Showing papers in "Plant and Cell Physiology in 2008"
TL;DR: It is found that the expression of sense TUs is necessary for the stress- or ABA-inducible expression of the antisense TUs in the fSATs (AGI code/non-AGI TU).
Abstract: Plants respond and adapt to drought, cold and high-salinity stresses in order to survive. In this study, we applied Arabidopsis Affymetrix tiling arrays to study the whole genome transcriptome under drought, cold, high-salinity and ABA treatment conditions. The bioinformatic analysis using the tiling array data showed that 7,719 non-AGI transcriptional units (TUs) exist in the unannotated "intergenic" regions of Arabidopsis genome. These include 1,275 and 181 TUs that are induced and downregulated, respectively, by the stress or ABA treatments. Most of the non-AGI TUs are hypothetical non-protein-coding RNAs. About 80% of the non-AGI TUs belong to pairs of the fully overlapping sense-antisense transcripts (fSATs). Significant linear correlation between the expression ratios (treated/untreated) of the sense TUs and the ratios of the antisense TUs was observed in the SATs of AGI code/non-AGI TU. We studied the biogenesis mechanisms of the stress- or ABA-inducible antisense RNAs and found that the expression of sense TUs is necessary for the stress- or ABA-inducible expression of the antisense TUs in the fSATs (AGI code/non-AGI TU).
487 citations
TL;DR: The results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.
Abstract: Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.
455 citations
TL;DR: The expression divergence of duplicated WRKY genes under normal or stressed conditions showed that high expression divergence has occurred not only among fragmentally but also among tandemly duplicated genes, suggesting that the highexpression divergence could be one of the mechanisms for the retention of these duplicatedWRKY genes.
Abstract: WRKY transcription factors play important roles in the regulation of various biological processes. We have analyzed the publicly available rice genome sequence databases and predicted 103 genes encoding WRKY transcription factors. Among them, the majority of rice WRKY genes (77.7%) were located in duplicated regions; 45.6% of WRKY genes were fragmentally duplicated and 35% of them were tandemly duplicated. These results suggested that genome duplications might be regarded as a major mechanism for expansion of this family in the rice genome. Subsequently, we analyzed their expression profiles under normal and abiotic stress, as well as various hormone treatments. Under normal growth conditions, 65 WRKY genes were expressed differentially either in their transcript abundance or in their expression patterns. Under abiotic (cold, drought and salinity) stresses and various phytohormone treatments, 54 WRKY genes exhibited significant differences in their transcript abundance; among them three genes were expressed only in stressed conditions. Among the stress-inducible genes, 13 genes were regulated only by abiotic stresses, another set of 13 genes were responsive to only phytohormone treatments and the remaining 28 genes were regulated by both factors, suggesting an interaction between abiotic stress and hormone signaling. On the other hand, we have also surveyed the expression divergence of duplicated genes under normal or stressed conditions, and the results showed that high expression divergence has occurred not only among fragmentally but also among tandemly duplicated genes. These results suggested that the high expression divergence could be one of the mechanisms for the retention of these duplicated WRKY genes.
336 citations
TL;DR: The results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.
Abstract: Nitric oxide (*NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous *NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of *NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO(2)-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total *NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO(2)-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.
307 citations
TL;DR: The results indicate that histone modifications on the H3 N-tail are altered with gene activation on the coding regions of drought stress-responsive genes under drought stress conditions and that several patterns of nucleosome changes function in the drought stress response.
