Plant Physiology

WAVY LEAF 1, an Ortholog of Arabidopsis HEN1, Regulates Shoot Development by Maintaining microRNA and trans-acting siRNA Accumulation in Rice.

Publication Date: 2010 Aug 30 PMID: 20805329
Authors: Abe, M. - Yoshikawa, T. - Nosaka, M. - Sakakibara, H. - Sato, Y. - Nagato, Y. - Itoh, J. I.
Journal: Plant Physiol

In rice, trans-acting siRNA (ta-siRNA) is essential for shoot development, including SAM formation and leaf morphogenesis. The rice wavy leaf 1 (waf1) mutant has been identified as an embryonic mutant resembling shoot organization 1 (sho1) and sho2, homologs of a loss-of-function mutant of DCL4 and a hypomorphic mutant of AGO7, respectively, which both act in the ta-siRNA production pathway. About half of the waf1 mutants showed seedling lethality due to defects in SAM maintenance, but the rest survived to the reproductive phase and exhibited pleiotropic phenotypes in leaf morphology and floral development. Map-based cloning of WAF1 revealed that it encodes an RNA methyltransferase, a homolog of Arabidopsis HEN1. The reduced accumulation of small RNAs in waf1 indicated that the stability of the small RNA was decreased. Despite the greatly reduced level of miRNAs and ta-siRNA, microarray and RT-PCR experiments revealed that the expression levels of their target genes were not always enhanced. A double mutant between sho and waf1 showed an enhanced SAM defect, suggesting that the amount and/or quality of ta-siRNA is crucial for SAM maintenance. Our results indicate that stabilization of small RNAs by WAF1 is indispensable for rice development, especially for SAM maintenance and leaf morphogenesis governed by the ta-siRNA pathway. In addition, the inconsistent relationship between the amount of small RNAs and the level of the target mRNA in waf1 suggest that there is a complex regulatory mechanism that modifies the effects of miRNA/ta-siRNA on the expression of the target gene.

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Arabidopsis Calcium-Dependent Protein Kinase AtCPK10 Functions in ABA and Ca2+-Mediated Stomatal Regulation in Response to Drought Stress.

Publication Date: 2010 Aug 30 PMID: 20805328
Authors: Zou, J. J. - Wei, F. J. - Wang, C. - Wu, J. J. - Ratnasekera, D. - Liu, W. X. - Wu, W. H.
Journal: Plant Physiol

Plant calcium-dependent protein kinases (CDPKs) may function as calcium sensors and play important roles in regulation of plant growth and development and in plant responses to biotic and abiotic stresses. The Arabidopsis genome encodes 34 CDPKs and most of them have not been functionally characterized. Here we report the functional characterization of CPK10 in Arabidopsis response to drought stress. The cpk10 mutant, a T-DNA insertion mutant for Arabidopsis CPK10 gene, showed much more sensitive phenotype to drought stress compared with wild-type plants, while the CPK10 overexpression lines displayed enhanced tolerance to drought stress. Induction of stomatal closure and inhibition of stomatal opening by ABA and Ca(2+) were impaired in the cpk10 mutants. Using the yeast two-hybrid methods, a heat shock protein HSP1 was identified as a CPK10-interacting protein. The interaction between CPK10 and HSP1 was further confirmed by pull-down and bimolecular fluorescence complementation (BiFC) assays. The HSP1 knock-out mutant (hsp1) plants showed similar sensitive phenotype under drought stress as the cpk10 mutant plants, and were similarly less sensitive to ABA and Ca(2+) in regulation of stomatal movements. Electrophysiological experiments showed that ABA- and Ca(2+)-inhibition of the inward K(+) currents in stomatal guard cells were impaired in the cpk10 and hsp1 mutants. All presented data demonstrated that CPK10, possibly via interacting with HSP1, plays important roles in ABA and Ca(2+)-mediated regulation of stomatal movements.

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Arabidopsis bZIP transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development.

Publication Date: 2010 Aug 30 PMID: 20805327
Authors: Murmu, J. - Bush, M. J. - Delong, C. - Li, S. - Xu, M. - Khan, M. - Malcolmson, C. - Fobert, P. R. - Zachgo, S. - Hepworth, S. R.
Journal: Plant Physiol

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis anther development. We show here that plants lacking the bZIP transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within non-dehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

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Lack of {alpha}-xylosidase activity in Arabidopsis alters xyloglucan composition and results in growth defects.

Publication Date: 2010 Aug 26 PMID: 20801759
Authors: Sampedro, J. - Pardo, B. - Gianzo, C. - Guitian, E. - Revilla, G. - Zarra, I.
Journal: Plant Physiol

Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose sidechains block the degradation of the backbone and alpha-xylosidase activity is necessary to remove them. Two Arabidopsis thaliana mutant lines with insertions in the alpha-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of alpha-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in accumulation of XXXG and XXLG in the liquid growth media of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan beta-galactosidase. In addition the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the non-reducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.

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SHORT-ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S-phase progression of the cell cycle.

