Search

Following cytokinesis in plants, Endoplasmic MTs (EMTs) assemble on the nuclear surface, forming a radial network that extends out to the cell cortex, where they attach and incorporate into the cortical microtubule (CMT) array. We found that in these post-cytokinetic cells, the MT-associated protein CLASP is enriched at sites of EMT-cortex attachment, and is required for stable EMT tethering and growth into the cell cortex. Loss of EMT-cortex anchoring in clasp-1 mutants results in destabilized EMT arrays, and is accompanied by enhanced mobility of the cytoplasm, premature vacuolation, and precocious entry into cell elongation phase. Thus, EMTs appear to maintain cells in a meristematic state by providing a structural scaffold that stabilizes the cytoplasm to counteract actomyosin-based cytoplasmic streaming forces, thereby preventing premature establishment of a central vacuole and rapid cell elongation. [ABSTRACT FROM AUTHOR]

In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST 3 (ZYG3) in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG3 encodes the transfer RNA (tRNA) ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL::cAtRNL-sYFP-complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis. [ABSTRACT FROM AUTHOR]

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca2+ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca2+ levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca2+ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca2+ channel blockers or phospholipase C inhibitors. These results indicate that Ca2+ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca2+ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes. [ABSTRACT FROM AUTHOR]

Wood biomass is mainly made of secondary cell walls, whose formation is controlled by a multilevel network. The tandem CCCH zinc finger ( TZF) proteins involved in plant secondary wall formation are poorly understood., Two TZF genes, Pd C3 H17 and Pd C3 H18, were isolated from Populus deltoides and functionally characterized in Escherichia coli, tobacco, Arabidopsis and poplar., Pd C3 H17 and Pd C3 H18 are predominantly expressed in cells of developing wood, and the proteins they encode are targeted to cytoplasmic foci. Transcriptional activation assays showed that Pd MYB2/3/20/21 individually activated the Pd C3 H17 and Pd C3 H18 promoters, but Pd MYB3/21 were most significant. Electrophoretic mobility shift assays revealed that Pd MYB3/21 bound directly to the Pd C3 H17/18 promoters. Overexpression of Pd C3 H17/ 18 in poplar increased secondary xylem width and secondary wall thickening in stems, whereas dominant repressors of them had the opposite effects on these traits. Similar alteration in secondary wall thickening was observed in their transgenic Arabidopsis plants. q RT- PCR results showed that Pd C3 H17/ 18 regulated the expression of cellulose, xylan and lignin biosynthetic genes, and several wood-associated MYB genes., These results demonstrate that Pd C3 H17 and Pd C3 H18 are the targets of Pd MYB3 and Pd MYB21 and are an additional two components in the regulatory network of secondary xylem formation in poplar. [ABSTRACT FROM AUTHOR]

