Plant Physiology

Constitutive Activation of Transcription Factor OsbZIP46 Improves Drought Tolerance in Rice.

Publication Date: 2012 Feb 1 PMID: 22301130
Authors: Tang, N. - Zhang, H. - Li, X. - Xiao, J. - Xiong, L.
Journal: Plant Physiol

OsbZIP46 is one member of the third subfamily bZIP transcription factors in rice. It has high sequence similarity to ABF/AREB transcription factors ABI5 and OsbZIP23, two transcriptional activators positively regulating stress tolerance in Arabidopsis and rice, respectively. Expression of OsbZIP46 was strongly induced by drought, heat, H2O2, and abscisic acid (ABA) treatment; however, it was not induced by salt and cold stresses. Overexpression of the native OsbZIP46 gene increased ABA sensitivity but had no positive effect on drought resistance. The activation domain of OsbZIP46 was defined by a series of deletions, and a region (domain D) was identified as having a negative effect on the activation. We produced a constitutive active form of OsbZIP46 (OsbZIP46CA1) with a deletion of domain D. Overexpression of OsbZIP46CA1 in rice significantly increased tolerance to drought and osmotic stresses. Gene chip analysis of the two overexpressors (native OsbZIP46 and constitutive active form OsbZIP46CA1) revealed that a large number of stress-related genes, many of them predicted to be downstream genes of ABF/AREBs, were activated in the OsbZIP46CA1 overexpressor but not (even down-regulated) in the OsbZIP46 overexpressor. OsbZIP46 can interact with homologs of SnRK2 protein kinases that phosphorylate ABFs in Arabidopsis. These results suggest that OsbZIP46 is a positive regulator of ABA signaling and drought stress tolerance of rice depending on its activation. The stress-related genes activated by OsbZIP46CA1 are largely different from those activated by the other rice ABF/AREB homologs (such as OsbZIP23), further implying the value of OsbZIP46CA1 in genetic engineering of drought tolerance.

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Mutant flower morphologies in Neofinetia falcata (Wind orchid), a novel orchid model species.

Publication Date: 2012 Feb 1 PMID: 22301129
Authors: Duttke, S. - Zoulias, N. - Kim, M.
Journal: Plant Physiol

N/A- Science Correspondence format.

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Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast.

Publication Date: 2012 Jan 31 PMID: 22298683
Authors: Wang, K. - Senthil-Kumar, M. - Ryu, C. M. - Kang, L. - Mysore, K. S.
Journal: Plant Physiol

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane in order to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana squalene synthase (SQS), a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv. tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis sterol methyltransferase mutant (smt2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The gene AtCYP710A1, which encodes an enzyme that converts beta-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.

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Loss of function of FATTY ACID DESATURASE 7 in tomato enhances basal aphid resistance in a salicylate-dependent manner.

Publication Date: 2012 Jan 30 PMID: 22291202
Authors: Avila, C. A. - Arevalo-Soliz, L. M. - Jia, L. - Navarre, D. A. - Chen, Z. - Howe, G. A. - Meng, Q. W. - Smith, J. E. - Goggin, F. L.
Journal: Plant Physiol

We report here that disruption of function of the omega-3-fatty acid desaturase FAD7 enhances plant defenses against aphids. The spr2 mutation in tomato (Solanum lycopersicum), which eliminates function of FAD7, reduces the settling behavior, survival and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, antisense suppression of LeFAD7 expression in wild-type (WT) tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the NahG transgene, which suppresses SA accumulation, restores WT levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show WT levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants fad7-2 and fad7-1fad8-1 also show enhanced resistance to the green peach aphid (Myzus persicae) compared to WT controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families.

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A novel method of transgene delivery into triticale plants using the Agrobacterium T-DNA-derived nano-complex.

Publication Date: 2012 Jan 30 PMID: 22291201
Authors: Ziemienowicz, A. - Shim, Y. S. - Matsuoka, A. - Eudes, F. - Kovalchuk, I.
Journal: Plant Physiol

Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists, since monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particle-mediated transformation, result in poor transgene expression due to integration of truncated DNA molecules. We developed a novel method of transgene delivery into monocots. This method relies on the use of in vitro prepared nano-complex consisting of T-DNA, VirD2 and RecA proteins delivered to triticale microspores with the help of Tat2 cell penetrating peptide. We showed that this approach (i) allowed for single transgene copy integration events, and (ii) prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with full T-DNA/protein complex. This approach can easily substitute bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.

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Reactive Oxygen Species are Involved in Gibberellin/Abscisic Acid Signaling in Barley Aleurone Cells.