Publication Date: 2010 Aug 25 PMID: 20739610
Authors: Dhondt, S. - Coppens, F. - De Winter, F. - Swarup, K. - Merks, R. M. - Inze, D. - Bennett, M. J. - Beemster, G. T.
Journal: Plant Physiol

SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis thaliana root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the cessation of root growth. This manuscript reports on the role of SHR and SCR in the development of leaves, which, in contrast to the root, have a determinate growth pattern and lack a persistent stem-cell niche. Our results demonstrate that inhibition of leaf growth in shr and scr mutants is not a secondary effect of the compromised root development, but is caused by an effect on cell division in the leaves: a reduced cell division rate and early exit of proliferation phase. Consistent with the observed cell division phenotype, the expression of SHR and SCR genes in leaves is closely associated with cell division activity in most cell types. The increased cell cycle duration is due to a prolonged S-phase duration, which is mediated by up-regulation of cell cycle inhibitors known to restrain the activity of the transcription factor, E2Fa. Therefore, we conclude that, in contrast to their specific role in cortex/endodermis differentiation and stem cell maintenance in the root, SHR and SCR primarily function as general regulators of cell proliferation in leaves.

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Plant cell growth in tissue.

Publication Date: 2010 Aug 25 PMID: 20739609
Authors: Ortega, J. K.
Journal: Plant Physiol

Cell walls are part of the apoplasm pathway that transports water, solutes, and nutrients to cells within plant tissue. Pressures within the apoplasm (cell walls and xylem) are often times different from atmospheric pressure during expansive growth of plant cells in tissue. The previously established Augmented Growth Equations are modified to evaluate the turgor pressure, water uptake, and expansive growth of plant cells in tissue when pressures within the apoplasm are lower and higher than atmospheric pressure. Analyses indicate that a step-down and step-up in pressure within the apoplasm will cause an exponential decrease and increase in turgor pressure, respectively, and the rates of water uptake and expansive growth each undergo a rapid decrease and increase, respectively, followed by an exponential return to their initial magnitude. Other analyses indicate that pressure within the apoplasm decreases exponentially to a lower value after a step-down in turgor pressure, which simulates its behavior after an increase in expansive growth rate. Also, analyses indicate that the turgor pressure decays exponentially to a constant value which is the sum of the critical turgor pressure and pressure within the apoplasm during stress-relaxation experiments in which pressures within the apoplasm are not atmospheric pressure. Additional analyses indicate that when the turgor pressure is constant (clamped), a decrease in pressure within the apoplasm elicits an increase in elastic expansion followed by an increase in irreversible expansion rate. Some analytical results are supported by prior experimental research and other analytical results can be verified with existing experimental methods.

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Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato.

Publication Date: 2010 Aug 24 PMID: 20736383
Authors: Bhaskar, P. B. - Wu, L. - Busse, J. S. - Whitty, B. R. - Hamernik, A. J. - Jansky, S. H. - Buell, C. R. - Bethke, P. C. - Jiang, J.
Journal: Plant Physiol

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4 degrees C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

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ATP-binding cassette transporter G26 (ABCG26) is required for male fertility and pollen exine formation in Arabidopsis thaliana.

Publication Date: 2010 Aug 23 PMID: 20732973
Authors: Quilichini, T. D. - Friedmann, M. C. - Samuels, A. L. - Douglas, C. J.
Journal: Plant Physiol

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires transport of one or more sporopollenin constituents to the surface of developing microspores. Here we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis thaliana. abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early in pollen wall formation, sporopollenin biosynthesis and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development, and is consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

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Antisense down-regulation of 4CL expression alters lignification, tree growth and saccharification potential of field-grown poplar.

Publication Date: 2010 Aug 23 PMID: 20729393
Authors: Voelker, S. L. - Lachenbruch, B. - Meinzer, F. C. - Jourdes, M. - Ki, C. - Patten, A. M. - Davin, L. B. - Lewis, N. G. - Tuskan, G. A. - Gunter, L. - Decker, S. R. - Selig, M. J. - Sykes, R. - Himmel, M. E. - Kitin, P. - Shevchenko, O. - Strauss, S. H.
Journal: Plant Physiol

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplars (Populus), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below ~50% of the non-transgenic control. Relatively small lignin reductions of greater, similar10% were also associated with reduced productivity, decreased wood S/G (syringyl/guaiacyl) lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than ~50% 4CL RNA expression were also characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

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Reproductive development modulates gene expression and metabolite levels with possible feedback inhibition of artemisinin in Artemisia annua L.

Publication Date: 2010 Aug 19 PMID: 20724645
Authors: Arsenault, P. R. - Vail, D. R. - Wobbe, K. K. - Erickson, K. - Weathers, P. J.
Journal: Plant Physiol

The relationship between the transition to budding and flowering in A. annua and the production of the antimalarial sesquiterpene, artemisinin (AN), the dynamics of artemisinic metabolite changes, AN related transcriptional changes, and plant and trichome developmental changes were measured. Maximum production of AN occurs during full flower stage within floral tissues, but that changes in the leafy bracts and non-bolt leaves as the plant shifts from budding to full flower. Expression levels of early pathway genes known to be involved in isopentenyl diphosphate and farnesyl diphosphate biosynthesis leading to AN were not immediately positively correlated with either AN or its precursors. However, we found that the later AN pathway genes, amorpha-4,11-diene synthase (ADS) and the P450, CYP71AV1 (CYP), were more highly correlated with AN's immediate precursor DHAA within all leaf tissues tested. In addition, leaf trichome formation throughout the developmental phases of the plant also appears to be more complex than originally thought. Trichome changes correlated closely with the levels of AN but not its precursors. Differences were observed in trichome densities that are dependant both on developmental stage (vegetative, budding, and flowering) and on position (upper and lower leaf tissue). AN levels declined significantly as plants matured as did ADS and CYP transcripts. Spraying leaves with AN or artemisinic acid (AA) inhibited CYP transcription; AA also inhibited ADS transcription. These data allow us to present a novel model for differential control of AN biosynthesis as it results to developmental stage and trichome maturation and collapse.