Abstract: Post-translational modification of histone N-tails affects eukaryotic gene activity. In Arabidopsis, the histone modification level correlates with gene activation and repression in vernalization and flowering processes, but there is little information on changes in histone modification status and nucleosome structure under abiotic stresses. We determined the temporal and spatial changes in nucleosome occupancy and levels of H3K4me3, H3K9ac, H3K14ac, H3K23ac and H3K27ac in the histone H3 N-tail on the regions of four Arabidopsis drought stress-inducible genes, RD29A, RD29B, RD20 and RAP2.4, under drought stress conditions by chromatin immunoprecipitation analysis. We found two types of regulatory mechanisms of nucleosome occupancy function in the drought stress response. For RD29A and RD29B genes, nucleosome occupancy of promoter regions is low compared with that of coding regions, and no notable nucleosome loss occurs under drought stress. In contrast, nucleosome density is gradually decreased in response to drought stress on RD20 and RAP2.4 genes. Enrichments of H3K4me3 and H3K9ac correlate with gene activation in response to drought stress in all four genes. Interestingly, establishment of H3K4me3 occurs after accumulation of RNAPII on the coding regions of RD29A and RAP2.4. Enrichment of H3K23ac and H3K27ac occurs in response to drought stress on the coding regions of RD29B, RD20 and RAP2.4, but not on the coding region of RD29A. Our results indicate that histone modifications on the H3 N-tail are altered with gene activation on the coding regions of drought stress-responsive genes under drought stress conditions and that several patterns of nucleosome changes function in the drought stress response.
302 citations
TL;DR: The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.
Abstract: Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the β-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.
286 citations
TL;DR: The requirement of specific defense pathways for powdery mildew resistance induced by the basidiomycete Piriformospora indica in Arabidopsis was analyzed and it was found that cytoplasmic but not nuclear localization of NPR1 is required for P. indica-induced resistance.
Abstract: We analyzed the requirement of specific defense pathways for powdery mildew (Golovinomyces orontii) resistance induced by the basidiomycete Piriformospora indica in Arabidopsis. Piriformospora indica root colonization reduced G. orontii conidia in wild-type (Col-0), npr1-3 (nonexpressor of PR genes 1-3) and NahG plants, but not in the npr1-1 null mutant. Therefore, cytoplasmic but not nuclear localization of NPR1 is required for P. indica-induced resistance. Two jasmonate signaling mutants were non-responsive to P. indica, and jasmonic acid-responsive vegetative storage protein expression was primed and thus elevated in response to powdery mildew, suggesting that P. indica confers resistance reminiscent of induced systemic resistance (ISR).
272 citations
TL;DR: This work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two.
Abstract: Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.
253 citations
TL;DR: It is shown that the observed cytokinin-associated phenotypes of arr1 arr10 arr12 were very severe and highly analogous to those observed for certain ahk2 ahk3 ahk4/cre1 triple mutants, which have virtually no cytokinIn receptor to propagate the phosphorelay signal transduction network.
Abstract: Arabidopsis thaliana has 11 members belonging to the typical type-B ARR (authentic response regulator) family. Among them, seven highly homologous members appear also to be conserved in rice (Oryza sativa), but others are not. It was suggested that these seven ARRs are commonly implicated as DNA-binding transcription factors in the phosphorelay-mediated cytokinin signal transduction network in higher plants. To gain an insight into the functions of the cytokinin-associated type-B ARRs, we previously investigated an arr1 arr10 arr12 triple mutant and reported that it exhibited stunted growth and abnormality in vascular development. Based on this fact, here we attempted to characterize the mutant intensively with reference to cytokinin-associated phenotypes through the life cycle. We showed that the observed cytokinin-associated phenotypes of arr1 arr10 arr12 were very severe and highly analogous to those observed for certain ahk2 ahk3 ahk4/cre1 triple mutants, which have virtually no cytokinin receptor to propagate the phosphorelay signal transduction network. Among the seven ARR members belonging to the cytokinin-associated type-B ARR subfamily, it was thus suggested that ARR1, ARR10 and ARR12 together play essential (or general) roles in cytokinin signal transduction. It is therefore conceivable that the other type-B ARRs (ARR2, ARR11, ARR14 and ARR18) might play more specific roles spatially and temporally in plants.
