Plant Physiology

Involvement of CBF Transcription Factors in Winter Hardiness in Birch.

Publication Date: 2008 May 8 PMID: 18467468
Authors: Welling, A. - Palva, E. T.
Journal: Plant Physiol

Cold acclimation of plants involves extensive reprogramming of gene expression. In Arabidopsis three cold-inducible transcriptional activators designated CBF1-3/DREB1a-c have been shown to play an important regulatory role in this acclimation process. Similarly to Arabidopsis, boreal zone trees can increase their freezing tolerance (FT) in response to low temperature (LT) during the growing season. However, maximal FT of these trees requires short daylength (SD) induced dormancy development followed by exposure to both low and freezing temperatures. To elucidate the molecular basis of FT in overwintering trees, we characterized the role of birch (Betula pendula Roth) CBF transcription factors in the cold acclimation process. We identified four putative CBF orthologs in a birch EST collection designated BpCBF1-4. Ectopic expression of birch CBFs in Arabidopsis resulted in constitutive expression of endogenous CBF target genes and increased freezing tolerance of non-acclimated transgenic plants. In addition, these plants showed stunted growth and delayed flowering, typical features for CBF overexpressing plants. Expression analysis in birch showed that BpCBF1-4 are LT responsive but differentially regulated in dormant and growing plants, the expression being delayed in dormant tissues. Freeze-thaw treatment, simulating winter-time conditions in nature, resulted in strong induction of BpCBF genes during thawing, followed by induction of a CBF target gene BpLTI36. These results suggest that in addition to their role in cold acclimation during the growing season birch CBFs appear to contribute to control of winter hardiness in birch.

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Invasion of the Arabidopsis genome by the tobacco retrotransposon Tnt1 is controlled by reversible transcriptional gene silencing.

Publication Date: 2008 May 14 PMID: 18467467
Authors: Perez-Hormaeche, J. - Potet, F. - Beauclair, L. - Le Masson, I. - Courtial, B. - Bouche, N. - Lucas, H.
Journal: Plant Physiol

LTR-retrotransposons are generally silent in plant genomes. However, they often constitute a large proportion of repeated sequences in plants. This suggests that their silencing is set up after a certain copy number is reached and/or that it can be released in some circumstances. We introduced the tobacco LTR-retrotransposon Tnt1 into Arabidopsis thaliana, thus mimicking the horizontal transfer of a retrotransposon into a new host species and allowing us to study the regulatory mechanisms controlling its amplification. Tnt1 is transcriptionally silenced in Arabidopsis in a copy-number dependent manner. This silencing is associated with 24 nucleotide short-interfering RNAs targeting the promoter localised in the LTR region and with the non-CG site methylation of these sequences. Consequently, the silencing of Tnt1 is not released in methyltransferase1 (met1) mutants in contrast to decrease in DNA methylation1 (ddm1) or PolIVa (nrpd1a) mutants. Stable reversion of Tnt1 silencing is obtained when the number of Tnt1 elements is reduced down to two by genetic segregation. Our results support a model in which Tnt1 silencing in Arabidopsis occurs via an RNA-directed DNA methylation process. We further show that silencing can be partially overcome by some stresses.

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The Arabidopsis Halophytic Relative, Thellungiella halophila, Tolerates Nitrogenlimiting Conditions by Maintaining Growth, Nitrogen Uptake and Assimilation.

Publication Date: 2008 May 8 PMID: 18467466
Authors: Kant, S. - Bi, Y. M. - Weretilnyk, E. - Barak, S. - Rothstein, S. J.
Journal: Plant Physiol

A comprehensive knowledge of mechanisms regulating nitrogen use efficiency (NUE) is required to reduce excessive input of nitrogen (N) fertilizers while maintaining acceptable crop yields under limited nitrogen supply. Studying plant species which are naturally adapted to low nitrogen conditions could facilitate identification of novel regulatory genes conferring better NUE. Here we show that Thellungiella halophila, a halophytic relative of Arabidopsis thaliana grows better than Arabidopsis under moderate (1 mM nitrate) and severe (0.4 mM nitrate) N-limiting conditions. Thellungiella exhibited a lower carbon to nitrogen ratio than Arabidopsis under N-limitation which was due to Thellungiella plants possessing higher nitrogen content, total amino acids, total soluble protein and lower starch content compared to Arabidopsis. Furthermore, Thellungiella had higher amounts of several metabolites such as soluble sugars and organic acids under N-sufficient conditions (4 mM nitrate). Nitrate reductase activity and NR2 gene expression in Thellungiella displayed less of a reduction in response to N-limitation than in Arabidopsis. Thellungiella shoot GS1 expression was more induced by low-N than in Arabidopsis, while in roots, Thellungiella GS2 expression was maintained under N-limitation whereas it decreased in Arabidopsis. Up-regulation of NRT2.1 and NRT3.1 expression was higher and repression of NRT1.1 was lower in Thellungiella roots under N-limiting conditions compared to Arabidopsis. Differential transporter gene expression was correlated with higher nitrate influx in Thellungiella at low (15)NO3(-) supply. Taken together, our results suggest that Thellungiella is tolerant to N-limited conditions and could act as a model system to unravel the mechanisms for low nitrogen tolerance.

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Inactive Methyl Indole-3-Acetic Acid Ester Can Be Hydrolyzed and Activated by Several Esterases Belonging to the AtMES Esterase Family of Arabidopsis thaliana.

Publication Date: 2008 May 8 PMID: 18467465
Authors: Yang, Y. - Xu, R. - Ma, C. J. - Vlot, A. C. - Klessig, D. F. - Pichersky, E.
Journal: Plant Physiol

The plant hormone auxin (indole-3-acetic acid, IAA) is found both free and conjugated to a variety of carbohydrates, amino acids and peptides. We have recently shown that IAA could be converted to its methyl ester (MeIAA) by the Arabidopsis thaliana enzyme IAMT1 (IAA carboxyl methyltransferase 1). However, the presence and function of MeIAA in vivo remains unclear. Recently it has been shown that the tobacco protein SABP2 (Salicylic acid binding protein-2) hydrolyzes methyl salicylate to salicylic acid. There are 20 homologs of SABP2 in the genome of Arabidopsis thaliana, which we have named AtMES (for methyl esterases). We tested 15 of the proteins encoded by these genes in biochemical assays with various substrates, and identified several candidate MeIAA esterases that could hydrolyze MeIAA. MeIAA, like IAA, exerts inhibitory activity on the growth of wild type roots when applied exogenously. However, the roots of Arabidopsis plants carrying T-DNA insertions in the putative MeIAA esterase gene AtMES17 (At3g10870) displayed significantly decreased sensitivity to MeIAA compared with wild type roots, while remaining as sensitive to free IAA as wild type roots. Incubating seedlings in the presence of [(14)C]-MeIAA for 30 min revealed that mes17 mutants hydrolyzed only 40% of the [(14)C]-MeIAA taken up by plants, whereas wildtype plants hydrolyzed 100% of absorbed [(14)C]-MeIAA. Roots of Arabidopsis plants overexpressing AtMES17 showed increased sensitivity to MeIAA, but not to IAA. Additionally, mes17 plants have longer hypocotyls, and display increased expression of the auxin-responsive DR5:GUS reporter gene, suggesting a perturbation in IAA homeostasis and/or transport. mes17-1/axr1-3 double mutant plants have the same phenotype as axr1-3, suggesting MES17 acts upstream of AXR1. The protein encoded by AtMES17 had a Km value of 13 microM, and a Kcat value of 0.18 sec(-1) for MeIAA. AtMES17 was expressed at the highest levels in shoot apex, stem and root of Arabidopsis. Our results demonstrate that MeIAA is an inactive form of IAA, and the manifestations of MeIAA in vivo activity are due to the action of free IAA that is generated from MeIAA upon hydrolysis by one or more plant esterases.

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A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase.

Publication Date: 2008 May 8 PMID: 18467464
Authors: Dayan, F. E. - Ferreira, D. - Wang, Y. H. - Khan, I. A. - McInroy, J. A. - Pan, Z.
Journal: Plant Physiol

Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogues, the mode of action of cyperin is not light-dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis thaliana enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. While cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR are similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of A. thaliana ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the pi-pi stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD(+). Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.

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The Effect of Iron on the Primary Root Elongation of Arabidopsis during Phosphate Deficiency.