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Non-reductive iron uptake mechanism in the marine alveolate Chromera velia.

Publication Date: 2010 Aug 19 PMID: 20724644
Authors: Sutak, R. - Slapeta, J. - San Roman, M. - Camadro, J. M. - Lesuisse, E.
Journal: Plant Physiol

Chromera velia is a newly cultured photosynthetic marine alveolate. This micro-alga has a high iron requirement for respiration and photosynthesis, although its natural environment contains less than 1 nM of this metal. We found that this organism uses a novel mechanism of iron uptake, differing from the classic reductive and siderophore-mediated iron uptake systems characterized in the model yeast Saccharomyces cerevisiae and present in most yeasts and terrestrial plants. C. velia has no trans-plasma membrane electron transfer system, and thus cannot reduce extracellular ferric chelates. It is also unable to use hydroxamate siderophores as iron sources. Iron uptake from ferric citrate by C. velia is not inhibited by a ferrous chelator, but the rate of uptake is strongly decreased by increasing the ferric ligand (citrate) concentration. The cell wall contains a large number of iron binding sites, allowing the cells to concentrate iron in the vicinity of the transport sites. We describe a model of iron uptake in which aqueous ferric ions are first concentrated in the cell wall before being taken up by the cells without prior reduction. We discuss our results in relation to the strategies used by the phytoplankton to take up iron in the oceans.

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13C tracer and GC-MS analysis reveal metabolic flux distribution in oleaginous microalga Chlorella protothecoides.

Publication Date: 2010 Aug 20 PMID: 20720172
Authors: Xiong, W. - Liu, L. - Wu, C. - Yang, C. - Wu, Q.
Journal: Plant Physiol

The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-(13)C]/[1-(13)C] glucose were performed with heterotrophic growth of C.protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. GC-CMS analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway and the TCA cycle with inactive glyoxylate shunt. Evidences of phosphoenol pyruvate carboxylase, phosphoenol pyruvate carboxykinase and malic enzyme activity were also obtained. It was demonstrated that relative activity of pentose phosphate pathway to glycolysis under nitrogen limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C.protothecoides maintained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bio-products.

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Stacking multiple transgenes at a selected genomic site via repeated recombinase mediated DNA cassette exchanges.

Publication Date: 2010 Aug 18 PMID: 20720171
Authors: Li, Z. - Moon, B. P. - Xing, A. - Liu, Z. B. - McCardell, R. P. - Damude, H. G. - Flaco, S. C.
Journal: Plant Physiol

Recombinase mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene silencing cassette, designed to simultaneously silence soybean genes omega-6 desaturase (FAD2) and thioesterase 2 (FATB) to improve oleic acid (18:1) content, was first inserted by RMCE at a pre-characterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis, and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley high lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS) to improve the contents of essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four over-expression cassettes to the previously integrated FAD2-FATB gene silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

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Analysis of the rice mitochondrial carrier family reveals anaerobic accumulation of a basic amino acid carrier involved in arginine metabolism during seed germination.

Publication Date: 2010 Aug 18 PMID: 20720170
Authors: Taylor, N. L. - Howell, K. A. - Heazlewood, J. L. - Tan, T. Y. - Narsai, R. - Huang, S. - Whelan, J. - Millar, A. H.
Journal: Plant Physiol

Given the substantial changes in mitochondrial gene expression, the mitochondrial proteome and respiratory function during rice germination under anaerobic or aerobic conditions, we have attempted to identify changes in mitochondrial membrane transport capacity during these processes. We have assembled a preliminary rice mitochondrial carrier gene family of 50 members, defined its orthology to carriers of known function, and observe significant changes in microarray expression data for these rice genes during germination under aerobic and anaerobic conditions and across rice development. To determine if these transcript changes reflect alteration of the carrier profile itself and to determine which members of the family encode the major mitochondrial carrier proteins, we analysed mitochondrial integral membrane protein preparations using SDS-PAGE and peptide mass spectrometry, identifying seven distinct carrier proteins. We have used mass spectrometry-based quantitative approaches to compare the abundance of these carriers between mitochondria from dry seed and those from aerobic- or anaerobic-germinated seeds. We highlight an anaerobic-enhanced basic amino acid carrier and show concomitant increases in mitochondrial arginase and the abundance of Arg and Orn in anaerobic-germinated seeds, consistent with an anaerobic role of this mitochondria carrier. The potential role of this carrier in facilitating mitochondrial involvement in arginine metabolism and the plant urea cycle during growth of rice coleoptiles and early seed nitrate assimilation under anaerobic conditions are discussed.

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The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia.