239 citations
TL;DR: The changes that occurred in the ASC-GSH cycle enzymes of the leaves suggest that the whole plant improved its antioxidant defense, even in those parts which had not yet been reached by Cd, and further highlight the tight regulation and the relevance of this cycle in the defense against heavy metals.
Abstract: Durum wheat plants (Triticum durum cv Creso) were grown in the presence of cadmium (0–40kM) and analysed after 3 and 7 d for their growth, oxidative stress markers, phytochelatins, and enzymes and metabolites of the ascorbate (ASC)–glutathione (GSH) cycle. Cd exposure produced a dose-dependent inhibition of growth in both roots and leaves. Lipid peroxidation, protein oxidation and the decrease in the ascorbate redox state indicate the presence of oxidative stress in the roots, where H2O2 overproduction and phytochelatin synthesis also occurred. The activity of the ASC–GSH cycle enzymes significantly increased in roots. Consistently, a dosedependent accumulation of Cd was evident in these organs. On the other hand, no oxidative stress symptoms or phytochelatin synthesis occurred in the leaves; where, at least during the time of our analysis, the levels of Cd remained irrelevant. In spite of this, enzymes of the ASC–GSH cycle significantly increased their activity in the leaves. When ASC biosynthesis was enhanced, by feeding plants with its last precursor, L-galactono-c-lactone (GL), Cd uptake was not affected. On the other hand, the oxidative stress induced in the roots by the heavy metal was alleviated. GL treatment also inhibited the Cd-dependent phytochelatin biosynthesis. These results suggest that different strategies can successfully cope with heavy metal toxicity. The changes that occurred in the ASC–GSH cycle enzymes of the leaves also suggest that the whole plant improved its antioxidant defense, even in those parts which had not yet been reached by Cd. This precocious increase in the enzymes of the ASC–GSH cycle further highlight the tight regulation and the relevance of this cycle in the defense against heavy metals.
227 citations
TL;DR: Results indicate that Cu-responsive proline synthesis is closely related to NO generation in C. reinhardtii, suggesting the regulatory function of NO in proline metabolism under heavy metal stress.
Abstract: Excess copper affects the growth and metabolism of plants and green algae. However, the physiological processes under Cu stress are largely unknown. In this study, we investigated Cu-induced nitric oxide (NO) generation and its relationship to proline synthesis in Chlamydomonas reinhardtii. The test alga accumulated a large amount of proline after exposure to relatively low Cu concentrations (2.5 and 5.0 microM Cu2+). A concomitant increase in the intracellular NO level was observed with increasing concentrations of Cu applied. Data analysis revealed that the endogenous NO generated was positively associated with the proline level in Cu-stressed algae. The involvement of NO in Cu-induced proline accumulation was confirmed by using an NO-specific donor, sodium nitroprusside (SNP), and an NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide]. Pre-treatment with 10 microM SNP increased the proline accumulation in Cu-treated cells by about 1.5-fold, while this effect could be blocked by addition of 10 microM cPTIO. We further investigated the effect of Cu and NO on the activity and transcript amount of Delta(1)-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11), the key enzyme of proline biosynthesis, and observed that application of SNP was able to stimulate the P5CS activity and up-regulate the expression of P5CS in the Cu-treated algae. These results indicate that Cu-responsive proline synthesis is closely related to NO generation in C. reinhardtii, suggesting the regulatory function of NO in proline metabolism under heavy metal stress.
TL;DR: Investigation of the relationship between the functions of PIP-type water channels and water relations of tobacco plants under drought stress suggests that NtPIP1;1 and NTPIP2;1 play an important role in water transport in roots, and that expression of NtAQP1 andNtPip2; 1 is down-regulated in order to reduce osmotic hydraulic conductance in the roots of tobacco Plants under drought Stress.