Publication Date: 2008 May 8 PMID: 18467463
Authors: Ward, J. T. - Lahner, B. - Yakubova, E. - Salt, D. E. - Raghothama, K. G.
Journal: Plant Physiol

Root architectures differences have been linked to the survival of plants on phosphate-deficient soils, as well as to the improved yields of phosphate-efficient crop cultivars. To understand how these differences arise, we have studied the root architectures of phosphate-deficient Arabidopsis thaliana (Col-0) plants. A striking aspect of the root architecture of these plants is that their primary root elongation is inhibited when grown on phosphate-deficient media. Here we present evidence suggesting that this inhibition is a result of iron toxicity. When the iron concentration in phosphate-deficient medium is reduced, we observe elongation of the primary root without an increase in phosphate availability or a corresponding change in the expression of phosphate-deficiency regulated genes. Recovery of the primary root elongation is associated with larger plant weights, improved ability to take up phosphate from the media, and increased tissue phosphate content. This suggests that manipulating iron availability to a plant could be a valuable strategy for improving a plant's ability to tolerate phosphate deficiency.

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The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells.

Publication Date: 2008 May 8 PMID: 18467462
Authors: Doi, M. - Shimazaki, K. I.
Journal: Plant Physiol

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response, but open in response to red light. We investigated this light response of Adiantum stomata, and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration (Ci) nor closed under high Ci. Stomata in Arabidopsis, which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K(+) accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). The stomatal opening was completely inhibited by CsCl, a K(+) channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by DCMU. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K(+) uptake.

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Engineering a Catabolic Pathway in Plants for the Degradation of 1,2-Dichloroethane.

Publication Date: 2008 May 14 PMID: 18467461
Authors: Mena Benitez, G. L. - Gandia-Herrero, F. - Graham, S. - Larson, T. R. - McQueen-Mason, S. J. - French, C. E. - Rylott, E. L. - Bruce, N. C.
Journal: Plant Physiol

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds which include 1,2-dichloroethane. Although plants lack the enzymatic activity required to metabolise this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics including 1,2-dichloroethane. We have engineered the dhlA and dhlB genes into Nicotiana tabacum cv Xanthi (tobacco) plants, and used 1,2-dichlorethane as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-dichloroethane to 2-chloroethanol, which is then metabolised to the phytotoxic 2-chloroacetaldehyde then chloroacetic acid by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing dhlA only, produced phytotoxic levels of chlorinated intermediates and died, whilst plants expressing dhlA together with dhlB thrived at levels of 1,2-dichloroethane that were toxic to dhlA-expressing plants. This represents a significant advance in the development of a low cost, phytoremediation approach towards the clean-up of halogenated organic pollutants from contaminated soil and groundwater.

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Nitrogen recycling and remobilisation are differentially controlled by leaf senescence and development stage in Arabidopsis thaliana under low nitrogen nutrition.

Publication Date: 2008 May 8 PMID: 18467460
Authors: Diaz, C. - Lemaitre, T. - Christ, A. - Azzopardi, M. - Kato, Y. - Sato, F. - Morot-Gaudry, J. F. - Le Dily, F. - Masclaux-Daubresse, C.
Journal: Plant Physiol

Five recombinant inbred lines (RIL) of Arabidopsis thaliana, previously selected by Diaz et al. (2005) from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early-senescing to late-senescing) when cultivated under nitrogen limiting conditions, were analyzed in order to monitor metabolic markers related to nitrogen assimilation and nitrogen remobilisation pathways. In each RIL, a decrease of total nitrogen, free amino acid and soluble protein contents with leaf ageing was observed. In parallel, the expression of markers for nitrogen remobilisation such as cytosolic glutamine synthetase, glutamate dehydrogenase and CND41-like protease was increased. This increase occurred earlier and was more rapidly in early-senescing lines than in late senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that nitrogen remobilisation from the source leaves to the sink leaves, was more efficient in the early senescing lines. The N-remobilisation rate was correlated with leaf-senescence severity at vegetative stage. Experiments of (15)N tracing at reproductive stage showed however that the rate of nitrogen remobilisation from the rosettes to the flowering organs and to the seeds, was similar in early and late senescing lines. At reproductive stage, N-remobilisation efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.

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An evaluation of the basis and consequences of a stay-green mutation in the navel negra (nan) citrus mutant using transcriptomic and proteomic profiling and metabolite analysis.

Publication Date: 2008 May 8 PMID: 18467459
Authors: Alos, E. - Roca, M. - Iglesias, D. J. - Minguez-Mosquera, M. I. - Damasceno, C. M. - Thannhauser, T. W. - Rose, J. K. - Talon, M. - Cercos, M.
Journal: Plant Physiol

A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown colored flavedo during ripening. Analysis of pigment composition in the wild type (WT) and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis, was impaired in the mutant, identifying nan as a Type C stay-green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes and chlorophyllase activity, but no accumulation of dephytylated chlorophyll compounds during ripening, suggesting that the mutation is not related to a lesion in any of the principal enzymatic steps in Chl catabolism. Transcript profiling using a citrus microarray indicated that a citrus ortholog of a number of SGR (stay green) genes was expressed at substantially lower levels in nan, both prior to, and during, ripening. However, the pattern of catabolite accumulation and SGR sequence analysis suggested that the nan mutation is distinct from those in previously described stay-green mutants and is associated with an upstream regulatory step, rather than directly influencing a specific component of Chl catabolism. Transcriptomic and comparative proteomic profiling further indicated that the nan mutation resulted in the suppressed expression of numerous photosynthesis-related genes and in the induction of genes that are associated with oxidative stress. These data, in addition to metabolite analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate for the elevated Chl levels and associated photooxidative stress.

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AUXIN RESPONSES IN MUTANTS OF THE ARABIDOPSIS COP9 SIGNALOSOME.

Publication Date: 2008 May 8 PMID: 18467458
Authors: Dohmann, E. M. - Levesque, M. P. - Isono, E. - Schmid, M. - Schwechheimer, C.
Journal: Plant Physiol

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex that interacts with cullin-RING type E3 ubiquitin ligases (CRLs). CSN subunit 5 (CSN5), which when incorporated into CSN can deconjugate the NEDD8-modification from the cullin subunit of CRLs, is essential for CSN's role in controlling CRL activity. Whether the CSN5 monomer, which is maintained in csn mutants such as csn3 or csn4, has a functional role, remained to be established. We performed a comparative gene expression profiling experiment with Arabidopsis (Arabidopsis thaliana) csn3, csn4, and csn5 mutants, and we show here that these mutants cannot be distinguished at the transcriptional level. Furthermore, we show that csn3 csn5 mutants are morphologically indistinguishable from csn3 or csn5 mutants. Taken together these data suggest that the CSN5 monomer does not have a function that leads to transcriptional or morphological changes in the csn mutants. We further examined auxin responses in csn mutants. While CSN had previously been shown to be required for the auxin response-regulatory E3 complexes, specifically SCF(TIR1), the csn mutant phenotype suggests that CSN is not essential for auxin responses. We present physiological and genetic data, which indicate that auxin responses are indeed only partially impaired in csn mutants and that this is not the result of maternally contributed CSN. Finally, we discuss these findings in the context of the current understanding of the role of neddylation and CSN-mediated deneddylation for CRL activity.

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The {beta}-glucosidases responsible for bio-activation of hydroxynitrile glucosides in Lotus japonicus.

Publication Date: 2008 May 8 PMID: 18467457
Authors: Morant, A. V. - Bjarnholt, N. - Kragh, M. E. - Kjargaard, C. H. - Jorgensen, K. - Paquette, S. M. - Piotrowski, M. - Imberty, A. - Olsen, C. E. - Moller, B. L. - Bak, S.
Journal: Plant Physiol

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bio-activated by hydrolysis by specific beta-glucosidases. A mixture of two hydroxynitrile glucoside cleaving beta-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside cleaving beta-glucosidases preferentially hydrolyzed rhodiocyanoside A and lotaustralin, whereas linamarin was only slowly hydrolyzed in agreement with measurements of their rate of degradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 and LjBGD4 had nearly identical overall topologies and substrate binding pockets. Heterologous expression of LjBGD2 and LjBGD4 in Arabidopsis thaliana enabled analysis of their individual substrate specificity profiles and confirmed that both LjBGD2 and LjBGD4 preferentially hydrolyze the hydroxynitrile glucosides present in L. japonicus. Phylogenetic analyses revealed a third L. japonicus putative hydroxynitrile glucoside cleaving beta-glucosidase, LjBGD7. RT-PCR analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7 corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450 that catalyze the first committed step in biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.