Background Ca2+, a versatile intracellular second messenger in various signaling pathways, initiates many responses involved in growth, defense and tolerance to biotic and abiotic stress. Endogenous and exogenous signals induce cytoplasmic Ca2+ ([Ca2+]cyt) elevation, which are responsible for the appropriate downstream responses. Results Here we report on an ethyl-methane sulfonate-mediated Arabidopsis mutant that fails to induce [Ca2+]cyt elevation in response to exudate preparations from the pathogenic mibrobes Alternaria brassicae, Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The cytoplasmic Ca2+ elevation mutant1 (cycam1) is susceptible to infections by A. brassicae, its toxin preparation and sensitive to abiotic stress such as drought and salt. It accumulates high levels of reactive oxygen species and contains elevated salicylic acid, abscisic acid and bioactive jasmonic acid iso-leucine levels. Reactive oxygen species- and phytohormone-related genes are higher in A. brassicae-treated wild-type and mutant seedlings. Depending on the analysed response, the elevated levels of defense-related compounds are either caused by the cycam mutation and are promoted by the pathogen, or they are mainly due to the pathogen infection or application of pathogen-associated molecular patterns. Furthermore, cycam1 shows altered responses to abscisic acid treatments: ABA inhibits germination and growth of the mutant. Conclusions We isolated an Arabidopsis mutant which fails to induce [Ca2+]cyt elevation in response to exudate preparations from various microbes. The higher susceptibility of the mutant to pathogen infections correlates with the higher accumulation of defense-related compounds, such as phytohormones, reactive oxygen-species, defense-related mRNA levels and secondary metabolites. Therefore, CYCAM1 couples [Ca2+]cyt elevation to biotic, abiotic and oxidative stress responses. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) signaling plays important roles in plant growth, development and adaptation to various stresses. RCAR1/PYL9 has been known as a cytoplasm and nuclear ABA receptor in Arabidopsis. To obtain further insight into the regulatory mechanism of RCAR1/PYL9, a yeast two-hybrid approach was performed to screen for RCAR1/PYL9-interacting proteins and an R2R3-type MYB transcription factor, AtMYB44, was identified. The interaction between RCAR1/PYL9 and AtMYB44 was further confirmed by glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays. Gene expression analysis showed that AtMYB44 negatively regulated the expression of ABA-responsive gene RAB18, in contrast to the opposite role reported for RCAR1/PYL9. Competitive GST pull-down assay and analysis of phosphatase activity demonstrated that AtMYB44 and ABI1 competed for binding to RCAR1/PYL9 and thereby reduced the inhibitory effect of RCAR1/PYL9 on ABI1 phosphatase activity in the presence of ABA in vitro. Furthermore, transient activation assay in protoplasts revealed AtMYB44 probably also decreased RCAR1/PYL9-mediated inhibition of ABI1 activity in vivo. Taken together, our work provides a reasonable molecular mechanism of AtMYB44 in ABA signaling. [ABSTRACT FROM AUTHOR]

Although heat-shock transcription factors are well characterized in the heat stress-related pathway, they are poorly understood in other stress responses. Here, we functionally characterized AtHsfA6a in the presence of exogenous abscisic acid ( ABA) and under high salinity and dehydration conditions. AtHsfA6a expression under normal conditions is very low, but was highly induced by exogenous ABA, NaCl and drought. Unexpectedly, the levels of AtHsfA6a transcript were not significantly altered under heat and cold stresses. Electrophoretic mobility shift assays and transient transactivation assays indicated that AtHsfA6a is transcriptionally regulated by ABA-responsive element binding factor/ ABA-responsive element binding protein, which are key regulators of the ABA signalling pathway. Additionally, fractionation and protoplast transient assays showed that AtHsfA6a was in cytoplasm and nucleus simultaneously; however, under conditions of high salinity the majority of AtHsfA6A was in the nucleus. Furthermore, at both seed germination and seedlings stage, plants overexpressing AtHsfA6a were hypersensitive to ABA and exhibited enhanced tolerance against salt and drought stresses. Finally, the microarray and qRT- PCR analyses revealed that many stress-responsive genes were up-regulated in the plants overexpressing AtHsfA6a. Taken together, the data strongly suggest that AtHsfA6a acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signalling pathway. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is essential for de novo DNA methylation in higher plants, and recent reports established novel elements of this silencing pathway in the model organism Arabidopsis thaliana. INVOLVED IN DE NOVO DNA METHYLATION 2 (IDN2) and the closely related FACTOR OF DNA METHYLATION (FDM) are members of a plant-specific family of dsRNA-binding proteins characterized by conserved XH/XS domains and implicated in the regulation of RdDM at chromatin targets. Genetic analyses have suggested redundant as well as non-overlapping activities for different members of the gene family. However, detailed insights into the function of XH/XS-domain proteins are still elusive. By the generation and analysis of higher-order mutant combinations affected in IDN2 and further members of the gene family, we have provided additional evidence for their redundant activity. Distinct roles for members of the XH/XS-domain gene family were indicated by differences in their expression and subcellular localization. Fluorescent protein-tagged FDM genes were expressed either in nuclei or in the cytoplasm, suggestive of activities of XH/XS-domain proteins in association with chromatin as well as outside the nuclear compartment. In addition, we observed altered location of a functional FDM1–VENUS reporter from the nucleus into the cytoplasm under conditions when availability of further FDM proteins was limited. This is suggestive of a mechanism by which redistribution of XH/XS-domain proteins could compensate for the loss of closely related proteins.Activity of Arabidopsis XH/XS-domain proteins has been implicated in late events of RdDM, in close association with chromatin. Functional expression analysis now indicates partially redundant activities in nuclei and cytoplasm. [ABSTRACT FROM PUBLISHER]