Abstract: Plasma membrane intrinsic proteins (PIPs), a type of aquaporins, mediate water transport in many plant species. In this study, we investigated the relationship between the functions of PIP-type water channels and water relations of tobacco plants (Nicotiana tabacum cv. Samsun) under drought stress. Drought stress treatments have led to reductions in the stomatal conductance, transpiration, water potential and turgor pressure in leaves, and also the sap flow rate and osmotic hydraulic conductance in roots. In contrast, leaf osmotic pressure was increased in response to drought stress. Interestingly, the accumulation of NtPIP1;1 and NtPIP2;1 transcripts was significantly decreased, but only that of the NtAQP1 transcript was increased under drought stress. Functional analysis using Xenopus laevis oocytes revealed that NtPIP2;1 shows marked water transport activity, but the activities of NtAQP1 and NtPIP1;1 are weak or almost negligible, respectively, when expressed alone. However, co-expression of NtPIP1;1 with NtPIP2;1 significantly enhanced water transport activity compared with that of NtPIP1;1- or NtPIP2;1-expressing oocytes, suggesting that these two aquaporins may function as a water channel, forming a heterotetramer. Heteromerization of NtPIP1;1 and NtPIP2;1 was also suggested by co-expression analyses of NtPIP1;1-GFP (green fluorescent protein) and NtPIP2;1 in Xenopus oocytes. Re-watering treatments recovered water relation parameters and the accumulation of the three NtPIP transcripts to levels similar to control conditions. These results suggest that NtPIP1;1 and NtPIP2;1 play an important role in water transport in roots, and that expression of NtPIP1;1 and NtPIP2;1 is down-regulated in order to reduce osmotic hydraulic conductance in the roots of tobacco plants under drought stress.
TL;DR: It is proposed that PR-3 mediates ABA-dependent salt stress signals that affect seed germination in Arabidopsis and is found that individual PR genes are temporally and spatially regulated in distinct patterns, supporting the contention that the PR proteins play a role in plant developmental processes other than disease resistance response.
Abstract: Pathogenesis-related (PR) proteins are a group of heterogeneous proteins encoded by genes that are rapidly induced by pathogenic infections and by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). They are widely used as molecular markers for resistance response to pathogens and systemic acquired response (SAR). However, recent studies have shown that the PR genes are also regulated by environmental factors, including light and abiotic stresses, and by developmental cues, suggesting that they also play a role in certain stress responses and developmental processes. In this work, we systematically examined the expression patterns of Arabidopsis PR genes. We also investigated the effects of environmental stresses and growth hormones on the expression of PR genes. We found that individual PR genes are temporally and spatially regulated in distinct patterns. In addition, they are differentially regulated by plant growth hormones, including SA, ABA, JA, ET and brassinosteroid (BR), and by diverse abiotic stresses, supporting the contention that the PR proteins play a role in plant developmental processes other than disease resistance response. Interestingly, PR-3 was induced significantly by high salt in an ABA-dependent manner. Consistent with this, a T-DNA insertional knockout plant with disruption of the PR-3 gene showed a significantly reduced rate of seed germination in the presence of high salt. It is thus proposed that PR-3 mediates ABA-dependent salt stress signals that affect seed germination in Arabidopsis. PR-4 and PR-5 also contributed to salt regulation of seed germination, although their effects were not as evident as those of PR-3.
TL;DR: Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a), indicating that the major effect of theWx transgene on amylopectin structure was ELC formation.
Abstract: A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx a ) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx a .
TL;DR: LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of I AA and GA4 in the mature anther.
Abstract: To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profi les of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA 4 accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LMarray analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA 4 in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was downregulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.
TL;DR: It is suggested that MeJa induces ROS production and alterations of mitochondrial dynamics as well as subsequent photosynthetic collapse, which occur upstream of cell death and are necessary components of the cell death process.