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CressExpress: A tool for large-scale mining of expression data from Arabidopsis thaliana.

Publication Date: 2008 May 8 PMID: 18467456
Authors: Srinivasasainagendra, V. - Page, G. - Mehta, T. - Coulibaly, I. - Loraine, A. E.
Journal: Plant Physiol

CressExpress is a user-friendly, on-line co-expression analysis tool for Arabidopsis microarray expression data that computes patterns of correlated expression between user-entered query genes and the rest of the genes in the genome. Unlike other co-expression tools, CressExpress allows characterization of tissue-specific co-expression networks through user-driven filtering of input data based on sample tissue type. CressExpress also performs Pathway-Level Co-expression (PLC) analysis on each set of query genes, identifying and ranking genes based on their common connections with two or more query genes. This allows identification of novel candidates for involvement in common processes and functions represented by the query group. Users launch experiments using an easy-to-use web-based interface and then receive the full complement of results, along with a record of tool settings and parameters, via an email link to the CressExpress Web site. Data sets featured in CressExpress are strictly versioned, and include expression data from MAS5, GCRMA, and RMA array processing algorithms. To demonstrate applications for CressExpress, we present co-expression analyses of cellulose synthase (CESA) genes, indolic glucosinolate biosynthesis, and flowering. We show that sub-selecting sample types produces a richer network for genes involved in flowering in Arabidopsis. CressExpress provides direct access to expression values via an easy-to-use URL-based web service, allowing users to determine quickly if their query genes are co-expressed with each other and likely to yield informative PLC results. The tool is available at http://www.cressexpress.org.

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Biochemical and Genomic Characterization of Terpene Synthases in Magnolia grandiflora.

Publication Date: 2008 May 8 PMID: 18467455
Authors: Lee, S. - Chappell, J.
Journal: Plant Physiol

Southern Magnolia (Magnolia grandiflora) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominately to beta-cubebene, while Mg17 converted GPP (C5) to alpha-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron-exon organizations for the 3 Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthase, but Mg11 contains only 4 introns, and Mg25 has only a single intron located near the 5' terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from an common ancestor TPS gene, or by a gain of introns into an intron-deficienct progenote TPS gene.

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Functional characterization of HFR-1, a high mannose N-glycan-specific wheat lectin induced by Hessian fly larvae.

Publication Date: 2008 May 14 PMID: 18467454
Authors: Subramanyam, S. - Smith, D. F. - Clemens, J. C. - A Webb, M. - Sardesai, N. - Williams, C. E.
Journal: Plant Physiol

We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum L.) plants that responds to infestation by Hessian fly [Mayetiola destructor (Say)] larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (Hessian fly-responsive gene 1) transcripts compared to uninfested or susceptible plants. Here we characterize the soluble and active recombinant His6-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His6-HFR1 to Manalpha1-6(Manalpha1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR-1 at the larval feeding sites as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percent mortality increased with progressively higher concentrations of His6-HFR1 in the diet. Thus, HFR-1 is an anti-nutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants.

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An external {delta}-Carbonic Anhydrase in a free-living marine dinoflagellate may circumvent diffusion limited carbon acquisition.

Publication Date: 2008 May 8 PMID: 18467453
Authors: Lapointe, M. - Mackenzie, T. D. - Morse, D.
Journal: Plant Physiol

The oceans globally constitute an important sink for carbon dioxide due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO2 and bicarbonate is pH dependent, and in normal slightly alkaline seawater, [CO2] is typically low ( approximately 10 microM). Second, the rate of CO2 diffusion in seawater is slow, so for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon-limited due to depletion of CO2 from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco, as this form is less oxygen-tolerant than the usually found form I enzyme. We have identified a Carbonic Anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with delta-class CAs, an isoform so far found only in marine algae. Immunoelectron microscopy indicates this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole cell CA assays and vectorial labeling of cell surface proteins with (125)I. The fixation of (14)CO2 is strongly pH dependant, suggesting preferential uptake of CO2 rather than bicarbonate, and photosynthetic rates decrease in the presence of 1 mM acetazolamide, a non-membrane permeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and taken together, our results suggest this enzyme may help to increase CO2 availability at the cell surface.

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An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis and expression in Arabidopsis thaliana.

Publication Date: 2008 May 8 PMID: 18467452
Authors: Meesapyodsuk, D. - Qiu, X.
Journal: Plant Physiol

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate PCR, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) which has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis thaliana wild type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase and CpFAH, the first Delta(12) oleate hydroxylase of non-plant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

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Functional analysis of a predicted flavonol synthase gene family in Arabidopsis.

Publication Date: 2008 May 8 PMID: 18467451
Authors: Owens, D. K. - Alerding, A. B. - Crosby, K. C. - Bandara, A. B. - Westwood, J. H. - Winkel, B. S.
Journal: Plant Physiol

The genome of Arabidopsis thaliana contains five sequences with high similarity to AtFLS1, a previously-characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically-competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under non-stressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell-type specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus it is possible that these "pseudogenes" have alternative, non-catalytic functions that have not yet been uncovered.

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The AP2/ERF-domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense.

Publication Date: 2008 May 8 PMID: 18467450
Authors: Pre, M. - Atallah, M. - Champion, A. - De Vos, M. - Pieterse, C. M. - Memelink, J.
Journal: Plant Physiol

Plant defense against pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene. In certain defense responses, JA and ethylene signaling pathways synergize to activate a specific set of defense genes. Here we describe the role of the Arabidopsis thaliana AP2/ERF-domain transcription factor ORA59 in JA and ethylene signaling and in defense. JA- and ethylene-responsive expression of several defense genes, including the plant defensin gene PDF1.2, depended on ORA59. As a result, overexpression of ORA59 caused increased resistance against the fungus Botrytis cinerea, whereas ORA59-silenced plants were more susceptible. Several AP2/ERF-domain transcription factors have been suggested to be positive regulators of PDF1.2 gene expression based on overexpression in stably transformed plants. Using two different transient overexpression approaches we found that only ORA59 and ERF1 were able to activate PDF1.2 gene expression in contrast to the related proteins AtERF1 and AtERF2. Our results demonstrate that ORA59 is an essential integrator of the JA and ethylene signal transduction pathways, and thereby provide new insight in the nature of the molecular components involved in the crosstalk between these two hormones.

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THE 'OLD' EUONYMUS EUROPAEUS AGGLUTININ REPRESENTS A NOVEL FAMILY OF UBIQUITOUS PLANT PROTEINS.

Publication Date: 2008 May 1 PMID: 18451263
Authors: Fouquaert, E. - Peumans, W. J. - Smith, D. F. - Proost, P. - Savvides, S. N. - Damme, E. J.
Journal: Plant Physiol

Molecular cloning of the 'old' but still unclassified Euonymus europaeus agglutinin (EEA) demonstrated that the lectin is a homodimeric protein composed of 152 residue subunits. Analysis of the deduced sequence indicated that EEA is synthesized without a signal peptide and undergoes no post-translational processing apart from the removal of a 6-residue N-terminal peptide. Glycan array screening confirmed the previously reported high reactivity of EEA towards blood group B oligosaccharides, but also revealed binding to high mannose N-glycans, providing firm evidence for the occurrence of a plant carbohydrate-binding domain that can interact with structurally different glycans. BLAST searches indicated that EEA shares no detectable sequence similarity with any other lectin but is closely related evolutionarily to a domain that was first identified in some abscisic acid and salt stress-responsive rice proteins, and according to the available sequence data might be ubiquitous in Spermatophyta. Hence, EEA can be considered the prototype of a novel family of presumably cytoplasmic/nuclear proteins that are apparently ubiquitous in plants. Taking into account that some of these proteins are definitely stress-related, the present identification of the EEA lectin domain might be a first step in the recognition of the involvement and importance of protein-glycoconjugate interactions in some essential cellular processes in Embryophyta.