Publication Date: 2012 Feb 1 PMID: 22291200
Authors: Ishibashi, Y. - Tawaratsumida, T. - Kondo, K. - Kasa, S. - Sakamoto, M. - Aoki, N. - Zheng, S. H. - Yuasa, T. - Iwaya-Inoue, M.
Journal: Plant Physiol

Reactive oxygen species (ROS) act as signal molecules for a variety of processes in plants. However, many questions about the roles of ROS in plants remain to be clarified. Here, we report the role of ROS in gibberellin (GA) and abscisic acid (ABA) signaling in barley (Hordeum vulgare) aleurone cells. The production of hydrogen peroxide (H2O2), a type of ROS, was induced by GA in aleurone cells but suppressed by ABA. Furthermore, exogenous H2O2 appeared to promote the induction of alpha-amylases by GA. In contrast, antioxidants suppressed the induction of alpha-amylases. Therefore, H2O2 seems to function in GA and ABA signaling, and in regulation of alpha-amylase production, in aleurone cells. To identify the target of H2O2 in GA and ABA signaling, we analyzed the interrelationships between H2O2 and DELLA protein SLN1, GAMyb, and PKABA, and their roles in GA and ABA signaling in aleurone cells. In the presence of GA, exogenous H2O2 had little effect on the degradation of SLN1, the primary transcriptional repressor mediating GA signaling, but it promoted the production of the mRNA encoding GAMyb, which acts downstream of SLN1 and involves in induction of alpha-amylase mRNA. Additionally, H2O2 suppressed the production of PKABA mRNA, which is induced by ABA: PKABA represses the production of GAMyb mRNA. From these observations, we concluded that H2O2 released the repression of GAMyb mRNA by PKABA, and consequently promoted the production of alpha-amylase mRNA, thus suggesting that the H2O2 generated by GA in aleurone cells isignal molecule that antagonizes ABA signaling.

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The maize tapetum employs diverse mechanisms to synthesize and store proteins and flavonoids and transfer them to the pollen surface.

Publication Date: 2012 Jan 30 PMID: 22291199
Authors: Li, Y. - Suen, D. - Huang, C. Y. - Kung, S. Y. - Huang, A. H.
Journal: Plant Physiol

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage and transfer of these pollen-coat materials in maize (Zea mays, L.) differs completely from that reported in Arabidopsis, which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, 3 proteins--glucanase, xylanase and a novel protease, ZmPCP, -- are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid-developmental stage, whereas protease transcript emerged at a late-developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanse transcript distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative N-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All 3 proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid-late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the 3 proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or pollen surface.

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The ubiquitin E3 ligase LOSS OF GDU 2 is required for GLUTAMINE DUMPER 1- induced amino secretion in Arabidopsis.

Publication Date: 2012 Jan 30 PMID: 22291198
Authors: Pratelli, R. - Guerra, D. D. - Yu, S. - Wogulis, M. - Kraft, E. - Frommer, W. B. - Callis, J. - Pilot, G.
Journal: Plant Physiol

Amino acids serve as transport forms for organic nitrogen in the plant and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, over-expression of GLUTAMINE DUMPER 1 (GDU1) in Arabidopsis thaliana led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS of GDU 2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by GST pull-down, in vitro ubiquitination, and in planta co-immunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant, log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial miRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

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Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity and fruit development.

Publication Date: 2012 Jan 27 PMID: 22286183
Authors: Pabon-Mora, N. - Ambrose, B. - Litt, A.
Journal: Plant Physiol

Several MADS-box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity, but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core-eudicot euAP1 and euFUL genes, but notably the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae-specific. Understanding how functional divergence of the core-eudicot duplicates occurred requires a careful examination of the function of pre-duplication (FUL-like) genes. Using virus induced gene silencing (VIGS), we show that FUL-like genes in Papaver somniferum (opium poppy) and Eschscholzia californica (California poppy) function in axillary meristem growth and in floral meristem and sepal identity, and play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal organ identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.

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Identification of high temperature responsive genes in cereals.

Publication Date: 2012 Jan 25 PMID: 22279145
Authors: Hemming, M. N. - Walford, S. A. - Fieg, S. - Dennis, E. S. - Trevaskis, B.
Journal: Plant Physiol

High temperature influences plant development and can reduce crop yields. We examined how ambient temperature influences reproductive development in the temperate cereals wheat (Triticum aestivum) and barley (Hordeum vulgare). High temperature resulted in rapid progression through reproductive development in long-days, but inhibited early stages of reproductive development in short-days. Activation of the long-day flowering response pathway through daylength-insensitive alleles of the PHOTOPERIOD1 gene, which result in high FLOWERING LOCUS T-like1 transcript levels, did not allow rapid early reproductive development at high temperature in short-days. Furthermore, high temperature did not increase transcript levels of FLOWERING LOCUS T-like genes. These data suggest that genes or pathways other than the long-day response pathway mediate developmental responses to high temperature in cereals. Transcriptome analyses suggested a possible role for vernalization-responsive genes in the developmental response to high temperature. The MADS-box floral repressor HvODDSOC2 is expressed at elevated levels at high temperature in short-days, and might contribute to the inhibition of early reproductive development under these conditions. FLOWERING PROMOTING FACTOR1-like, RNAse-like genes and VER2-like genes were also identified as candidates for high temperature responsive developmental regulators. Overall, these data suggest that rising temperatures might elicit different developmental responses in cereal crops at different latitudes or times of year, due to the interaction between temperature and daylength. Additionally, we suggest that different developmental regulators might mediate the response to high temperature in cereals compared to Arabidopsis.