Publication Date: 2010 Aug 16 PMID: 20713618
Authors: Anderson, J. P. - Lichtenzveig, J. - Gleason, C. - Oliver, R. P. - Singh, K. B.
Journal: Plant Physiol

The fungal necrotrophic pathogen Rhizoctonia solani (Kuhn) is a significant constraint to a range of crops as diverse as cereals, canola and legumes. Despite wide ranging germplasm screens in many of these crops, no strong genetic resistance has been identified suggesting alternative strategies to improve resistance are required. In this study we characterise moderate resistance to R. solani AG8 identified in Medicago truncatula. The activity of the ethylene and jasmonate responsive GCC box promoter element was associated with the moderate resistance as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Over-expression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis but not root knot nematode. These results indicate that targeting specific regulators of ethylene defence may enhance resistance to an important subset of root pathogens. We also demonstrate that over-expression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background while over-expression in skl reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis ubiquitously over-expressing B-3 ERFs, over-expression of MtERF-1 in M. truncatula roots did not show adverse effects on plant development.

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Tethering factors required for cytokinesis in Arabidopsis.

Publication Date: 2010 Aug 16 PMID: 20713617
Authors: Thellmann, M. - Rybak, K. - Thiele, K. - Wanner, G. - Assaad, F. F.
Journal: Plant Physiol

At the end of the cell cycle, the nascent cross wall is laid down within a transient membrane compartment referred to as the cell plate. Tethering factors, which act by capturing vesicles and holding them in the vicinity of their target membranes, are likely to play an important role in the first stages of cell plate assembly. Factors required for cell plate biogenesis, however, remain to be identified. In this study, we used a reverse genetic screen to isolate tethering factors required for cytokinesis in Arabidopsis. We focused on the TRAPPI and TRAPPII tethering complexes, which are thought to be required for the flow of traffic through the Golgi and for Trans Golgi Network (TGN) function, as well as on the GARP complex, thought to be required for the tethering of endocytotic vesicles to the TGN. We found weak cytokinesis defects in some TRAPPI mutants and strong cytokinesis defects in all the TRAPPII lines we surveyed. Indeed, four insertion lines at the TRAPPII locus AtTRS120 had canonical cytokinesis defective seedling lethal phenotypes, including cell wall stubs and incomplete cross walls. Confocal and electron microscopy showed that in trs120 mutants, vesicles accumulated at the equator of dividing cells yet failed to assemble into a cell plate. This shows that AtTRS120 is required for cell plate biogenesis. In contrast to the TRAPP complexes, we found no conclusive evidence for cytokinesis defects in seven GARP insertion lines. We discuss the implications of these findings for the origin and identity of cell plate membranes.

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Co-expression analysis identifies Rice Starch Regulator1 (RSR1), a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator.

Publication Date: 2010 Aug 16 PMID: 20713616
Authors: Fu, F. F. - Xue, H. W.
Journal: Plant Physiol

Starch biosynthesis is important for plant development and is a critical factor in crop quality and nutrition. As a complex metabolic pathway, regulation of starch biosynthesis is still poorly understood. We here present the identification of candidate regulators for starch biosynthesis by gene co-expression analysis in rice (Oryza sativa). Starch synthesis genes can be grouped into type I (in seeds, sink tissues) and type II (in vegetative tissues, source tissues), and 307 and 621 co-expressed genes are putatively involved in the regulation of starch biosynthesis in rice seeds and vegetative tissues, respectively. Among these genes, Rice Starch Regulator1 (RSR1), an AP2/EREBP family transcription factor, was found to negatively regulate the expression of type I starch synthesis genes, and RSR1 deficiency results in the enhanced expression of starch synthesis genes in seeds. Seeds of the knockout mutant rsr1 consistently show the increased amylose content and altered fine structure of amylopectin, and consequently form the round and loosely packed starch granules, resulting in decreased gelatinization temperature. In addition, rsr1 mutants have a larger seed size and increased seed mass and yield. In contrast, RSR1-overexpression suppresses expression of starch synthesis genes, resulting in the altered amylopectin structure and increased gelatinization temperature. Interestingly, decreased proportion of A chains in rsr1 results in abnormal starch granules but reduced gelatinization temperature, whereas increased proportions of A chains in RSR1-overexpressing plants lead to higher gelatinization temperatures, which is novel and different from the previous reports, further indicating the complicated regulation of starch synthesis and determination of the physico-chemical properties of starch. These results demonstrate the potential of co-expression analysis for studying rice starch biosynthesis and the regulation of a complex metabolic pathway, and provide informative clue, including the characterization of RSR1, to facilitate the improvement of rice quality and nutrition.

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Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning, Sugar Signalling and Seed Yield in Arabidopsis.

Publication Date: 2010 Aug 13 PMID: 20709831
Authors: Wingenter, K. - Schulz, A. - Wormit, A. - Wic, S. - Trentmann, O. - Hoermiller, I. I. - Heyer, A. G. - Marten, I. - Hedrich, R. - Neuhaus, E.
Journal: Plant Physiol

The extent to which vacuolar sugar transport activity affects molecular, cellular and developmental processes in Arabidopsis is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton coupled monosaccharide import into isolated mesophyll vacuoles, in comparison to wild type vacuoles. TMT1 overexpressor mutants grew faster than wild type plants on soil and in high glucose containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by non-aqueous fractionation, and by CAB1 and NR1 gene expression studies. Soil grown TMT1 overexpressor plants respired less glucose than wild type plants, and only about half the amount of glucose respired by tmt1-2::tDNA mutants. In sum, these data showed that TMT activity in wild type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO(2) release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as glucose repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic glucose levels than wild type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased biomass of Arabidopsis seeds and accelerated early plant development.

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ABA-deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Botrytis cinerea.