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Characterization and biological function of the ISOCHORISMATE SYNTHASE 2 gene of Arabidopsis thaliana.

Publication Date: 2008 May 1 PMID: 18451262
Authors: Garcion, C. - Lohmann, A. - Lamodiere, E. - Catinot, J. - Buchala, A. - Doermann, P. - Metraux, J. P.
Journal: Plant Physiol

Salicylic acid (SA) is an important mediator of plant defense response. In Arabidopsis thaliana, this compound was proposed to derive mainly from isochorismate, itself produced from chorismate through the activity of ICS1 (Isochorismate Synthase1). Null ics1 mutants still accumulate some SA, suggesting the existence of an enzymatic activity redundant with ICS1 or of an alternative ICS-independent SA biosynthetic route. Here we studied the role of ICS2, second ICS gene of the Arabidopsis genome, in the production of SA. We have shown that ICS2 encodes a functional ICS enzyme and that, similarly to ICS1, ICS2 is targeted to the plastids. Comparison of SA accumulation in the ics1, ics2 and ics1 ics2 mutants indicates that ICS2 participates in the synthesis of SA but in limited amounts, that become clearly detectable only when ICS1 is lacking. This unequal redundancy relationship was also observed for phylloquinone, another isochorismate-derived end-product. Furthermore, detection of SA in the double ics1 ics2 double mutant that is completely devoid of phylloquinone provides genetic evidence of the existence of an ICS-independent SA biosynthetic pathway in Arabidopsis.

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Corrections.

Publication Date: 2008 May PMID: 18443105
Authors:
Journal: Plant Physiol

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Activity Range of Arabidopsis Small RNAs Derived from Different Biogenesis Pathways.

Publication Date: 2008 May PMID: 18443104
Authors: Tretter, E. M. - Alvarez, J. P. - Eshed, Y. - Bowman, J. L.
Journal: Plant Physiol

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Regulating the regulators: the future prospects for transcription-factor-based agricultural biotechnology products.

Publication Date: 2008 May PMID: 18443103
Authors: Century, K. - Reuber, T. L. - Ratcliffe, O. J.
Journal: Plant Physiol

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Targets for Crop Biotechnology in a Future High-CO2 and High-O3 World.

Publication Date: 2008 May PMID: 18443102
Authors: Ainsworth, E. A. - Rogers, A. - Leakey, A. D.
Journal: Plant Physiol

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The outlook for protein engineering in crop improvement.

Publication Date: 2008 May PMID: 18443101
Authors: Rao, A. G.
Journal: Plant Physiol

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Editor's Choice Series: The Next Generation of Biotech Crops.

Publication Date: 2008 May PMID: 18443100
Authors: Martino-Catt, S. J. - Sachs, E. S.
Journal: Plant Physiol

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Gene Expression and Metabolism in Tomato Fruit Surface Tissues.

Publication Date: 2008 Apr 25 PMID: 18441227
Authors: Mintz-Oron, S. - Mandel, T. - Rogachev, I. - Feldberg, L. - Lotan, O. - Yativ, M. - Wang, Z. - Jetter, R. - Venger, I. - Adato, A. - Aharoni, A.
Journal: Plant Physiol

The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive ones such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative Transcriptome and Metabolome analyses were carried out on peel and flesh tissues during tomato fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin and phenylpropanoids. Orthologues of the Arabidopsis SHINE2 and MIXTA-like regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. UPLC-QTOF-MS and GC-MS/FID analyses identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism, and protection against photo-oxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.

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Evidence for Abscisic Acid Biosynthesis in Cuscuta reflexa, a Parasitic Plant Lacking Neoxanthin.

Publication Date: 2008 Apr 25 PMID: 18441226
Authors: Qin, X. - Yang, S. H. - Kepsel, A. C. - Schwartz, S. H. - Zeevaart, J. A.
Journal: Plant Physiol

Abscisic acid (ABA) is a plant hormone found in all higher plants; it plays an important role in seed dormancy, embryo development, and adaptation to environmental stresses, most notably drought. The regulatory step in ABA synthesis is the cleavage reaction of a 9-cis--epoxy-carotenoid catalyzed by the 9-cis-epoxy-carotenoid dioxygenases (NCEDs). The parasitic angiosperm Cuscuta reflexa lacks neoxanthin, one of the common precursors of ABA in all higher plants. Thus, is C. reflexa capable of synthesizing ABA, or does it acquire ABA from its host plants? Stem tips of C. reflexa were cultured in vitro and found to accumulate ABA in the absence of host plants. This demonstrates that this parasitic plant is capable of synthesizing ABA. Dehydration of detached stem tips caused a big rise in ABA content. During dehydration, (18)O was incorporated into ABA from (18)O2, indicating that ABA was synthesized de novo in C. reflexa. Two 9-cis-epoxy-carotenoid dioxygenase genes, CrNCED1 and CrNCED2, were cloned from C. reflexa. Expression of CrNCEDs was upregulated significantly by dehydration. In vitro enzyme assays with recombinant CrNCED1 protein showed that the protein is able to cleave both 9-cis-violaxanthin and 9'-cis-neoxanthin to give xanthoxin. Thus, despite the absence of neoxanthin in C. reflexa, the biochemical activity of CrNCED1 is similar to that of NCEDs from other higher plants. These results provide evidence for conservation of the ABA biosynthesis pathway among members of the plant kingdom.

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Functional identification of Arabidopsis stress regulatory genes using the Controlled cDNA Overexpression System, COS.

Publication Date: 2008 Apr 25 PMID: 18441225
Authors: Papdi, C. - Abraham, E. - Joseph, M. P. - Popescu, C. - Koncz, C. - Szabados, L.
Journal: Plant Physiol

Responses to environmental stresses in higher plants are controlled by a complex web of ABA dependent and independent signalling pathways. To perform genetic screens for identification of novel Arabidopsis loci involved in the control of abiotic stress responses, a cDNA expression library was created in a Gateway version of estradiol-inducible XVE binary vector (Controlled cDNA Overexpression System, COS). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance and activation of a stress-responsive alcohol dehydrogenase-luciferase (ADH1-LUC) reporter gene. Twenty seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by PCR amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat-shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase (2AER) cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.

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Knocking Out Cytosolic Cysteine Synthesis Compromises the Antioxidant Capacity of the Cytosol to Maintain Discrete Concentrations of Hydrogen Peroxide in Arabidopsis.

Publication Date: 2008 Apr 25 PMID: 18441224
Authors: Lopez-Martin, M. C. - Becana, M. - Romero, L. C. - Gotor, C.
Journal: Plant Physiol

Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. Evidences that the mutation caused a perturbation in H2O2 homeostasis are that in the knockout, H2O2 production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H2O2 homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis.

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Tocotrienols, the unsaturated forms of vitamin E, can function as antioxidants and lipid protectors in tobacco leaves.

Publication Date: 2008 Apr 25 PMID: 18441223
Authors: Matringe, M. - Ksas, B. - Rey, P. - Havaux, M.
Journal: Plant Physiol

Vitamin E is a generic term for a group of lipid-soluble antioxidant compounds, the tocopherols and tocotrienols. While tocotrienols are considered as important vitamin E components in humans with functions in health and disease, the protective functions of tocotrienols have never been investigated in plants, contrary to tocopherols. We took advantage of the strong accumulation of tocotrienols in leaves of double transgenic tobacco plants that co-expressed the yeast prephenate dehydrogenase gene (PDH) and the Arabidopsis hydroxyphenylpyruvate dioxygenase gene (HPPD) to study in vivo the antioxidant function of those compounds. In young leaves of wild-type and transgenic tobacco plants, the majority of vitamin E was stored in thylakoid membranes, while plastoglobules contained mainly delta-tocopherol, a very minor component of vitamin E in tobacco. However, the vitamin E composition of plastoglobules was observed to change substantially during leaf aging, with alpha-tocopherol becoming the major form. Tocotrienol accumulation in young transgenic HPPD-PDH leaves occurred without any significant perturbation of photosynthetic electron transport. Tocotrienols noticeably reinforced the tolerance of HPPD-PDH leaves to high light stress at chilling temperature, with PSII photoinhibition and lipid peroxidation being maintained low relative to wild-type leaves. Very young leaves of wild-type tobacco plants turned yellow during chilling stress, because of a strongly reduced level of chlorophylls and carotenoids, and this phenomenon was attenuated in transgenic HPPD-PDH plants. While sugars accumulated similarly in young WT and HPPD-PDH leaves exposed to chilling stress in high light, a substantial decrease in tocotrienols was observed in the transgenic leaves only, suggesting vitamin E consumption during oxy-radical scavenging. Our results demonstrate that tocotrienols can function in vivo as efficient antioxidants protecting membrane lipids from peroxidation.