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The functional network of the Arabidopsis thaliana plastoglobule proteome based on quantitative proteomics and genome-wide co-expression analysis.

Publication Date: 2012 Jan 24 PMID: 22274653
Authors: Lundquist, P. - Poliakov, A. - Bhuiyan, N. H. - Zybailov, B. - Sun, Q. - van Wijk, K. J.
Journal: Plant Physiol

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl- and neutral lipids, and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared to previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two ABC1 atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including 6 ABC1 kinases and 7 fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins located predominantly in the stroma or thylakoid and not PG; we discovered that this partitioning can be predicted by their isoeletric point and hydrophobicity. A genome-wide co-expression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis and plastid redox and kinase regulators of electron flow (e.g. thioredoxins, STN7, RAP38). We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions.

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The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

Publication Date: 2012 Jan 23 PMID: 22271748
Authors: Bulgakov, V. P. - Gorpenchenko, T. Y. - Veremeichik, G. N. - Shkryl, Y. N. - Tchernoded, G. K. - Bulgakov, D. V. - Aminin, D. L. - Zhuravlev, Y. N.
Journal: Plant Physiol

The rolB oncogene has previously been identified as a key player in the formation of hairy roots during the plant-Agrobacterium rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng and Arabidopsis thaliana cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.

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Gradual increase of miR156 regulates temporal expression changes of numerous genes during leaf development in rice.

Publication Date: 2012 Jan 23 PMID: 22271747
Authors: Xie, K. - Shen, J. - Hou, X. - Yao, J. - Li, X. - Xiao, J. - Xiong, L.
Journal: Plant Physiol

The highly conserved plant microRNA, miR156, is an essential regulator for plant development. In Arabidopsis, miR156 modulates phase changing through its temporal expression in the shoot. In contrast to the gradual decrease over time in the shoot (or whole plant), we found that the miR156 level in rice gradually increased from young leaf to old leaf after juvenile stage. However, the miR156-targeted rice SQUAMOSA-promoter binding-like (SPL) transcription factors were either dominantly expressed in young leaves or not changed over the time of leaf growth. A comparison of the transcriptomes of early emerged old and later emerged young leaves from wild-type and miR156-overexpression (miR156-OE) rice lines found that expression levels of 3008 genes were affected in miR156-OE leaves. Analysis of temporal expression changes of these genes suggested that miR156 regulates gene expression in a leaf age-dependent manner, and miR156-OE attenuated the temporal changes of 2660 genes. Interestingly, seven conserved plant microRNAs also showed temporal changes from young to old leaves and miR156-OE also attenuated the temporal changes of six microRNAs. Consistent with global gene expression changes, miR156-OE plants resulted in dramatic changes including precocious leaf maturation and rapid leaf/tiller initiation. Our results indicate that another gradient of miR156 is present over time, a gradual increase during leaf growth, in addition to the gradual decrease during shoot growth. Gradually increased miR156 expression in the leaf might be essential for regulating the temporal expression of genes involved in leaf development.

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A Mutant in the ADH1 Gene of Chlamydomonas reinhardtii Elicits Metabolic Restructuring during Anaerobiosis.

Publication Date: 2012 Jan 23 PMID: 22271746
Authors: Magneschi, L. - Catalanotti, C. - Subramanian, V. - Dubini, A. - Yang, W. - Mus, F. - Posewitz, M. C. - Seibert, M. - Perata, P. - Grossman, A. R.
Journal: Plant Physiol

The green alga Chlamydomonas reinhardtii (Chlamydomonas) has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde deydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO2 was significantly reduced, but fermentative H2 production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.

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AtBGAL10 is the main xyloglucan beta-galactosidase in Arabidopsis and its abscence results in unusual xyloglucan subunits and growth defects.

Publication Date: 2012 Jan 20 PMID: 22267505
Authors: Sampedro, J. - Gianzo, C. - Iglesias, N. - Guitian, E. - Revilla, G. - Zarra, I.
Journal: Plant Physiol

In growing cells xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis thaliana a significant proportion of xyloglucan sidechains contain beta-galactose linked to alpha-xylose at O2. In this work we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of beta-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both beta-galactosidase and alpha-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on non-reducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of non-reducing ends leading to a reinforced xyloglucan network. Additionally AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones or developing vasculature, showing good correlation with alpha-xylosidase expression.

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Exotic Plant Invasion in the Context of Plant Defense Against Herbivores.

Publication Date: 2012 Jan 19 PMID: 22257854
Authors: -, I.
Journal: Plant Physiol

This manuscript is submitted under the category UPDATE, therefore not requires an abstract.