Publication Date: 2010 Aug 13 PMID: 20709830
Authors: Curvers, K. - Seifi, H. - Mouille, G. - De Rycke, R. - Asselbergh, B. - Van Hecke, A. - Vanderschaeghe, D. - Hofte, H. R. - Callewaert, N. - Van Breusegem, F. - Hofte, M. M.
Journal: Plant Physiol

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells which is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared to the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological and ultrastructural level. We demonstrate that ABA-deficiency in the mutant leads to increased cuticle permeability which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. Sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.

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Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis thaliana mesophyll protoplasts.

Publication Date: 2010 Aug 13 PMID: 20709829
Authors: Kim, J. - Somers, D. E.
Journal: Plant Physiol

Rapid assessment of the effect of reduced levels of gene products is often a bottleneck in determining how to proceed with an interesting gene candidate. Additionally, gene families with closely related members can confound determination of the role of even a single one of the group. We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs (amiRNAs) in Arabidopsis thaliana mesophyll protoplasts. We use a luciferase-based reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently co-transfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than six days. Using both amiRNA plasmids available through the ABRC, as well as custom-design of constructs using the Weigel amiRNA design algorithm, we show that transient knockdown of known clock genes recapitulates the same circadian phenotypes reported in the literature for loss-of-function mutant plants. We additionally show that amiRNA designed to knockdown expression of the casein kinase 2 beta subunit (CKB) gene family lengthens period, consistent with previous reports of a short period in CKB overexpressors. Our results demonstrate that this system can facilitate a much more rapid analysis of gene function by obviating the need to initially establish stably transformed transgenics to assess the phenotype of gene knockdowns. This approach will be useful in a wide range of plant disciplines when an endogenous cell-based phenotype is observable or can be devised, as done here using a luciferase reporter.

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Towards in vivo chemical imaging of epicuticular waxes.

Publication Date: 2010 Aug 13 PMID: 20709828
Authors: Weissflog, I. - Vogler, N. - Akimov, D. - Dellith, A. - Schachtschabel, D. - Svatos, A. - Boland, W. - Dietzek, B. - Popp, J.
Journal: Plant Physiol

Epicuticular waxes, which are found on the outer surface of plant cuticles, are difficult to study in vivo. To monitor the growth, development and structural alterations of epicuticular wax layers, coherent anti-Stokes Raman scattering (CARS) might be used. CARS, as a Raman based technique, not only provides structural insight but also chemical information by imaging the spatial distribution of Raman-active vibrations. Here, we present a comparative study using CARS and scanning electron microscopy (SEM) to characterize the structure of epicuticular waxes. The ability of CARS to provide detailed structural information on the biologically important wax layer was detailed on the examples of Prunus laurocerasus, Hoya carnosa and Monstera sp. aff. deliciosa Liebm. We anticipate that the work presented will open a doorway for online monitoring of formation and alterations of epicuticular wax layers.

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The seed composition of Arabidopsis mutants for the group 3 sulfate transporters indicates a role in sulfate translocation within developing seeds.

Publication Date: 2010 Aug 11 PMID: 20702726
Authors: Zuber, H. - Davidian, J. C. - Aubert, G. - Aime, D. - Belghazi, M. - Lugan, R. - Heintz, D. - Wirtz, M. - Hell, R. - Thompson, R. - Gallardo, K.
Journal: Plant Physiol

Sulfate is required for the synthesis of sulfur-containing amino acids and numerous other compounds essential for the plant life cycle. The delivery of sulfate to seeds and its translocation between seed tissues is likely to require specific transporters. In Arabidopsis, the group 3 plasmalemma-predicted sulfate transporters (SULTR3) comprise five genes, all expressed in developing seeds, especially in the tissues surrounding the embryo. Here, we show that sulfur supply to seeds is unaffected by T-DNA insertions in the SULTR3 genes. However, remarkably, an increased accumulation of sulfate was found in mature seeds of four mutants out of five. In these mutant seeds, the ratio of sulfur in sulfate form versus total sulfur was significantly increased, accompanied by a reduction in free cysteine content which varied depending on the gene inactivated. These results demonstrate a reduced capacity of the mutant seeds to metabolize sulfate and suggest these transporters may be involved in sulfate translocation between seed compartments. This was further supported by sulfate measurements of the envelopes separated from the embryo of the sultr3;2 mutant seeds, which showed differences in sulfate partitioning compared to wild-type. A dissection of the seed proteome of the sultr3 mutants revealed protein changes characteristic of a sulfur-stress response, supporting a role for these transporters in providing sulfate to the embryo. The mutants were affected in 12S globulin accumulation, demonstrating the importance of intra-seed sulfate transport for synthesis and maturation of embryo proteins. Metabolic adjustments were also revealed, some of which could release sulfur from glucosinolates.

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A Suite of Lotus japonicus Starch Mutants Reveals both Conserved and Novel Features of Starch Metabolism.

Publication Date: 2010 Aug 10 PMID: 20699404
Authors: Vriet, C. - Welham, T. - Brachmann, A. - Pike, M. - Pike, J. - Perry, J. - Parniske, M. - Sato, S. - Tabata, S. - Smith, A. M. - Wang, T. L.
Journal: Plant Physiol

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterised a comprehensive collection of forward and TILLING reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADPglucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12 h photoperiod. In contrast, the loss of GWD1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative re-growth.