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G{gamma}1 + G{gamma}2 ≠ G{beta}. Heterotrimeric G protein G{gamma}-deficient mutants do not recapitulate all phenotypes of G{beta}-deficient mutants.

Publication Date: 2008 May 8 PMID: 18441222
Authors: Trusov, Y. - Zhang, W. - Assmann, S. M. - Botella, J. R.
Journal: Plant Physiol

Heterotrimeric G proteins are signaling molecules ubiquitous among all eukaryotes. The Arabidopsis genome contains one Galpha (GPA1) , one Gbeta ( (AGB1) and two Ggamma subunit genes (AGG1 and AGG2). Gbeta requirement of a functional Ggamma subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotypically resemble mutants lacking AGB1 in all respects. We have previously reported that Gbeta- and Ggamma-deficient mutants coincide during plant pathogen interaction, lateral root development, gravitropic response and some aspects of seed germination. Here we report a number of phenotypic discrepancies between Gbeta- and Ggamma-deficient mutants, including the double mutant lacking both Ggamma subunits. While Gbeta-deficient mutants are hypersensitivie to ABA-inhibition of seed germination and are hyposensitive to ABA-inhibition of stomatal opening and guard-cell inward K(+) currents, none of the available Ggamma-deficient mutants show any deviation from wild type in these responses, nor do they show the hypocotyl elongation and hook development defects that are characteristic of Gbeta-deficient mutants. In addition, striking discrepancies were observed in the aerial organs of Gbeta- versus Ggamma-deficient mutants. In fact, none of the distinctive traits observed in Gbeta-deficient mutants (such as reduced size of cotyledons, leaves, flowers and siliques), are present in any of the Ggamma single and double mutants. Despite the considerable amount of phenotypic overlap between Gbeta- and Ggamma-deficient mutants, confirming the tight relationship between Gbeta and Ggamma subunits in plants, considering the significant differences reported here we hypothesize the existence of new and as yet unknown elements in the heterotrimeric G protein signaling complex.

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Artificial trans-acting siRNAs confer consistent and effective gene silencing.

Publication Date: 2008 Apr 25 PMID: 18441221
Authors: de la Luz Gutierrez-Nava, M. - Aukerman, M. J. - Sakai, H. - Tingey, S. V. - Williams, R. W.
Journal: Plant Physiol

Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing and artificial microRNAs take advantage of endogenous post-transcriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs (ata-siRNAs) in Arabidopsis thaliana. Replacing the endogenous siRNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with a miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of siRNAs from the TAS1c locus.

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The wheat MAP-kinases TaMPK3 and TaMPK6 are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola.

Publication Date: 2008 Apr 25 PMID: 18441220
Authors: Rudd, J. J. - Keon, J. - Hammond-Kosack, K.
Journal: Plant Physiol

Many race- or isolate-specific disease resistance responses of plants towards pathogens (incompatible interactions) invoke hypersensitive response (HR)-like programmed cell death (PCD), and the co-ordinate activation of mitogen-activated protein kinases homologous to Arabidopsis AtMPK6 and AtMPK3 (or Tobacco SIPK and WIPK), respectively. Resistance of wheat leaves to the necrotrophic fungal pathogen Mycosphaerella graminicola can also operate at an isolate-cultivar specific level. We confirm here that resistance is achieved without any sign of HR-like PCD during the incompatible interaction. Instead PCD is strictly associated with the compatible interaction and is triggered during disease symptom expression. A strong transcriptional activation of TaMPK3, the wheat homologue of Arabidopsis AtMPK3, was observed immediately preceding PCD and symptom development in the compatible interaction. Generation and use of TaMPK3 and TaMPK6 -specific antibodies in western blots and in coupled immunoprecipitation-protein kinase assays demonstrated that the TaMPK3 protein also accumulated, and was subsequently post-translationally activated, during the compatible interaction in parallel to PCD. In contrast, no increase in expression, protein levels or post-translational activation of TaMPK6 was observed at any stage of either compatible or incompatible interactions. However, the protein levels of TaMPK6 became markedly reduced during the compatible interaction coincident with the onset of TaMPK3 protein accumulation. These data highlight the emerging similarity between the signalling pathways triggered in a host plant during successful infection by a necrotrophic fungal pathogen with the resistance responses normally effective against biotrophs.

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pur4 mutations are lethal to the male, but not the female gametophyte and affect sporophyte development in Arabidopsis.

Publication Date: 2008 Apr 25 PMID: 18441219
Authors: Berthome, R. - Thomasset, M. - Maene, M. - Bourgeois, N. - Froger, N. - Budar, F.
Journal: Plant Physiol

Purine metabolism is crucial in living cells and involves three complex pathways in plants: the de novo synthesis, the salvage, and the degradation pathways. The relative importance of each pathway in plant development and reproduction, however, is still unclear. We identified two T-DNA insertions in the Arabidopsis PUR4 gene (At1g74260) that encodes formylglycinamidine ribonucleotide synthase (FGAMS; EC 6.3.5.3), the fourth enzyme in the de novo purine biosynthesis pathway. The mutated alleles were never transmitted through the pollen of heterozygous plants but could be inherited through the female gametophyte indicating that de novo purine synthesis is specifically necessary for pollen development. Because the pur4 mutations were lethal to the male gametophyte, homozygous pur4 plants could not be obtained. However, the reproductive phenotype of hetererozygous plants carrying the pur4-2 mutated allele was more severe than that carrying the pur4-1 mutated allele, and pur4-2/+ plants showed slightly delayed early development. We showed that the pur4-2 allele produces an antisense transcript and that the amount of PUR4 mRNA is reduced in these plants. Transient expression of a translational fusion with the green fluorescent protein (GFP) in Arabidopsis plantlets showed that the FGAMS protein is dually targeted to chloroplast and mitochondria, suggesting that at least some steps of the de novo purine biosynthesis pathway can take place in both organelles in Arabidopsis, a dual location previously thought to be a peculiarity of ureide-forming tropical legumes.

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A pentapeptide motif related to a pigment binding site in the major light-harvesting protein of photosystem II, LHCII, governs substrate-dependent plastid import of NADPH:proto-chlorophyllide oxidoreductase (POR) A.

Publication Date: 2008 Apr 25 PMID: 18441218
Authors: Reinbothe, C. - Pollmann, S. - Phetsarath-Faure, P. - Quigley, F. - Weisbeek, P. - Reinbothe, S.
Journal: Plant Physiol

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only thus far known example of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex (PTC). Here we demonstrate that a pentapeptide motif, Thr-Thr-Ser-Pro-Gly (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate-dependent. An additional motif was identified in the NH2-terminal part of transA that was needed for binding of the precursor to the PTC complex. Point mutations of the TTSPG motif identified Gly as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of Gly may bind directly or via a water molecule to the central Mg atom of the pigment.

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Photosynthetic utilization of HCO3- in Zostera marina is reduced by inhibitors of mitochondrial ATPase and electron transport.

Publication Date: 2008 Apr 23 PMID: 18434609
Authors: Carr, H. - Axelsson, L.
Journal: Plant Physiol

When Zostera marina (Linnaeus) was irradiated after a period of darkness, initiation of photosynthetic O2 evolution occurred in two phases. During a lag phase, lasting 4-5 minutes, photosynthesis was supported by a diffusive entry of CO2. Photosynthesis then rapidly increased to its full rate. Tris buffer, at a concentration of 50 mM, completely inhibited this increase without affecting CO2-supported photosynthesis during the lag phase. These results verify that the increase in photosynthesis after the lag phase depended on an activation of HCO3(-) utilization through acid zones generated by proton pumps located to the outer cell membrane. In similar experiments, 6.25 microM of the mitochondrial ATPase blocker oligomycin inhibited photosynthetic HCO3(-) utilization by more than 60 %. Antimycin A, a selective blocker of mitochondrial electron transport, caused a similar inhibition of HCO3(-) utilization. Measurements at elevated CO2 concentrations verified that neither oligomycin nor antimycin interfered with linear photosynthetic electron transport or with CO2 fixation. Thus, a major part of the ATP used for the generation of acid zones involved in HCO3(-) utilization in Zostera marina was derived from mitochondrial respiration.