Abstract: Methyl jasmonate (MeJa) is a well-known plant stress hormone. Upon exposure to stress, MeJa is produced and causes activation of programmed cell death (PCD) and defense mechanisms in plants. However, the early events and the signaling mechanisms of MeJa-induced cell death have yet to be fully elucidated. To obtain some insights into the early events of this cell death process, we investigated mitochondrial dynamics, chloroplast morphology and function, production and localization of reactive oxygen species (ROS) at the single-cell level as well as photosynthetic capacity at the whole-seedling level under MeJa stimulation. Our results demonstrated that MeJa induction of ROS production, which first occurred in mitochondria after 1 h of MeJa treatment and subsequently in chloroplasts by 3 h of treatment, caused a series of alterations in mitochondrial dynamics including the cessation of mitochondrial movement, the loss of mitochondrial transmembrane potential (MPT), and the morphological transition and aberrant distribution of mitochondria. Thereafter, photochemical efficiency dramatically declined before obvious distortion in chloroplast morphology, which is prior to MeJa-induced cell death in protoplasts or intact seedlings. Moreover, treatment of protoplasts with ascorbic acid or catalase prevented ROS production, organelle change, photosynthetic dysfunction and subsequent cell death. The permeability transition pore inhibitor cyclosporin A gave significant protection against MPT loss, mitochondrial swelling and subsequent cell death. These results suggested that MeJa induces ROS production and alterations of mitochondrial dynamics as well as subsequent photosynthetic collapse, which occur upstream of cell death and are necessary components of the cell death process.
TL;DR: The results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.
Abstract: A large amount of gamma-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar 'DG03-9' which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of 'Micro-Tom' and 'DG03-9' fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of alpha-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In 'DG03-9' fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.
TL;DR: It is found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination.
Abstract: MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II) Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80 We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1 Our results identify new components in miRNA biogenesis
TL;DR: A shotgun analysis of the proteome of phloem and xylem saps from rice suggests that proteomics on a nano scale is a potent tool for investigation of biological processes in plants.
Abstract: The main physiological roles of phloem and xylem in higher plants involve the transport of water, nutrients and metabolites. They are also involved, however, in whole plant events including stress responses and long-distance signaling. Phloem and xylem saps therefore include a variety of proteins. In this study, we have performed a shotgun analysis of the proteome of phloem and xylem saps from rice, taking advantage of the complete and available genomic information for this plant. Xylem sap was prepared using the root pressure method, whereas phloem sap was prepared with a unique method with the assistance of planthoppers to ensure the robustness of the detected proteins. The technical difficulties caused by the very limited availability of rice samples were overcome by the use of nano-flow liquid chromatography linked to a mass spectrometer. We identified 118 different proteins and eight different peptides in xylem sap, and 107 different proteins and five different peptides in phloem sap. Signal transduction proteins, putative transcription factors and stress response factors as well as metabolic enzymes were identified in these saps. Interestingly, we found the presence of three TERMINAL FLOWER 1/FLOWERING LOCUS T (FT)-like proteins in phloem sap. The detected FT-like proteins were not rice Hd3a (OsFTL2) itself that acted as a non-cell-autonomous signal for flowering control, but they were members of distinct subfamilies of the FT family with differential expression patterns. These results imply that proteomics on a nano scale is a potent tool for investigation of biological processes in plants.
TL;DR: The partial inhibition of the hypoxia-induced alanine accumulation in roots of these mutants suggests that the GABA shunt is, in part, responsible for the alanines accumulation under hypoxic conditions.