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On the discordance of metabolomics with proteomics and transcriptomics: coping with increasing complexity in logic, chemistry and network interactions.

Publication Date: 2012 Jan 17 PMID: 22253257
Authors: Fernie, A. R. - Stitt, M.
Journal: Plant Physiol

N/A.

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The TGB1 movement protein of potato virus X re-organises actin and endomembranes into the 'X-body', a viral replication factory.

Publication Date: 2012 Jan 17 PMID: 22253256
Authors: Tilsner, J. - Linnik, O. - Wright, K. M. - Bell, K. - Roberts, A. G. - Lacomme, C. - Santa Cruz, S. - Oparka, K. J.
Journal: Plant Physiol

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum (ER)-derived vesicles. Here we show that TGB1 organises the PVX 'X-body', a virally-induced inclusion structure, by remodelling host actin and endomembranes (ER and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granules within this reservoir, in the same region as non-encapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organised virus 'factory'. TGB1 is both necessary and sufficient to remodel host actin and endomembranes and recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane reorganising properties of TGB1 function to compartmentalise the viral gene products of PVX infection.

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Transcriptional regulation of Arabidopsis MIR168a and ARGONAUTE1 homeostasis in ABA and abiotic stress responses.

Publication Date: 2012 Jan 13 PMID: 22247272
Authors: Li, W. - Cui, X. - Meng, Z. - Huang, X. - Xie, Q. - Wu, H. - Jin, H. - Zhang, D. - Liang, W.
Journal: Plant Physiol

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here we revealed a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis thaliana. Plants overexpressing MIR168a and the AGO1 loss-of-function mutant (ago1-27) display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four ABA-responsive element (ABRE) binding factors (ABF1-4), which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and post-transcriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants.

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Plant defense mechanisms are activated during biotrophic and necrotrophic development of Colletotricum graminicola in maize.

Publication Date: 2012 Jan 12 PMID: 22247271
Authors: Vargas, W. A. - Sanz-Martin, J. M. - Rech, G. E. - Rivera, L. P. - Benito, E. P. - Diaz-Minguez, J. M. - Thon, M. R. - Sukno, S. A.
Journal: Plant Physiol

Hemibiotrophic, plant pathogens first establish a biotrophic interaction with the host plant and later switch to a destructive necrotrophic lifestyle. Studies of biotrophic pathogens have shown that they actively suppress plant defenses after an initial MAMP triggered activation. In contrast, studies of the hemibiotrophs suggest that they do not suppress plant defenses during the biotrophic phase indicating that while there are similarities between the biotrophic phase of hemibiotrophs and biotrophic pathogens, the two lifestyles are not analogous. We performed transcriptomic, histological and biochemical studies of the early events during the infection of maize with C. graminicola, a model pathosystem for the study of hemibiotrophy. Time-course experiments revealed that mRNA of several defense related genes, reactive oxygen species, and antimicrobial compounds all begin to accumulate early in the infection process and continue to accumulate, during the biotrophic stage. We also discovered the production of maize-derived vesicular bodies containing H2O2 targeting the fungal hyphae. We describe fungal respiratory burst during host infection, paralleled by superoxide ion production in specific fungal cells during the transition from biotrophy into necrotrophic lifestyle. We also identified several novel putative fungal effectors and study their expression during anthracnose development in maize. Our results demonstrate a strong induction of defense mechanisms occurring in maize cells during C. graminicola infection, even during the biotrophic development of the pathogen. We hypothesize that the switch to necrotrophic growth enables the fungus to evade the effects of the plant immune system and allow for full fungal pathogenicity.

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CYP701A8: A rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism.

Publication Date: 2012 Jan 12 PMID: 22247270
Authors: Wang, Q. - Hillwig, M. L. - Wu, Y. - Peters, R. J.
Journal: Plant Physiol

All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 CYP701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA biosynthesis derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A sub-family members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A sub-family members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4alpha-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3alpha position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3alpha-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide novel evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.

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The developmental trajectory of leaflet morphology in wild tomato species.

Publication Date: 2012 Jan 12 PMID: 22247269
Authors: Chitwood, D. H. - Headland, L. R. - Kumar, R. - Peng, J. - Maloof, J. N. - Sinha, N. R.
Journal: Plant Physiol

Leaves between species vary in their size, serration, complexity, and shape. However, phylogeny is not the only predictor of leaf morphology. The shape of a leaf is the result of intricate developmental processes, including heteroblastic progression (changes in leaf size and shape at different nodes) and the developmental stage of an organ. The leaflets that arise from complex leaves are additionally modified by their positioning along the proximal-distal axis of a leaf and whether they fall on the left or right side of leaves. Even further, leaves are environmentally responsive, and their final shape is influenced by environmental inputs. Here, we comprehensively describe differences in leaflet shape between wild tomato species using a Principal Component Analysis (PCA) on Elliptical Fourier Descriptors (EFD) arising from > 11,000 sampled leaflets. We leverage differences in developmental rate to approximate a developmental series, which allows us to resolve the confounding differences in intrinsic leaflet form and developmental stage along positions of the heteroblastic leaf series and proximal-distal axis of leaves. We find that the resulting "developmental trajectory" of organs at different positions along these axes are useful for describing the changes in leaflet shape that occur during the shade avoidance response in tomato. We argue that it is the developmental trajectory, the changes in shape that occur over developmental time in organs reiterated at multiple positions, that is the relevant phenotype for discerning differences between populations and species, and to understand the underlying developmental processes that change during evolution.