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RUPTURED POLLEN GRAIN 1 (RPG1), a Member of MtN3 /Saliva Gene Family, is Crucial for Exine Pattern Formation and Cell Integrity of Microspores in Arabidopsis thaliana.

Publication Date: 2008 Apr 23 PMID: 18434608
Authors: Guan, Y. F. - Huang, X. Y. - Zhu, J. - Gao, J. F. - Zhang, H. X. - Yang, Z. N.
Journal: Plant Physiol

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, Ruptured Pollen Grain 1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis thaliana. The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the post-meiotic abortion of microspores. Scanning Electron Microscopy (SEM) examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission Electron Microscopy (TEM) examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.

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Reduction of plastid-localized CA activity results in reduced Arabidopsis thaliana seedling survivorship.

Publication Date: 2008 Apr 23 PMID: 18434607
Authors: Ferreira, F. J. - Guo, C. - Coleman, J. R.
Journal: Plant Physiol

Carbonic anhydrase (CA; EC 4.2.1.1) catalyses the interconversion of CO2 and HCO3(-) and is a major protein constituent of the C3 higher plant chloroplast where it is presumed to play a role in photosynthetic carbon assimilation. In this study we have used both RNA antisense and gene knockout lines to specifically reduce the activity of the chloroplast betaCA1 polypeptide (At3g01500) in the model plant Arabidopsis. Although able to germinate, seedling establishment of transgenic plants is significantly reduced relative to wild type plants when grown at ambient levels of CO2. Growth at elevated (1500 microl.L-1) CO2 or on plates supplemented with sucrose restores seedling establishment rates to wild type levels. Seed from wild type and transgenic plants exhibited no significant differences in seed protein, and lipid content, or reserve mobilization during seedling growth. betaCA1 deficient seedlings do, however, exhibit reduced capacity for light dependent (14)CO2 assimilation prior to the development of true leaves. The small number of surviving seedlings able to grow and develop are phenotypically similar to wild type plants, even when subsequently grown at sub-ambient levels of CO2. Microarray analysis of mature leaves of betaCA1 deficient plants show some differences in transcript abundance, particularly with genes involved in ethylene signalling and response. The data suggest that reduced levels of seedling establishment by betaCA1 deficient plants could be the result of poor cotyledon photosynthetic performance at the onset of phototrophic growth and prior to the development of true leaves.

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Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSTIVE 1-dependent pathway.

Publication Date: 2008 Apr 23 PMID: 18434606
Authors: Ribot, C. - Zimmerli, C. - Farmer, E. E. - Reymond, P. - Poirier, Y.
Journal: Plant Physiol

Expression of AtPHO1;H10, a member of the Arabidopsis thaliana PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE 1 (COI1). The expression of AtPHO1;H10 in the OPDA reductase 3-deficient (opr3) mutant was comparable to wild type following both wounding and dehydration, and was also dependent on COI1. In contrast, induction of AtPHO1;H10 expression by exogenous ABA was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, while a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in co-regulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration.

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A genome-wide functional investigation into the roles s of receptor-like proteins in Arabidopsis.

Publication Date: 2008 Apr 23 PMID: 18434605
Authors: Wang, G. - Ellendorff, U. - Kemp, B. - Mansfield, J. W. - Forsyth, A. - Mitchell, K. - Bastas, K. - Liu, C. M. - Woods-Tor, A. - Zipfel, C. - de Wit, P. J. - Jones, J. D. - Tor, M. - Thomma, B. P.
Journal: Plant Physiol

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular LRR-domain, a transmembrane domain and a short cytoplasmatic tail. In several plant species RLPs have been found to play a role in disease resistance, such as the tomato Cf and Ve proteins, and the apple HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp. and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis TMM regulates stomatal distribution, while Arabidopsis CLV2 and its functional maize ortholog FEA2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses including susceptibility towards pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate ABA sensitivity and another AtRLP gene was found to influence non-host resistance towards Pseudomonas syringae pv. phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.

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Light regulation and daytime dependency of inducible plant defences in Arabidopsis: phytochrome signalling controls systemic acquired resistance rather than local defence.

Publication Date: 2008 Apr 23 PMID: 18434604
Authors: Griebel, T. - Zeier, J.
Journal: Plant Physiol

We have examined molecular and physiological principles underlying the light-dependency of defence activation in Arabidopsis plants challenged with the bacterial pathogen Pseudomonas syringae. Within a fixed light/dark cycle, plant defence responses and disease resistance significantly depend on the time of day when pathogen contact takes place. Morning and midday inoculations result in higher salicylic acid (SA) accumulation, faster expression of pathogenesis-related (PR) genes, and a more pronounced hypersensitive response than inoculations in the evening or at night. Rather than to the plants' circadian rhythm, this increased plant defence capability upon day inoculations is attributable to the availability of a prolonged light period during the early plant-pathogen interaction. Moreover, pathogen responses of Arabidopsis double mutants affected in light perception, i.e. cryptochrome1cryptochrome2 (cry1cry2), phototropin1phototropin2 (phot1phot2), and phytochromeAphyto-chromeB (phyAphyB) were assessed. Induction of defence responses by either avirulent or virulent P. syringae at inoculation sites is relatively robust in leaves of photoreceptor mutants, indicating little cross-talk between local defence and light signalling. In addition, the blue-light receptor mutants cry1cry2 and phot1phot2 are both capable to establish a full systemic acquired resistance (SAR) response. Induction of SAR and SA-dependent systemic defence reactions, however, are compromised in phyAphyB mutants. Phytochrome regulation of SAR involves the essential SAR component FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Our findings highlight the importance of phytochrome photoperception during systemic rather than local resistance induction. The phytochrome system seems to accommodate the supply of light energy to the energetically costly increase in whole plant resistance.

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Transcript profiling reveals new insights into the acclimation of the mesophilic fresh-water cyanobacterium Synechococcus elongatus PCC 7942 to iron starvation.

Publication Date: 2008 Apr 18 PMID: 18424627
Authors: Nodop, A. - Pietsch, D. - Hocker, R. - Becker, A. - Pistorius, E. K. - Forchhammer, K. - Michel, K. P.
Journal: Plant Physiol

The regulatory network for acclimation of the obligate photoautotrophic fresh water cyanobacterium Synechococcus elongatus PCC 7942 to iron limitation was studied by transcript profiling with an oligonucleotide whole genome DNA microarray. Six regions on the chromosome with several iron-regulated genes each were identified. The irpAB and fut region encode putative iron uptake systems, the suf region participates in [Fe-S] cluster assembly under oxidative stress and iron limitation, the isiAB region encodes CP43' and flavodoxin, the idiCB region encodes the NuoE-like electron transport associated protein IdiC and the transcriptional activator IdiB, and the ackA/pgam region encodes an acetate kinase and a phosphoglycerate mutase. We also investigated the response of two Synechococcus elongatus PCC 7942 mutants to iron starvation. These were mutant K10, lacking IdiB but containing IdiC, and mutant MuD, representing a idiC-merodiploid mutant with a strongly reduced amount of IdiC as well as IdiB. The absence of IdiB in mutant K10 or the strongly reduced amount of IdiB in mutant MuD allowed for the identification of additional members of the iron-responsive IdiB regulon. Besides idiA and the irpAB operon also somB(1), somA(2), ftr1, ackA, pgam, and nat seem to be regulated by IdiB. In addition to the reduced amount of IdiB in MuD, the low concentration of IdiC may be responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to wild-type and mutant K10. This fact may explain, why it has been impossible to obtain a fully segregated IdiC-free mutant, while it was possible to obtain a fully segregated IdiB-free mutant.

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The absence of Alternative Oxidase 1a in Arabidopsis thaliana results in acute sensitivity to combined light and drought stress.