Abstract: When subjected to low oxygen stress, plants accumulate alanine and gamma-aminobutyric acid (GABA). To investigate the function of GABA metabolism under hypoxia and its contribution to alanine accumulation, we studied the genes that encode the two key enzymes of the GABA shunt, glutamate decarboxylase (GAD) and GABA transaminase (GABA-T). Among the five homologous GAD genes found in Arabidopsis thaliana, GAD1 expression was predominantly found in roots, while GAD2 expression was evident in all organs. Expression of the other three GAD genes was generally weak. In response to hypoxia, transcriptional induction was observed for GAD4 only. For GABA-T1, its expression was detected in all organs, but there was no significant transcriptional change under hypoxic conditions. Moreover, we have isolated and characterized Arabidopsis mutants defective in GAD1 and GABA-T1. In gad1 mutants, GAD activity was significantly reduced in roots but was not affected in shoots. In the gaba-t1 mutant, GABA-T activity was decreased to negligible levels in both shoots and roots. These mutants were phenotypically normal under normal growth conditions except for the reduced seed production of the pop2 mutants as described previously. However, metabolite analysis revealed significant changes in GABA content in gad1 and gaba-t1 mutants. The levels of alanine under hypoxic conditions were also affected in the roots of gad1 and gaba-t1 mutants. The partial inhibition of the hypoxia-induced alanine accumulation in roots of these mutants suggests that the GABA shunt is, in part, responsible for the alanine accumulation under hypoxia.
TL;DR: The lower membrane ion leakage and malondialdehyde level in these transgenic plants suggest that overexpression of H(+)-PPase causes the accumulation of Na(+) and Cl(-) in vacuoles instead of in the cytoplasm, thus reducing their toxic effects.
Abstract: Salinity is one of the major environmental factors limiting plant growth and productivity. An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into cotton (Gossypium hirsutum) in sense and antisense orientations under control of the cauliflower mosaic virus (CaMV) 35S promoter. Southern and Northern blotting analysis showed that the sense or antisense TsVP were integrated into the cotton genome and expressed. Transgenic plants overexpressing the vacuolar H(+)-PPase were much more resistant to 150 and 250 mM NaCl than the isogenic wild-type plants. In contrast, the plants from the antisense line (L-2), with lower H(+)-PPase activity, were more sensitive to salinity than the wild-type plants. Overexpressing TsVP in cotton improved shoot and root growth and photosynthetic performance. These transgenic plants accumulated more Na(+), K(+), Ca(2+), Cl(-) and soluble sugars in their root and leaf tissues under salinity conditions compared with the wild-type plants. The lower membrane ion leakage and malondialdehyde (MDA) level in these transgenic plants suggest that overexpression of H(+)-PPase causes the accumulation of Na(+) and Cl(-) in vacuoles instead of in the cytoplasm, thus reducing their toxic effects. On the other hand, the increased accumulation of ions and sugars decreases the solute potential in cells, and facilitates water uptake under salinity, which is an important mechanism for the increased salt tolerance in TsVP-overexpressing cotton.
TL;DR: A strategy for improving the ability of cereals to adapt to low P environments is proposed that involves alteration in partitioning of carbohydrates into organic acids and amino acids to enable more efficient utilization of carbon in P-deficient plants.
Abstract: Plants modify metabolic processes for adaptation to low phosphate (P) conditions. Whilst transcriptomic analyses show that P deficiency changes hundreds of genes related to various metabolic processes, there is limited information available for global metabolite changes of P-deficient plants, especially for cereals. As changes in metabolites are the ultimate 'readout' of changes in gene expression, we profiled polar metabolites from both shoots and roots of P-deficient barley (Hordeum vulgare) using gas chromatography-mass spectrometry (GC-MS). The results showed that mildly P-deficient plants accumulated di- and trisaccharides (sucrose, maltose, raffinose and 6-kestose), especially in shoots. Severe P deficiency increased the levels of metabolites related to ammonium metabolism in addition to di- and trisaccharides, but reduced the levels of phosphorylated intermediates (glucose-6-P, fructose-6-P, inositol-1-P and glycerol-3-P) and organic acids (alpha-ketoglutarate, succinate, fumarate and malate). The results revealed that P-deficient plants modify carbohydrate metabolism initially to reduce P consumption, and salvage P from small P-containing metabolites when P deficiency is severe, which consequently reduced levels of organic acids in the tricarboxylic acid (TCA) cycle. The extent of the effect of severe P deficiency on ammonium metabolism was also revealed by liquid chromatography-mass spectrometry (LC-MS) quantitative analysis of free amino acids. A sharp increase in the concentrations of glutamine and asparagine was observed in both shoots and roots of severely P-deficient plants. Based on these data, a strategy for improving the ability of cereals to adapt to low P environments is proposed that involves alteration in partitioning of carbohydrates into organic acids and amino acids to enable more efficient utilization of carbon in P-deficient plants.