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The miR159 regulated GAMYB-like genes inhibit growth and promote Programmed Cell Death in Arabidopsis.

Publication Date: 2010 Aug 11 PMID: 20699403
Authors: Alonso-Peral, M. M. - Li, J. - Li, Y. - Allen, R. S. - Schnippenkoetter, W. - Ohms, S. - White, R. L. - Millar, A. A.
Journal: Plant Physiol

The microRNA159 family represses the conserved GAMYB-like genes that encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) signalling in anthers and germinating seeds. In Arabidopsis the two major miR159 family members, miR159a and miR159b are functionally specific for two GAMYB-like genes, MYB33 and MYB65. These transcription factors have been shown to be involved in anther development, but there are conflicting reports about their role in the promotion of flowering and little is known about their function in seed germination. To understand the function of this pathway, we identified the genes and processes controlled by these GAMYB-like genes. Firstly, we demonstrate that miR159 completely represses MYB33 and MYB65 in vegetative tissues. We show that GA does not release this repression and that these transcription factors are not required for flowering or growth. In contrast, the de-regulation of MYB33 and MYB65 in vegetative tissues in the absence of miR159 up-regulates genes that are GA-induced and highly expressed in the aleurone during seed germination. Confirming these genes are GAMYB-like regulated, their expression was dramatically reduced in myb33.myb65.myb101 seeds. Aleurone vacuolation, a GA-mediated programmed cell death (PCD) process required for germination, was impaired in these seeds. Finally, the de-regulation of MYB33 and MYB65 in vegetative tissues inhibits growth by reducing cell proliferation. Therefore, we conclude that miR159 acts as a molecular switch only permitting the expression of GAMYB-like genes in anthers and seeds. In seeds, these transcription factors participate in GA-induced pathways required for aleurone development and death.

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Leaf senescence signaling: The Ca2+-conducting Arabidopsis cyclic nucleotide gated channel2 acts through nitric oxide to repress senescence programming.

Publication Date: 2010 Aug 24 PMID: 20699402
Authors: Ma, W. - Smigel, A. - Walker, R. K. - Moeder, W. - Yoshioka, K. - Berkowitz, G. A.
Journal: Plant Physiol

Ca(2+) and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca(2+). The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca(2+) modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of an NO donor. At present, the interrelationship between Ca(2+) and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca(2+) uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca(2+) accumulation is reduced in dnd1 leaves compared to wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence associated genes, H(2)O(2) generation, lipid peroxidation, tissue necrosis, and salicylic acid levels) were more prominent in dnd1 leaves compared to wild type. Application of a Ca(2+) channel blocker hastened senescence of detached wild type leaves maintained in the dark; increasing the rate of chlorophyll loss, expression of a senescence associated gene, and lipid peroxidation. Pharmacological manipulation of Ca(2+) signaling provides evidence consistent with genetic studies of the relationship between Ca(2+) signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild type plants. Application of an NO donor effectively rescues many dnd1 senescence related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca(2+) uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream 'basal' NO production in addition to its role (also linked to NO signaling) in pathogen defense responses, and that this NO generation acts as a negative regulator during plant leaf senescence signaling.

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A gain-of-function mutation in the Arabidopsis disease resistance gene RPP4 confers sensitivity to low temperature.

Publication Date: 2010 Aug 11 PMID: 20699401
Authors: Huang, X. - Li, J. - Bao, F. - Zhang, X. - Yang, S.
Journal: Plant Physiol

How plants adapt to low temperature is not well understood. To identify components involved in low temperature signaling, we characterized the previously isolated chilling-sensitive 2 mutant (chs2). This mutant grew normally at 22 degrees C, but showed phenotypes similar to activation of defense responses when shifted to temperatures below 16 degrees C. These phenotypes include yellowish and collapsed leaves, increased electrolyte leakage, up-regulation of PATHOGENESIS RELATED genes and accumulation of excess H(2)O(2) and salicylic acid (SA). Moreover, the chs2 mutant was seedling-lethal when germinated at or shifted for more than three days to low temperatures of 4-12 degrees C. Map-based cloning revealed that a single amino acid substitution occurred in the TIR-NB-LRR (Toll/Interleukin-1 receptor- nucleotide-binding leucine-rich repeat) type resistance (R) protein RPP4, which causes a deregulation of the R protein in a temperature-dependent manner. The chs2 mutation led to an increase in the mutated RPP4 mRNA transcript, activation of defense responses, and an induction of cell death at low temperatures. In addition, a chs2 intragenic suppressor, in which the mutation occurs in the conserved NB domain, abolished defense responses at lower temperatures. Genetic analyses of chs2 in combination with known SA pathway and immune signaling mutants indicate that the chs2-conferred temperature sensitivity requires EDS1, RAR1 and SGT1b, but does not require PAD4, NPR1 or SA. This study reveals that an activated TIR-NB-LRR protein has a large impact on temperature sensitivity in plant growth and survival.

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Endogenous ABA as a Key Switch for Natural Variation in Flooding-Induced Shoot Elongation.