Publication Date: 2008 Apr 18 PMID: 18424626
Authors: Giraud, E. - Ho, L. H. - Clifton, R. - Carroll, A. - Estavillo, G. - Tan, Y. F. - Howell, K. A. - Ivanova, A. - Pogson, B. J. - Millar, A. H. - Whelan, J.
Journal: Plant Physiol

Treatment of alternative oxidase 1a mutant plants (aox1a) with moderate light under drought conditions resulted in a phenotypic difference compared to Col-0, evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, increased O2(-) and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, sucrose and starch metabolism changed indicating that effects were not confined to mitochondria where the AOX1a protein is located. Microarray and QRT-PCR analysis revealed that transcripts typically induced upon stress treatment or involved in anti-oxidant defence systems, especially chloroplast located anti-oxidant defence components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signalling pathways that regulate these components. Taken together, these results indicate aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal REDOX balance in the cell.

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Two GATA transcription factors are downstream effectors of floral homeotic gene action in Arabidopsis.

Publication Date: 2008 Apr 16 PMID: 18417639
Authors: Mara, C. D. - Irish, V. F.
Journal: Plant Physiol

Floral organogenesis is dependent on the combinatorial action of MADS box transcription factors, which in turn control the expression of suites of genes required for growth, patterning, and differentiation. In Arabidopsis, the specification of petal and stamen identity depends on the action of two MADS box gene products, APETALA3 (AP3) and PISTILLATA (PI). In a screen for genes whose expression was altered in response to the induction of AP3 activity, we identified GNC (GATA, nitrate-inducible, carbon-metabolism-involved) as being negatively regulated by AP3 and PI. The GNC gene encodes a member of the Arabidopsis GATA transcription factor family and has been implicated in the regulation of chlorophyll biosynthesis as well as carbon and nitrogen metabolism. In addition, we found that the GNC paralog, GNL (GNC-like), is also negatively regulated by AP3 and PI. Using chromatin immunoprecipitation, we showed that promoter sequences of both GNC and GNL are bound by PI protein, suggesting a direct regulatory interaction. Analyses of single and double gnc and gnl mutants indicated that the two genes share redundant roles in promoting chlorophyll biosynthesis, suggesting that in repressing GNC and GNL, AP3/PI have roles in negatively regulating this biosynthetic pathway in flowers. In addition, co-expression analyses of genes regulated by AP3, PI, GNC and GNL indicate a complex regulatory interplay between these transcription factors in regulating a variety of light and nutrient responsive genes. Together, these results provide new insights into the transcriptional cascades controlling the specification of floral organ identities.

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A diurnal component to the variation in sieve tube amino acid content in wheat (Triticum aestivum L.).

Publication Date: 2008 Apr 16 PMID: 18417638
Authors: Gattolin, S. - Newbury, H. J. - Bale, J. S. - Tseng, H. M. - Barrett, D. A. - Pritchard, J.
Journal: Plant Physiol

We have used a high sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection (CE-LIF) method to quantify 16 amino acids in wheat (Triticum aestivum L.) sieve tube (ST) samples as small as 2 nl collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual ST. Some of the total concentrations of amino acids recorded are higher than those previously reported. The results obtained show variation in the concentrations of phenylalanine, histidine/valine, leucine/isoleucine, arginine, asparagine, glutamine, tyrosine and lysine across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (tyrosine, lysine, phenylalanine, histidine/valine, leucine/isoleucine) showed striking co-variation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of tyrosine, lysine, phenylalanine, leucine/isoleucine, histidine/valine, asparagine, arginine and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analysing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed; samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.

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Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon.

Publication Date: 2008 Apr 16 PMID: 18417637
Authors: Hartati, S. - Sudarmonowati, E. - Park, Y. W. - Kaku, T. - Kaida, R. - Baba, K. - Hayashi, T.
Journal: Plant Physiol

In this study, poplar cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

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3-D gas exchange pathways in pome fruit characterised by synchrotron X-ray computed tomography.

Publication Date: 2008 Apr 16 PMID: 18417636
Authors: Verboven, P. - Kerckhofs, G. - Mebatsion, H. K. - Ho, Q. T. - Temst, K. - Wevers, M. - Cloetens, P. - Nicolai, B. M.
Journal: Plant Physiol

Our understanding of the gas exchange mechanisms in plant organs critically depends on insights in the 3-D structural arrangement of cells and voids. Using synchrotron radiation X-ray tomography, we obtained for the first time high contrast 3-D absorption images of in vivo fruit tissues of high moisture content at 1.4 microm resolution and 3-D phase contrast images of cell assemblies at a resolution as low as 0.7 microm, enabling visualization of individual cell morphology, cell walls and entire void networks that were previously unknown. Intercellular spaces were always clear of water. The apple cortex contains considerably larger parenchyma cells and voids than pear parenchyma. Voids in apple often are larger than the surrounding cells and some cells are not connected to void spaces. The main voids in apple stretch hundreds of micrometers but are disconnected. Voids in pear cortex tissue are always smaller than parenchyma cells, but each cell is surrounded by a tight and continuous network of voids, except near brachyssclereid groups. Vascular and dermal tissues were also measured. The visualised network achitecture was consistent over different picking dates and shelf life. The differences in void fraction (5.1 % for pear cortex and 23.0 % for apple cortex) and in gas network architecture helps explain the ability of tissues to facilitate or impede gas exchange. Structural changes and anisotropy of tissues may eventually lead to physiological disorders. A combined tomography and internal gas analysis during growth are needed to make progress on the understanding of void formation in fruit.

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Nitric Oxide Triggers Phosphatidic Acid Accumulation via Phospholipase D during Auxin-Induced Adventitious Root Formation in Cucumber.

Publication Date: 2008 May PMID: 18375601
Authors: Lanteri, M. L. - Laxalt, A. M. - Lamattina, L.
Journal: Plant Physiol

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transduction leading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR) formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- and NO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with (32)P-inorganic phosphate and treated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in the presence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs were separated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Both auxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or 30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation via phospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR number compared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally, exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA is an early signaling event during AR formation induced by auxin and NO in cucumber explants.

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Principal transcriptional programs regulating plant amino Acid metabolism in response to abiotic stresses.

Publication Date: 2008 May PMID: 18375600
Authors: Less, H. - Galili, G.
Journal: Plant Physiol

Using a bioinformatics analysis of public Arabidopsis (Arabidopsis thaliana) microarray data, we propose here a novel regulatory program, combining transcriptional and posttranslational controls, which participate in modulating fluxes of amino acid metabolism in response to abiotic stresses. The program includes the following two components: (1) the terminal enzyme of the module, responsible for the first catabolic step of the amino acid, whose level is stimulated or repressed in response to stress cues, just-in-time when the cues arrive, principally via transcriptional regulation of its gene; and (2) the initiator enzyme of the module, whose activity is principally modulated via posttranslational allosteric feedback inhibition in response to changes in the level of the amino acid, just-in-case when it occurs in response to alteration in its catabolism or sequestration into different intracellular compartments. Our proposed regulatory program is based on bioinformatics dissection of the response of all biosynthetic and catabolic genes of seven different pathways, involved in the metabolism of 11 amino acids, to eight different abiotic stresses, as judged from modulations of their mRNA levels. Our results imply that the transcription of the catabolic genes is principally more sensitive than that of the biosynthetic genes to fluctuations in stress-associated signals. Notably, the only exception to this program is the metabolic pathway of Pro, an amino acid that distinctively accumulates to significantly high levels under abiotic stresses. Examples of the biological significance of our proposed regulatory program are discussed.

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The Pea DELLA Proteins LA and CRY Are Important Regulators of Gibberellin Synthesis and Root Growth.

Publication Date: 2008 May PMID: 18375599
Authors: Weston, D. E. - Elliott, R. C. - Lester, D. R. - Rameau, C. - Reid, J. B. - Murfet, I. C. - Ross, J. J.
Journal: Plant Physiol

The theory that bioactive gibberellins (GAs) act as inhibitors of inhibitors of plant growth was based originally on the slender pea (Pisum sativum) mutant (genotype la cry-s), but the molecular nature of this mutant has remained obscure. Here we show that the genes LA and CRY encode DELLA proteins, previously characterized in other species (Arabidopsis [Arabidopsis thaliana] and several grasses) as repressors of growth, which are destabilized by GAs. Mutations la and cry-s encode nonfunctional proteins, accounting for the fact that la cry-s plants are extremely elongated, or slender. We use the la and cry-s mutations to show that in roots, DELLA proteins effectively promote the expression of GA synthesis genes, as well as inhibit elongation. We show also that one of the DELLA-regulated genes is a second member of the pea GA 3-oxidase family, and that this gene appears to play a major role in pea roots.