TL;DR: It is shown that roots of COI1-silenced tobacco plants are insensitive to growth inhibition by methyl jasmonate, and do not activate nicotine biosynthesis genes after jASmonate treatment or wounding of leaves, and results indicate that jasMonate-triggered, COi1-mediated degradation of JAZ repressors activates transcriptional regulation of nicotine biosynthetic genes in tobacco roots.
Abstract: Biosynthesis of many plant alkaloids is enhanced by endogenous accumulation and exogenous application of jasmonates, but the general and specific signaling components are not well understood. In Arabidopsis, jasmonate-induced ZIM-domain-containing (JAZ) proteins have recently been found to be critical transcriptional repressors linking CORONATINE INSENSTIVE1 (COI1)-mediated jasmonate perception and jasmonate-regulated transcriptional regulation. Insect herbivory on tobacco leaves activates the jasmonate signaling pathway, leading to up-regulation of nicotine biosynthesis genes in roots. We show here that roots of COI1-silenced tobacco plants are insensitive to growth inhibition by methyl jasmonate, and do not activate nicotine biosynthesis genes after jasmonate treatment or wounding of leaves. Tobacco JAZ proteins appeared to be rapidly degraded after jasmonate treatment, whereas a C-terminally truncated form lacking the conserved Jas motif did not. When the non-degradable JAZ forms were expressed in tobacco hairy roots, jasmonate induction of nicotine biosynthesis was strongly inhibited. Formation of tobacco alkaloids in jasmonate-elicited tobacco BY-2 cells was also effectively suppressed by the COI1 RNAi (RNA interference) construct and by the dominant-negative truncated JAZ constructs. In addition, jasmonate-mediated induction of nicotine biosynthesis genes was diminished by treatment with a proteasome inhibitor MG132. These results indicate that jasmonate-triggered, COI1-mediated degradation of JAZ repressors activates transcriptional regulation of nicotine biosynthesis genes in tobacco roots.
TL;DR: Analysis of recombinant AtPAO4 protein indicated that it is a flavoprotein that catalyzed the oxidative conversion of spermine to spermidine and that it participates in polyamine catabolism.
Abstract: We characterized three Arabidopsis polyamine oxidase genes, AtPAO2, AtPAO3 and AtPAO4. Transient expression of these genes as monomeric red fluorescent protein fusion proteins in Arabidopsis root cells revealed that all are peroxisomal proteins. Quantitative analysis of their transcripts in various organs suggested that AtPAO4 is the major isoform in root peroxisomes. Analysis of recombinant AtPAO4 protein indicated that it is a flavoprotein that catalyzed the oxidative conversion of spermine to spermidine. AtPAO4-deficient mutants established by using T-DNA insertion and RNA interference techniques had markedly increased spermine and decreased spermidine levels in the roots. These results suggest that AtPAO4 is a root peroxisomal polyamine oxidase that participates in polyamine catabolism. Microarray analysis showed that AtPAO4 deficiency induced alterations in the expression of genes related to the drought stress response and flavonoid biosynthesis.
TL;DR: It is found that thermospermine was detected in wild-type seedlings but was not detectable in the acl5-1 mutant and produced through the action of ACL5 and required for stem elongation in Arabidopsis.