Multistep phosphorelays involve a phosphate transfer from sensor histidine-aspartate kinases (HKs) to response regulators (RRs), via histidine-containing phosphotransfer proteins (HPts). InArabidopsis, some AHK receptors are organized as homodimers and able to interact with HPts (AHPs). However, there are no data available concerning the dimerization of theArabidopsisosmosensor AHK1. Although only AHP2 is able to interact with AHK1 in yeast, validation of this interaction remains to be clarified in planta. The ability of poplar HK1 osmosensor, homologous to AHK1, to homodimerize and interact with three HPts (HPt2, 7 and 9) as preferential partners has been previously shown by yeast two-hybrid assay. However, protein interaction studies need to use complementary approaches to avoid interaction artifacts. Here, we confirmedin plantahomodimerization of the cytoplasmic part of HK1 (HK1-CP) and the functional relevance of HK1-CP/HPt interactions by bimolecular fluorescence complementation assays. This work led us to validate these partnerships and to propose them as probably involved in osmosensing pathway inPopulus. [ABSTRACT FROM PUBLISHER]

Oxidative stress caused by ozone ( O3) affects plant development, but the roles of specific redox-homeostatic enzymes in O3 responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal-time exposures to O3 is not known. In Arabidopsis Col-0, day length affected the outcome of O3 exposure. In short-days ( SD), few lesions were elicited by treatments that caused extensive lesions in long days ( LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP( H) or NAD( H). To investigate roles of two genes potentially underpinning this redox stability, O3 responses of mutants for cytosolic NADP-isocitrate dehydrogenase ( icdh) and glutathione reductase 1 ( gr1) were analysed. Loss of ICDH function did not affect O3-induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH-producing enzymes and pathogenesis-related gene 1 ( PR1). In gr1, O3-triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col-0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal-time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR-glutathione system seems to play novel signalling roles during O3 exposure. [ABSTRACT FROM AUTHOR]

Programmed cell death ( PCD) in plants plays a key role in defense response and is promoted by the release of compartmentalized proteases to the cytoplasm. Yet the exact identity and control of these proteases is poorly understood. Serpins are an important group of proteins that uniquely curb the activity of proteases by irreversible inhibition; however, their role in plants remains obscure. Here we show that during cell death the Arabidopsis serpin protease inhibitor, At Serpin1, exhibits a pro-survival function by inhibiting its target pro-death protease, RD21. At Serpin1 accumulates in the cytoplasm and RD21 accumulates in the vacuole and in endoplasmic reticulum bodies. Elicitors of cell death, including the salicylic acid agonist benzothiadiazole and the fungal toxin oxalic acid, stimulated changes in vacuole permeability as measured by the changes in the distribution of marker dye. Concomitantly, a covalent At Serpin1- RD21 complex was detected indicative of a change in protease compartmentalization. Furthermore, mutant plants lacking RD21 or plants with At Serpin1 over-expression exhibited significantly less elicitor-stimulated PCD than plants lacking At Serpin1. The necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum secrete oxalic acid as a toxin that stimulates cell death. Consistent with a pro-death function for RD21 protease, the growth of these necrotrophs was compromised in plants lacking RD21 but accelerated in plants lacking At Serpin1. The results indicate that AtSerpin1 controls the pro-death function of compartmentalized protease RD21 by determining a set-point for its activity and limiting the damage induced during cell death. [ABSTRACT FROM AUTHOR]