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Increased Air Temperature during Simulated Autumn Conditions Impairs Photosynthetic Electron Transport between Photosystem II and Photosystem I.

Publication Date: 2008 May PMID: 18375598
Authors: Busch, F. - Huner, N. P. - Ensminger, I.
Journal: Plant Physiol

Changes in temperature and daylength trigger physiological and seasonal developmental processes that enable evergreen trees of the boreal forest to withstand severe winter conditions. Climate change is expected to increase the autumn air temperature in the northern latitudes, while the natural decreasing photoperiod remains unaffected. As shown previously, an increase in autumn air temperature inhibits CO(2) assimilation, with a concomitant increased capacity for zeaxanthin-independent dissipation of energy exceeding the photochemical capacity in Pinus banksiana. In this study, we tested our previous model of antenna quenching and tested a limitation in intersystem electron transport in plants exposed to elevated autumn air temperatures. Using a factorial design, we dissected the effects of temperature and photoperiod on the function as well as the stoichiometry of the major components of the photosynthetic electron transport chain in P. banksiana. Natural summer conditions (16-h photoperiod/22 degrees C) and late autumn conditions (8-h photoperiod/7 degrees C) were compared with a treatment of autumn photoperiod with increased air temperature (SD/HT: 8-h photoperiod/22 degrees C) and a treatment with summer photoperiod and autumn temperature (16-h photoperiod/7 degrees C). Exposure to SD/HT resulted in an inhibition of the effective quantum yield associated with a decreased photosystem II/photosystem I stoichiometry coupled with decreased levels of Rubisco. Our data indicate that a greater capacity to keep the primary electron donor of photosystem I (P700) oxidized in plants exposed to SD/HT compared with the summer control may be attributed to a reduced rate of electron transport from the cytochrome b(6)f complex to photosystem I. Photoprotection under increased autumn air temperature conditions appears to be consistent with zeaxanthin-independent antenna quenching through light-harvesting complex II aggregation and a decreased efficiency in energy transfer from the antenna to the photosystem II core. We suggest that models that predict the effect of climate change on the productivity of boreal forests must take into account the interactive effects of photoperiod and elevated temperatures.

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Disruption of the Arabidopsis circadian clock is responsible for extensive variation in the cold-responsive transcriptome.

Publication Date: 2008 May PMID: 18375597
Authors: Bieniawska, Z. - Espinoza, C. - Schlereth, A. - Sulpice, R. - Hincha, D. K. - Hannah, M. A.
Journal: Plant Physiol

In plants, low temperature causes massive transcriptional changes, many of which are presumed to be involved in the process of cold acclimation. Given the diversity of developmental and environmental factors between experiments, it is surprising that their influence on the identification of cold-responsive genes is largely unknown. A systematic investigation of genes responding to 1 d of cold treatment revealed that diurnal- and circadian-regulated genes are responsible for the majority of the substantial variation between experiments. This is contrary to the widespread assumption that these effects are eliminated using paired diurnal controls. To identify the molecular basis for this variation, we performed targeted expression analyses of diurnal and circadian time courses in Arabidopsis (Arabidopsis thaliana). We show that, after a short initial cold response, in diurnal conditions cold reduces the amplitude of cycles for clock components and dampens or disrupts the cycles of output genes, while in continuous light all cycles become arrhythmic. This means that genes identified as cold-responsive are dependent on the time of day the experiment was performed and that a control at normal temperature will not correct for this effect, as was postulated up to now. Time of day also affects the number and strength of expression changes for a large number of transcription factors, and this likely further contributes to experimental differences. This reveals that interactions between cold and diurnal regulation are major factors in shaping the cold-responsive transcriptome and thus will be an important consideration in future experiments to dissect transcriptional regulatory networks controlling cold acclimation. In addition, our data revealed differential effects of cold on circadian output genes and a unique regulation of an oscillator component, suggesting that cold treatment could also be an important tool to probe circadian and diurnal regulatory mechanisms.

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SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth.

Publication Date: 2008 May PMID: 18375596
Authors: Bhatia, S. - Gangappa, S. N. - Kuswaha, R. - Kundu, S. - Chattopadhyay, S.
Journal: Plant Physiol

Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis.

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Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity.

Publication Date: 2008 May PMID: 18359848
Authors: Nakamura, T. - Ishikawa, M. - Nakatani, H. - Oda, A.
Journal: Plant Physiol

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.

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Elucidating the germination transcriptional program using small molecules.

Publication Date: 2008 May PMID: 18359847
Authors: Bassel, G. W. - Fung, P. - Chow, T. F. - Foong, J. A. - Provart, N. J. - Cutler, S. R.
Journal: Plant Physiol

The transition from seed to seedling is mediated by germination, a complex process that starts with imbibition and completes with radicle emergence. To gain insight into the transcriptional program mediating germination, previous studies have compared the transcript profiles of dry, dormant, and germinating after-ripened Arabidopsis (Arabidopsis thaliana) seeds. While informative, these approaches did not distinguish the transcriptional responses due to imbibition, shifts in metabolism, or breaking of dormancy from those triggered by the initiation of germination. In this study, three mechanistically distinct small molecules that inhibit Arabidopsis seed germination (methotrexate, 2, 4-dinitrophenol, and cycloheximide) were identified using a small-molecule screen and used to probe the germination transcriptome. Germination-responsive transcripts were defined as those with significantly altered transcript abundance across all inhibitory treatments with respect to control germinating seeds, using data from ATH1 microarrays. This analysis identified numerous germination regulators as germination responsive, including the DELLA proteins GAI, RGA, and RGL3, the abscisic acid-insensitive proteins ABI4, ABI5, ABI8, and FRY1, and the gibberellin receptor GID1A. To help visualize these and other publicly available seed microarray data, we designed a seed mRNA expression browser using the electronic Fluorescent Pictograph platform. An overall decrease in gene expression and a 5-fold greater number of transcripts identified as statistically down-regulated in drug-inhibited seeds point to a role for mRNA degradation or turnover during seed germination. The genes identified in our study as responsive to germination define potential uncharacterized regulators of this process and provide a refined transcriptional signature for germinating Arabidopsis seeds.

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The Ionic Environment Controls the Contribution of the Barley HvHAK1 Transporter to Potassium Acquisition.

Publication Date: 2008 May PMID: 18359846
Authors: Fulgenzi, F. R. - Peralta, M. L. - Mangano, S. - Danna, C. H. - Vallejo, A. J. - Puigdomenech, P. - Santa-Maria, G. E.
Journal: Plant Physiol

The control of potassium (K(+)) acquisition is a critical requirement for plant growth. Although HAK1 (high affinity K(+) 1) transporters provide a pathway for K(+) acquisition, the effect exerted by the ionic environment on their contribution to K(+) capture remains essentially unknown. Here, the influence of the ionic environment on the accumulation of transcripts coding for the barley (Hordeum vulgare) HvHAK1 transporter as well as on HvHAK1-mediated K(+) capture has been examined. In situ mRNA hybridization studies show that HvHAK1 expression occurs in most root cells, being augmented at the outermost cell layers. Accumulation of HvHAK1 transcripts is enhanced by K(+) deprivation and transiently by exposure to high salt concentrations. In addition, studies on the accumulation of transcripts coding for HvHAK1 and its close homolog HvHAK1b revealed the presence of two K(+)-responsive pathways, one repressed and the other insensitive to ammonium. Experiments with Arabidopsis (Arabidopsis thaliana) HvHAK1-expressing transgenic plants showed that K(+) deprivation enhances the capture of K(+) mediated by HvHAK1. A detailed study with HvHAK1-expressing Saccharomyces cerevisiae cells also revealed an increase of K(+) uptake after K(+) starvation. This increase did not occur in cells grown at high Na(+) concentrations but took place for cells grown in the presence of NH(4)(+). 3,3'-Dihexyloxacarbocyanine iodide accumulation measurements indicate that the increased capture of K(+) in HvHAK1-expressing yeast cells cannot be explained only by changes in the membrane potential. It is shown that the yeast protein phosphatase PPZ1 as well as the halotolerance HAL4/HAL5 kinases negatively regulate the HvHAK1-mediated K(+) transport.

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