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A Comprehensive Dataset of Genes with a Loss-of-Function Mutant Phenotype in Arabidopsis thaliana.

Publication Date: 2012 Jan 13 PMID: 22247268
Authors: Lloyd, J. - Meinke, D.
Journal: Plant Physiol

Despite the widespread use of Arabidopsis thaliana as a model plant, a curated dataset of Arabidopsis genes with mutant phenotypes remains to be established. A preliminary list published nine years ago (Plant Physiol 131: 409-418) is outdated and genome-wide phenotype information remains difficult to obtain. We describe here a comprehensive dataset of 2,400 genes with a loss-of-function mutant phenotype in Arabidopsis. Phenotype descriptions were gathered primarily from manual curation of the scientific literature. Genes were placed into prioritized groups (essential, morphological, cellular-biochemical, and conditional) based on the documented phenotypes of putative knockout alleles. Phenotype classes (e.g. vegetative, reproductive, and timing, for the morphological group) and subsets (e.g. flowering time, senescence, circadian rhythms, and miscellaneous, for the timing class) were also established. Gene identities were classified as confirmed (through molecular complementation or multiple alleles) or not confirmed. Relationships between mutant phenotype and protein function, genetic redundancy, protein connectivity, and subcellular protein localization were explored. A complementary dataset of 401 genes that exhibit a mutant phenotype only when disrupted in combination with a putative paralog was also compiled. The importance of these genes in confirming functional redundancy and enhancing the value of single gene datasets is discussed. With further input and curation from the Arabidopsis community, these datasets should help to address a variety of important biological questions, provide a foundation for exploring the relationship between genotype and phenotype in angiosperms, enhance the utility of Arabidopsis as a reference plant, and facilitate comparative studies with model genetic organisms.

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Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability.

Publication Date: 2012 Jan 13 PMID: 22247267
Authors: Wingler, A. - Delatte, T. L. - O'Hara, L. E. - Primavesi, L. F. - Jhurreea, D. - Paul, M. J. - Schluepmann, H.
Journal: Plant Physiol

Trehalose 6-phosphate (T6P) is an important regulator of plant metabolism and development. T6P content increases when carbon availability is high and, in young growing tissue, T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Here, strong accumulation of T6P was found in senescing leaves of Arabidopsis (Arabidopsis thaliana), in parallel with a rise in sugar contents. To determine the role of T6P in senescence, T6P content was altered by expressing the bacterial T6P synthase gene, otsA, (to increase T6P) or the T6P phosphatase gene, otsB, (to decrease T6P). In otsB-expressing plants, T6P accumulated less strongly during senescence than in wild-type plants, while otsA-expressing plants contained more T6P throughout. Mature otsB-expressing plants showed a similar phenotype as described for plants overexpressing the SnRK1 gene, KIN10, including reduced anthocyanin accumulation and delayed senescence. This was confirmed by quantitative RT-PCR analysis of senescence-associated genes and genes involved in anthocyanin synthesis. To analyze if the senescence phenotype was due to decreased sugar sensitivity, the response to sugars was determined. In combination with low nitrogen supply, metabolizable sugars (glucose, fructose or sucrose) induced senescence in wild-type and otsA-expressing plants, but to a smaller extent in otsB-expressing plants. The sugar analogue 3-O-methyl glucose, on the other hand, did not induce senescence in any of the lines. Transfer of plants to and from glucose-containing medium suggested that glucose determines senescence during late development, but that the effects of T6P on senescence are established by the sugar response of young plants.

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Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis.

Publication Date: 2012 Jan 11 PMID: 22238422
Authors: Bedoya, L. - Martinez, F. - Orzaez, D. - Daros, J. A.
Journal: Plant Physiol

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids, 25.7 kDa) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco tissues turn bright red, demonstrating that in this context the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis thaliana and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

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Laticifer specific cis-prenyltransferase silencing affects the rubber, triterpene and inulin content of Taraxacum brevicorniculatum.

Publication Date: 2012 Jan 11 PMID: 22238421
Authors: Post, J. J. - van Deenen, N. - Fricke, J. - Kowalski, N. - Wurbs, D. - Schaller, H. - Eisenreich, W. - Huber, C. - Twyman, R. M. - Prufer, D. - Schulze Gronover, C.
Journal: Plant Physiol

Certain Taraxacum species such as T. koksaghyz (Rodin) and T. brevicorniculatum (Korol.) produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular-mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate (IPP) units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; E.C. 2.5.1.20) is a cis-prenyltransferase (CPT). Here we present the comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently-isolated CPTs known to be associated with rubber particles (TbCPT1-3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by NMR, size exclusion chromatography (SEC) and gas chromatography-mass spectrometry (GC/MS) indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy (TEM) revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-CoA reductase (HMGR), the key enzyme in the mevalonate (MVA) pathway, reflecting homeostatic control of the IPP pool. This is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.