Publication Date: 2010 Aug 10 PMID: 20699400
Authors: Chen, X. - Pierik, R. - Peeters, A. - Poorter, H. - Visser, E. J. - Huber, H. - de Kroon, H. - Voesenek, L. A.
Journal: Plant Physiol

Elongation of leaves and stem is a key trait for survival of terrestrial plants during shallow but prolonged floods that completely submerge the shoot. However, natural floods at different locations vary strongly in duration and depth, and, therefore, populations from these locations are subjected to different selection pressure, leading to intraspecific variation. Here, we identified the signal transduction component that causes response variation in shoot elongation among two accessions of the wetland plant Rumex palustris. These accessions differed twofold in petiole elongation rates upon submergence, with fast elongation found in a population from a river floodplain and slow elongation in plants from a lake bank. Fast petiole elongation under water consumes carbohydrates and depends on the (inter)action of the plant hormones ethylene, abscisic acid (ABA) and gibberellic acid (GA). We found that carbohydrate levels and dynamics in shoots did not differ between the fast and slow elongating plants, but that the level of ethylene-regulated ABA in petioles, and hence GA responsiveness of these petioles explained the difference in shoot elongation upon submergence. Since this is the exact signal transduction level that also explains the variation in flooding-induced shoot elongation among plant species (viz. R. palustris and R. acetosa), we suggest that natural selection results in similar modification of regulatory pathways within and between species.

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Evidence that High Activity of Vacuolar Invertase Is Required for Cotton Fiber and Arabidopsis Root Elongation through Osmotic Dependent and Independent Pathway, Respectively.

Publication Date: 2010 Aug 11 PMID: 20699399
Authors: Wang, L. - Li, X. R. - Lian, H. - Ni, D. A. - He, Y. K. - Chen, X. Y. - Ruan, Y. L.
Journal: Plant Physiol

Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due to, in part, the complexity of multi-cellular plant tissues. Here, we used cotton fibers, fast growing single-celled seed trichomes, to address this issue. VIN activity in fibers was ~~4-6-fold higher than that in leaves, stems and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Further, genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than that slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1 that displayed VIN sequence features and localized to vacuole. Once introduced to Arabidopsis, GhVIN1 complemented the short root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild-type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNAi or over-expression constructs reduced or enhanced fiber elongation, respectively. Together, the analyses provide a set of evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contribution of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively.

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Nitric Oxide Acts Downstream of Auxin to Trigger Root Ferric-Chelate Reductase Activity in Response to Iron Deficiency in Arabidopsis thaliana.

Publication Date: 2010 Aug 10 PMID: 20699398
Authors: Chen, W. W. - Yang, J. L. - Qin, C. - Jin, C. W. - Mo, J. H. - Ye, T. - Zheng, S. J.
Journal: Plant Physiol

In response to iron (Fe) deficiency, dicots employ a reduction-based mechanism by inducing ferric-chelate reductase (FCR) at the root plasma membrane to enhance Fe uptake. However, the signal pathway leading to FCR induction is still unclear. Here, we found that the Fe-deficiency-induced increase of auxin and nitric oxide (NO) levels in WT Arabidopsis was accompanied by up-regulation of root FCR activity, the bHLH transcription factor FIT and the ferric reductase FRO2 gene expression. This was further stimulated by application of exogenous auxin (NAA) or NO donor (GSNO), but suppressed by either polar auxin transport inhibition with NPA or NO scavenging with cPTIO, tungstate, or L-NAME. On the other hand, the root FCR activity, NO level and gene expression of FIT and FRO2 were higher in auxin-overproducing mutant yucca under Fe deficiency, which were sharply restrained by cPTIO treatment. The opposite response was observed in a basipetal auxin transport impaired mutant aux1-7, which was slightly rescued by exogenous GSNO application. Furthermore, Fe deficiency or NAA application failed to induce Fe deficiency responses in noa1 and nial nia2, two mutants with reduced NO synthesis, but root FCR activities in both mutants could be significantly elevated by GSNO. The inability to induce NO burst and FCR activity was further verified in a double mutant yucca noa1 with elevated auxin production and reduced NO accumulation. Therefore, we presented a novel signaling pathway where NO acts downstream of auxin to activate root FCR activity under Fe deficiency in Arabidopsis.

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The Plant-Specific SR45 Protein Negatively Regulates Glucose and ABA Signaling During Early Seedling Development in Arabidopsis.

Publication Date: 2010 Aug 10 PMID: 20699397
Authors: Carvalho, R. F. - Carvalho, S. D. - Duque, P.
Journal: Plant Physiol

The plant-specific SR45 belongs to the highly conserved family of serine/arginine-rich (SR) proteins, which play key roles in pre-mRNA splicing and other aspects of RNA metabolism. An Arabidopsis thaliana loss-of-function mutant, sr45-1, displays pleiotropic phenotypes, such as defects in flower and leaf morphology, root growth and flowering time. Here we show that the sr45-1 mutation confers hypersensitivity to glucose during early seedling growth in Arabidopsis. Unlike wild-type plants, the sr45-1 mutant displays impaired cotyledon greening and expansion as well as reduced hypocotyl elongation of dark grown seedlings when grown in the presence of low (3%) glucose concentrations. In addition, SR45 is involved in the control of glucose-responsive gene expression, as the mutant displays enhanced repression of photosynthetic and nitrogen metabolism genes and over induction of starch and anthocyanin biosynthesis genes. Like many other sugar-response mutants, sr45-1 also shows hypersensitivity to abscisic acid (ABA), but appears to be unaffected in ethylene signaling. Importantly, the sr45-1 mutant shows enhanced ability to accumulate ABA in response to glucose, and the ABA biosynthesis inhibitor fluridone partially rescues the sugar-mediated growth arrest. Moreover, three ABA biosynthesis genes and two key ABA signaling genes, ABI3 and ABI5, are markedly over induced by glucose in sr45-1. These results provide evidence that the SR45 protein defines a novel player in plant sugar response that negatively regulates glucose signaling during early seedling development by downregulating both glucose-specific ABA accumulation and ABA biosynthesis and signaling gene expression.

