plants.

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The Arabidopsis BET Bromodomain Factor GTE4 Is Involved in Maintenance of the Mitotic Cell Cycle during Plant Development.

Publication Date: 2010 Mar PMID: 20032077
Authors: Airoldi, C. A. - Rovere, F. D. - Falasca, G. - Marino, G. - Kooiker, M. - Altamura, M. M. - Citterio, S. - Kater, M. M.
Journal: Plant Physiol

Bromodomain and Extra Terminal domain (BET) proteins are characterized by the presence of two types of domains, the bromodomain and the extra terminal domain. They bind to acetylated lysines present on histone tails and control gene transcription. They are also well known to play an important role in cell cycle regulation. In Arabidopsis (Arabidopsis thaliana), there are 12 BET genes; however, only two of them, IMBIBITION INDUCIBLE1 and GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6), were functionally analyzed. We characterized GTE4 and show that gte4 mutant plants have some characteristic features of cell cycle mutants. Their size is reduced, and they have jagged leaves and a reduced number of cells in most organs. Moreover, cell size is considerably increased in the root, and, interestingly, the root quiescent center identity seems to be partially lost. Cell cycle analyses revealed that there is a delay in activation of the cell cycle during germination and a premature arrest of cell proliferation, with a switch from mitosis to endocycling, leading to a statistically significant increase in ploidy levels in the differentiated organs of gte4 plants. Our results point to a role of GTE4 in cell cycle regulation and specifically in the maintenance of the mitotic cell cycle.

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Tryptophan-Derived Metabolites Are Required for Antifungal Defense in the Arabidopsis mlo2 Mutant.

Publication Date: 2010 Mar PMID: 20023151
Authors: Consonni, C. - Bednarek, P. - Humphry, M. - Francocci, F. - Ferrari, S. - Harzen, A. - Ver Loren van Themaat, E. - Panstruga, R.
Journal: Plant Physiol

Arabidopsis (Arabidopsis thaliana) genes MILDEW RESISTANCE LOCUS O2 (MLO2), MLO6, and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defense responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyperaccumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady-state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single mutant and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defense against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE4/GLUCAN SYNTHASE-LIKE5 was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defense execution machinery as innate antifungal immune responses that restrict the invasion of nonadapted fungal pathogens.

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Glucanases and chitinases as causal agents in the protection of acacia extrafloral nectar from infestation by phytopathogens.

Publication Date: 2010 Mar PMID: 20023149
Authors: Gonzalez-Teuber, M. - Pozo, M. J. - Muck, A. - Svatos, A. - Adame-Alvarez, R. M. - Heil, M.
Journal: Plant Physiol

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

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Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome.

Publication Date: 2010 Mar PMID: 20023148
Authors: Oltmanns, H. - Frame, B. - Lee, L. Y. - Johnson, S. - Li, B. - Wang, K. - Gelvin, S. B.
Journal: Plant Physiol

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.

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Genomic Inventory and Transcriptional Analysis of Medicago truncatula Transporters.

Publication Date: 2010 Mar PMID: 20023147
Authors: Benedito, V. A. - Li, H. - Dai, X. - Wandrey, M. - He, J. - Kaundal, R. - Torres-Jerez, I. - Gomez, S. K. - Harrison, M. J. - Tang, Y. - Zhao, P. X. - Udvardi, M. K.
Journal: Plant Physiol

Transporters move hydrophilic substrates across hydrophobic biological membranes and play key roles in plant nutrition, metabolism, and signaling and, consequently, in plant growth, development, and responses to the environment. To initiate and support systematic characterization of transporters in the model legume Medicago truncatula, we identified 3,830 transporters and classified 2,673 of these into 113 families and 146 subfamilies. Analysis of gene expression data for 2,611 of these transporters identified 129 that are expressed in an organ-specific manner, including 50 that are nodule specific and 36 specific to mycorrhizal roots. Further analysis uncovered 196 transporters that are induced at least 5-fold during nodule development and 44 in roots during arbuscular mycorrhizal symbiosis. Among the nodule- and mycorrhiza-induced transporter genes are many candidates for known transport activities in these beneficial symbioses. The data presented here are a unique resource for the selection and functional characterization of legume transporters.

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The Missing Link in Plant Histidine Biosynthesis: Arabidopsis myoinositol monophosphatase-like2 Encodes a Functional Histidinol-Phosphate Phosphatase.

Publication Date: 2010 Mar PMID: 20023146
Authors: Petersen, L. N. - Marineo, S. - Mandala, S. - Davids, F. - Sewell, B. T. - Ingle, R. A.
Journal: Plant Physiol

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

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The involvement of lipid peroxide-derived aldehydes in aluminum toxicity of tobacco roots.

Publication Date: 2010 Mar PMID: 20023145
Authors: Yin, L. - Mano, J. - Wang, S. - Tsuji, W. - Tanaka, K.
Journal: Plant Physiol

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic alpha,beta-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl(3) on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl(3) treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl(3) treatment, the roots of AER-OE plants accumulated Al and H(2)O(2) to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl(3) treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.

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