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TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis-relative Thellungiella salsuginea, shows K+-specificity in the presence of NaCl.

Publication Date: 2012 Jan 11 PMID: 22238420
Authors: Ali, Z. - Park, H. C. - Ali, A. - Oh, D. H. - Aman, R. - Kropornicka, A. - Hong, H. - Choi, W. - Chung, W. S. - Kim, W. Y. - Bressan, R. A. - Bohnert, H. J. - Lee, S. Y. - Yun, D. J.
Journal: Plant Physiol

Cellular Na+/K+ ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na+/K+ co-transporters in the HKT family from the halophytic Arabidopsis-relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNAi interference in T. salsuginea resulted in Na+-sensitivity and K+-deficiency. The athkt1 mutant lines over-expressing TsHKT1;2 proved less sensitive to Na+ and showed less K+ deficiency than lines over-expressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na+ or K+ transporters revealed strong K+-transporter activity and selectivity for K+ over Na+. Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K+ transporter activity. We consider maintenance of K+-uptake through the TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

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Cross-talk between calcium, H2O2 and NO and activation of gene expression involving calmodulins and CDPKs in Ulva compressa exposed to copper excess.

Publication Date: 2012 Jan 10 PMID: 22234999
Authors: Gonzalez, A. - Cabrera, M. D. - Henriquez, M. J. - Contreras, R. A. - Morales, B. - Moenne, A.
Journal: Plant Physiol

In order to analyze the copper-induced cross-talk between calcium, NO and H2O2 and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine (rya) and xestospongin C (xes), of chloroplasts and mitochondrial electron transport chains, DCMU and antimycin A (AA), of piruvate dehydrogenase (PDH), moniliformin, of calmodulins (CaMs), W-7, and of calcium-dependent protein kinases (CDPKs), staurosporine, as well as with the scavengers of NO, cPTIO, and of H2O2, ascorbate, and exposed to a sub-lethal concentration of copper (10 microM) for 24 h. The level of NO increased at 2 and 12 h, the first peak was inhibited by ned-19 and DCMU and the second peak by ned-19 and AA indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3 and 12 h and was inhibited by ned-19, rya , xes and moniliformin indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, PDH, OGDH and IDH activities of the Krebs cycle increased at 2, 3, 12 and/or 14 h and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3 and 12 h was inhibited by cPTIO and ASC indicating activation by NO and H2O2. In addition, the level of antioxidant proteins gene transcripts decreased with W-7 and staurosporine. Thus, there is a copper-induced cross-talk between calcium, H2O2 and NO and a calcium-dependent activation of gene expression involving CaMs and CDPKs.

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Chemical quenching of singlet oxygen by carotenoids in plants.

Publication Date: 2012 Jan 10 PMID: 22234998
Authors: Ramel, F. - Birtic, S. - Cuine, S. - Triantaphylides, C. - Ravanat, J. L. - Havaux, M.
Journal: Plant Physiol

Carotenoids are considered to be the first line of defense of plants against singlet oxygen toxicity owing to their capacity to quench singlet oxygen as well as triplet chlorophylls through a physical mechanism involving transfer of excitation energy followed by thermal deactivation. Here we show that leaf carotenoids are also able to quench singlet oxygen by a chemical mechanism involving their oxidation. In vitro oxidation of beta-carotene, lutein and zeaxanthin by singlet oxygen generated various aldehydes and endoperoxides. A search for those molecules in Arabidopsis leaves (Arabidopsis thaliana) revealed the presence of singlet oxygen-specific carotenoid endoperoxides in low light-grown plants, indicating chronic oxidation of carotenoids by singlet oxygen. beta-Carotene endoperoxide, but not xanthophyll endoperoxide, rapidly accumulated during high light stress, and this accumulation was correlated with the extent of PSII photoinhibition and the expression of various singlet oxygen marker genes. The selective accumulation of beta-carotene endoperoxide points at the PSII reaction centers, rather than the PSII chlorophyll antennae, has a major site of singlet oxygen accumulation in plants under high light stress. beta-carotene endoperoxide was found to have a relatively fast turnover, decaying in the dark with a half time of about 6 h. This carotenoid metabolite provides an early index of singlet oxygen production in leaves, the occurrence of which precedes the accumulation of fatty acid oxidation products.

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WUSCHEL-RELATED HOMEOBOX5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation.