Abstract: Loss-of-function mutants of the ACAULIS5 (ACL5) gene in Arabidopsis thaliana have severe defects in stem elongation ACL5 was previously reported as encoding a spermine synthase A more recent study, however, showed that the bacterial expressed recombinant ACL5 protein catalyzes the conversion of spermidine to thermospermine, a structural isomer of spermine, rather than to spermine In the present study, we found that thermospermine was detected in wild-type seedlings but was not detectable in the acl5-1 mutant We further examined the effect of exogenous application of these isomers on the growth of acl5-1 Daily application of 01 mM thermospermine onto the shoot apex partially rescued the dwarf phenotype of acl5-1, while that of spermine had no effects on the morphology of the mutant The acl5-1 transcript level in acl5-1 seedlings, which is much higher than the ACL5 transcript level in wild-type seedlings, was reduced by exogenous thermospermine Thus we conclude that thermospermine is indeed produced through the action of ACL5 and required for stem elongation in Arabidopsis
TL;DR: The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.
Abstract: Adaptation to steady-state low-nutrient availability was investigated by comparing the Wassileskija (WS) accession of Arabidopsis thaliana grown on 2 or 10 mM nitrate. Low nitrogen conditions led to a limited rosette biomass and seed yield. The latter was mainly due to reduced seed number, while seed weight was less affected. However, harvest index was lower in high nitrate compared with limited nitrate conditions. Under nitrogen-limiting conditions, nitrate reductase activity was decreased while glutamine synthetase activity was increased due to a higher accumulation of the cytosolic enzyme. The level of nitrogen remobilization to the seeds was higher under low nitrogen, and the vegetative parts of the plants remaining after seed production stored very low residual nitrogen. Through promoting nitrogen remobilization and recycling pathways, nitrogen limitation modified plant and seed compositions. Rosette leaves contained more sugars and less free amino acids when grown under nitrogen-limiting conditions. Compared with high nitrogen, the levels of proline, asparagine and glutamine were decreased. The seed amino acid composition reflected that of the rosette leaves, thus suggesting that phloem loading for seed filling was poorly selective. The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.
TL;DR: Cd transfer from the root medium to the xylem in A. halleri is an energy-dependent process that is partly shared with Zn and/or Fe transport and is translocated from roots to shoots in inorganic forms.
Abstract: Arabidopsis halleri is a Cd hyperaccumulator; however, the mechanisms involved in the root to shoot translocation of Cd are not well understood. In this study, we characterized Cd transfer from the root medium to xylem in this species. Arabidopsis halleri accumulated 1,500 mg kg(-1) Cd in the shoot without growth inhibition. A time-course experiment showed that the release of Cd into the xylem was very rapid; by 2 h exposure to Cd, Cd concentration in the xylem sap was 5-fold higher than that in the external solution. The concentration of Cd in the xylem sap increased linearly with increasing Cd concentration in the external solution. Cd transfer to the xylem was completely inhibited by the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cd concentration in the xylem sap was decreased by increasing the concentration of external Zn, but enhanced by Fe deficiency treatment. Analysis with 113Cd-nuclear magnetic resonance (NMR) showed that the chemical shift of 113Cd in the xylem sap was the same as that of Cd(NO3)2. Metal speciation with Geochem-PC also showed that Cd occurred mainly in the free ionic form in the xylem sap. These results suggest that Cd transfer from the root medium to the xylem in A. halleri is an energy-dependent process that is partly shared with Zn and/or Fe transport. Furthermore, Cd is translocated from roots to shoots in inorganic forms.
TL;DR: Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance, but such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.
Abstract: Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.
TL;DR: It is suggested that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway.
Abstract: Arabidopsis sol2 mutants showed CLV3 peptide resistance. Twenty-six synthetic CLE peptides were examined in the clv1, clv2 and sol2 mutants. sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2. SOL2 encoded a receptor-like kinase protein which is identical to CORYNE (CRN). GeneChip analysis revealed that the expression of several genes was altered in the sol2 root tip. Here, we suggest that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway.
TL;DR: Results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.
Abstract: Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.