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The glabra1 mutation affects cuticle formation and plant responses to microbes.

Publication Date: 2010 Aug 11 PMID: 20699396
Authors: Xia, Y. - Yu, K. - Navarre, D. A. - Seebold, K. - Kachroo, A. - Kachroo, P.
Journal: Plant Physiol

SSystemic acquired resistance (SAR) is a form of defense that provides resistance against a broad-spectrum of pathogens in plants. Previous work indicates a role for plastidal glycerolipid biosynthesis in SAR. Specifically, mutations in FATTY ACID DESATURASE 7 (FAD7), which lead to reduced trienoic FA levels and compromised plastidal lipid biosynthesis, have been associated with defective SAR. We show that the defective SAR in fad7-1 plants is not associated with a mutation in FAD7, rather a second-site mutation in GLABRA (GL) 1, a gene well-known for its role in trichome formation. The compromised SAR in gl1 plants is associated with impairment in their cuticles. Furthermore, mutations in two other components of trichome development, GL3 and TRANSPARENT TESTA GLABRA 1, also impaired cuticle development and SAR. This suggests an overlap in the biochemical pathways leading to cuticle and trichome development. Interestingly, exogenous application of gibberellic acid (GA) not onlyenhanced SAR in wt plants, but also restored SAR in gl1 plants. In contrast to GA, the defense phytohoromes salicylic acid or jasmonic acid were unable to restore SAR in gl1 plants. GA application increased levels of cuticular components but not trichome formation on gl1 plants, thus, implicating cuticle, but not trichomes, as an important component of SAR. Our findings question the prudence of using mutant backgrounds for genetic screens and underscore a need to reevaluate phenotypes previously studied in the gl1 background.

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Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus.

Publication Date: 2010 Aug 5 PMID: 20688979
Authors: Rogato, A. - D'Apuzzo, E. - Barbulova, A. - Omrane, S. - Parlati, A. - Carfagna, S. - Costa, A. - Lo Schiavo, F. - Esposito, S. - Chiurazzi, M.
Journal: Plant Physiol

Plants respond to changes of the nutrients availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis thaliana root development represents a paradigm of these responses and nitrate transporters are involved both in local and systemic control. Ammonium (NH4+) represents an important nitrogen (N) source for plants although toxicity symptoms are often associated with high NH4+ concentration when this is present as the only N source. The reason for these effects is still controversial and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus, the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH4+ conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short root phenotype was phenocopied in transgenic lines, by LjAMT1;3 over-expression independently of ammonium supply and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

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Light quality-mediated petiole elongation in Arabidopsis during shade avoidance involves cell wall modification by XTHs.

Publication Date: 2010 Aug 5 PMID: 20688978
Authors: Sasidharan, R. - Chinnappa, C. C. - Staal, M. - Elzenga, J. T. - Yokoyama, R. - Nishitani, K. - Voesenek, L. A. - Pierik, R.
Journal: Plant Physiol

Some plants can avoid shaded conditions via rapid shoot elongation, thus growing into better lit areas in a canopy. Cell wall modifying mechanisms promoting this elongation response are therefore important regulatory points during shade avoidance. Two major cell wall modifying protein families are expansins and xyloglucan endotransglucosylase/hydrolases (XTHs). The role of these proteins during shade avoidance was studied in Arabidopsis thaliana. In response to two shade cues, low R/FR (implying neighbour proximity) and green shade (mimicking dense canopy conditions), Arabidopsis showed classic shade avoidance features: petiole elongation and leaf hyponasty. Measurement of the apoplastic proton flux in green shade-treated petioles revealed a rapid efflux of protons into the apoplast within minutes unlike white light controls. This apoplastic acidification probably provides the acidic pH required for the optimal activity of cell wall modifying proteins like expansins and XTHs. Acid-induced extension, expansin susceptibility and extractable expansin activity was similar in petioles from white light and shade treated plants. XTH activity, however, was high in petioles exposed to shade treatments. Five XTH (9, 15, 16, 17 and 19) genes were positively regulated by low red to far-red (R/FR) conditions while the latter four and XTH 22 showed a significant up regulation also in response to green shade. Consistently, knockout mutants for two of these XTH genes also had reduced or absent shade avoidance responses to these light signals. These results point toward the cell wall as a vital regulatory point during shade avoidance.

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The metabolic and developmental roles of carotenoid cleavage dioxygenase 4 from potato (Solanum tuberosum L).

Publication Date: 2010 Aug 5 PMID: 20688977
Authors: Campbell, R. - Ducreux, L. J. - Morris, W. L. - Morris, J. A. - Suttle, J. C. - Ramsay, G. - Bryan, G. J. - Hedley, P. E. - Taylor, M. A.
Journal: Plant Physiol

The factors that regulate storage organ carotenoid content remain to be fully elucidated despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether CCD4 activity affects potato (Solanum tuberosum L.) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNAi approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, two- to five-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses.

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