Publication Date: 2012 Jan 9 PMID: 22232385
Authors: Opisova, M. A. - Mortier, V. - Demchenko, K. N. - Tsyganov, V. E. - Tikhonovich, I. A. - Lutova, L. A. - Dolgikh, E. A. - Goormachtig, S.
Journal: Plant Physiol

In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLV1/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically control nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem "organizers". Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum L.) that form indeterminate nodules. Analysis of the temporal WOX5 expression during nodulation with quantitative reverse-transcription PCR and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants defective in AON the WOX5 expression was suppressed by the shoot-acting CLV-like receptor complex. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems, but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea might act downstream from or besides the CLE peptides during AON.

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The Arabidopsis thaliana TETRATRICO PEPTIDE THIOREDOXIN-LIKE gene family is required for osmotic stress tolerance and male sporogenesis.

Publication Date: 2012 Jan 9 PMID: 22232384
Authors: Lakhssassi, N. - Doblas, V. G. - Rosado, A. - Esteban Del Valle, A. - Pose, D. - Jimenez, A. J. - Castillo, A. G. - Valpuesta, V. - Borsani, O. - Botella, M. A.
Journal: Plant Physiol

Tetratricopeptide thioredoxin-Like (TTL) proteins are characterized by the presence of 6 tetratricopeptide repeats (TPR) in conserved positions and a C-terminal region known as thioredoxin-like (TRXL) domain with homology to thioredoxins. In Arabidopsis thaliana, the TTL gene family is composed by four members, and the founder member, TTL1, is required for osmotic stress tolerance. Analysis of sequenced genomes indicates that TTL genes are specific to land plants. In this study, we report the expression profiles of Arabidopsis TTL genes using data mining and promoter-reporter GUS fusions. Our results show that TTL1, TTL3, and TTL4 display ubiquitous expression in normal growing conditions but differential expression patterns in response to osmotic and NaCl stresses. TTL2 shows a very different expression pattern, being specific to pollen grains. Consistent with the expression data, ttl1, ttl3, and ttl4 mutants show reduced root growth under osmotic stress and the analysis of double and triple mutants indicate that TTL1, TTL3 and TTL4 have partially overlapping yet specific functions in abiotic stress tolerance while TTL2 is involved in male gametophytic transmission.

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Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development.

Publication Date: 2012 Jan 9 PMID: 22232383
Authors: Cho, Y. H. - Hong, J. W. - Kim, E. C. - Yoo, S. D.
Journal: Plant Physiol

Sucrose-non-fermentation1 (Snf1)-related protein kinase1 (SnRK1) is an evolutionarily conserved energy sensor protein that regulates gene expression in response to energy depletion in plants. Efforts to elucidate the functions and mechanisms of this protein kinase are hampered, however, by inherent growth defects of snrk1-null mutant plants. To overcome these limitations and study SnRK1 functions in vivo, we applied a method combining transient expression in leaf mesophyll protoplasts and stable expression in transgenic plants. We found that both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) SnRK1 activities critically influence stress-inducible gene expression and induction of stress tolerance. Genetic, molecular, and chromatin immunoprecipitation analyses further revealed that the nuclear SnRK1 modulated target gene transcription in a submergence-dependent manner. From early seedling development through late senescence, SnRK1 activities appeared to modulate developmental processes in the plants. Our findings offer insight into the regulatory functions of plant SnRK1 in stress-responsive gene regulation and in plant growth and development throughout the life cycle.

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Arabidopsis ETR1 and ERS1 Differentially Repress the Ethylene Response in Combination with Other Ethylene Receptor Genes.

Publication Date: 2012 Jan 6 PMID: 22227969
Authors: Liu, Q. - Wen, C. K.
Journal: Plant Physiol

The ethylene response is negatively regulated by a family of five ethylene receptor genes in Arabidopsis. The five members of the ethylene receptor family can physically interact and form complexes, which implies cooperativity for signaling may exist among the receptors. The ethylene receptor gene mutations etr1-1(C65Y), ers1-1(I62P), and ers1C65Y are dominant and each confers ethylene insensitivity. In this study, the repression of the ethylene response by these dominant mutant receptor genes was examined in receptor-defective mutants to investigate the functional significance of receptor cooperativity in ethylene signaling. We showed that etr1-1(C65Y), but not ers1-1(I62P), substantially repressed various ethylene responses independent of other receptor genes. In contrast, wild-type receptor genes differentially supported the repression of ethylene responses by ers1-1(I62P); ETR1 and EIN4 supported ers1-1(I62P) functions to a greater extent than did ERS2, ETR2, and ERS1. The lack of both ETR1 and EIN4 almost abolished the repression of ethylene responses by ers1C65Y, which implied that ETR1 and EIN4 have synergistic effects on ers1C65Y functions. Our data indicated that a dominant ethylene-insensitive receptor differentially repressed ethylene responses when coupled with a wild-type ethylene receptor, which supported the hypothesis that the formation of a variety of receptor complexes may facilitate differential receptor signal output, by which ethylene responses can be repressed to different extents. We hypothesize that plants can respond to a broad ethylene concentration range and exhibit tissue-specific ethylene responsiveness with differential cooperation of the multiple ethylene receptors.

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Untargeted metabolic quantitative trait loci (mQTL) analyses reveal a relationship between primary metabolism and potato tuber quality.

Publication Date: 2012 Jan 5 PMID: 22223596
Authors: Carreno-Quintero, N. - Acharjee, A. - Maliepaard, C. - Bachem, C. - Mumm, R. - Bouwmeester, H. - Visser, R. - Keurentjes, J.
Journal: Plant Physiol

Recent advances in ~omics technologies such as transcriptomics, metabolomics and proteomics along with genotypic profiling have permitted to dissect the genetics of complex traits represented by molecular phenotypes in non-model species. To identify the genetic factors underlying variation in primary metabolism in potato we have profiled primary metabolite content in a diploid potato mapping population, derived from crosses between Solanum tuberosum and wild relatives, using gas chromatography time of flight mass spectrometry (GC-TOF-MS). In total 139 polar metabolites were detected of which we identified metabolite quantitative trait loci (mQTLs) for ~72% of the detected compounds. In order to obtain an insight into the relationships between metabolic traits and classical phenotypic traits we also analysed statistical associations between them. The combined analysis of genetic information through QTL coincidence and the application of statistical learning methods provide information on putative indicators associated with the alterations in metabolic networks that affect complex phenotypic traits.

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Accumulation of beta-conglycinin in soybean cotyledon through formation of disulfide bonds between alpha' and alpha subunits.

Publication Date: 2012 Jan 18 PMID: 22218927
Authors: Wadahama, H. - Iwasaki, K. - Matsusaki, M. - Nishizawa, K. - Ishimoto, M. - Arisaka, F. - Takagi, K. - Urade, R.
Journal: Plant Physiol

beta-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean beta-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent oxidation of thiol groups and reduction or exchange of disulfide bonds. We found that approximately half of the alpha' and alpha subunit of beta-conglycinin were disulfide-linked, together or with P34, prior to N-terminal propeptide processing. Sedimentation velocity experiments, size exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with Blue native-PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the beta-conglycinin complexes containing the disulfide-linked alpha'/alpha subunits were complexes of >720 kDa. The alpha' or alpha subunits, when disulfide-linked with P34, were mostly present in approximately 480-kDa complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between alpha'/alpha subunits residing in different beta-conglycinin hexamers, but the binding of P34 to alpha' and alpha subunits reduces the linkage between beta-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when beta-conglycinin was expressed under nonreducing conditions.

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NtSCP1 from Nicotiana tabacum is an extracellular serine carboxypeptidase III that has an impact on cell elongation.

Publication Date: 2012 Jan 18 PMID: 22214816
Authors: Bienert, M. D. - Delannoy, M. - Navarre, C. - Boutry, M.
Journal: Plant Physiol

The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from Nicotiana tabacum, NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of N. tabacum. RT-PCR analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf and flower tissues. Expression analysis of the beta-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-GFP fusion protein showed that the protein is located in the extracellular space of N. tabacum leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic N. tabacum plants over-expressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.

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Silencing MPK4 in Nicotiana attenuata enhances photosynthesis and seed production but compromises abscisic acid-induced stomatal closure and guard cell-mediated resistance to Pseudomonas syringae pv. tomato DC3000.

Publication Date: 2012 Jan 20 PMID: 22147519
Authors: Hettenhausen, C. - Baldwin, I. - Wu, J.
Journal: Plant Physiol

Mitogen-activated protein kinases (MAPKs) play pivotal roles in development and environmental interactions in eukaryotes. Here we studied the function of a MAPK, NaMPK4, in a wild tobacco species Nicotiana attenuata. The NaMPK4-silenced N. attenuata (irNaMPK4) attained somewhat smaller statures, delayed senescence, and greatly enhanced stomatal conductance and photosynthetic rate, especially during late developmental stages. All these changes were associated with highly increased seed production. Using leaf epidermal peels, we demonstrate that guard cell closure in irNaMPK4 was strongly impaired in response to abscisic acid (ABA) and H2O2 and consistently, irNaMPK4 plants transpired more water and wilted sooner that did WT plants when they were deprived of water. We show that NaMPK4 plays an important role in the guard cell-mediated defense against surface-deposited bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst DC3000); in contrast, when bacteria directly entered leaves by pressure infiltration, NaMPK4 was found to be less important in the resistance to apoplast-located Pst DC3000. Salicylic acid (SA) also played an important role the guard-cell mediated resistance, as demonstrated by the high susceptibility to Pst DC3000 in plants expressing SA hydroxylase (NahG). However, SA was not involved in the defense against Pst DC3000 once it had entered leaf tissues. We conclude that NaMPK4 functions differently from that of AtMPK4 and AtMPK11 in Arabidopsis, despite their sequence similarities, suggesting complex functional divergence of MAPKs in different plant lineages. This work highlights the multifaceted functions of NaMPK4 in guard cells and underscores its role in mediating various ecologically important traits.

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