AtOPT6 transports glutathione derivatives and is induced by primisulfuron.

In their habitats, plants are increasingly subjected to a wide spectrum of biotic as well as abiotic stresses, which limit crop productivity. However, plants have an ability to sense the stress environment and activate appropriate responses to protect themselves. Understanding signalling events leading to plant defence against environmental challenges can lead to effective strategies to improve plant stress tolerance. Reactive oxygen species (ROS) produced under a variety of different stress conditions are known to play important signalling roles during adaptation of plants to biotic and abiotic challenges. Nevertheless, a major gap exists in our understanding about how ROS induce large-scale and coordinated expression from many genes. In addition, very little is known about the upstream signalling components which perceive and transduce ROS signals within the cell. Even though many genes including transcription factors (TFs) are known to be ROS-induced transcriptionally, only very few of these TFs have been functionally characterised. This PhD study had three main objectives. The first was to identify transcriptional regulators that modulate the early signalling events in the ROS signal transduction pathway. After screening for early and highly oxidative stress-induced TFs in Arabidopsis thaliana, the Ethylene Response Factor 6 (ERF6) gene was identified and chosen for further functional characterisation. The second aim was to decipher the upstream signalling events through which ROS are produced, perceived, and delivered for the ROS-mediated induction of ERF6. And the third objective was to identify and analyse potential downstream targets of ERF6 during ROS signalling. To fulfil the first aim of this study, the expression of ERF6 under different stresses has been investigated. Wild-type Arabidopsis plants were treated with the ROS-generating herbicide paraquat, H2O2 or inoculated with the bacterial pathogen Pseudomonas syringae and the fungal pathogen Fusarium oxysporum. In addition, Arabidopsis plants were subjected to water stress, heat shock, salt stress and cold treatment, all of which are known to induce ROS production. The results showed that ERF6 is strongly induced by oxidative stress imposed by paraquat and H2O2. The common finding that almost all types of stresses produce ROS in plants raised the possibility that ERF6 is very likely to be induced by ROS-inducing environmental challenges as well. However, the results presented here showed that ERF6 is repressed by drought and heat, but induced by cold. ERF6 is also induced by the biotrophic bacterial pathogen Pseudomonas syringae (DC3000) but not by the hemibiotrophic fungal pathogen Fusarium oxysporum. In addition, the treatment of Arabidopsis plants with the defence signalling compound salicylic acid (SA) led to the induction of ERF6 while treatment with the abiotic stress-related phytohormone abscisic acid (ABA) led to repression of ERF6. To achieve the second objective of this study, the dependence of ERF6 expression on signalling intermediates such as NADPH oxidases, G proteins and calcium ions was investigated using Arabidopsis mutants defective for each of these components. Firstly, comparing the expression of ERF6 and the NADPH oxidase D encoding gene RbohD under different stresses showed that the expression patterns of both genes were very similar, i.e. RbohD is induced by paraquat, H2O2, Pseudomonas and SA but repressed by water stress, heat and ABA. Additional results showed that ERF6 required RbohD for full induction by oxidative stress as ROS-dependent expression of ERF6 was compromised in the rbohD mutant. Also, this finding suggests that, via the differential expression of NADPH oxidases, ERF6 is induced by actively ROS-generating stresses such as pathogen infection, but repressed by passively-ROS generating abiotic stresses as a consequence of cell damage. In addition, our data suggest that NADPH oxidase D could represent a point at which the control of abiotic and biotic stress responses by ROS diverges. In addition to RbohD, the results show that G protein a (Ga) and calcium are required for full induction of ERF6 by H2O2. For the third objective of this project, the erf6 knockout mutant was used to investigate the function of ERF6 in modulating plant oxidative stress responses. Results presented here suggest that ERF6 is one of the important signalling intermediates that are required to maintain ROS levels under control. Compared to wild-type, erf6 mutant plants showed a growth reduction, inefficient photosynthesis and high levels of H2O2. Out of fifty potential ERF6 target genes tested, MDAR3, encoding an enzyme (monodehydroascorbate reductase) involved in ROS scavenging, was found to have reduced ROS-responsiveness in erf6 mutant plants. This indicates that ERF6 might control a subset of genes, including MDAR3, encoding proteins involved in controlling ROS levels. Therefore, when ERF6 is knocked out, the ROS controlling machinery is impaired, leading to accumulation of H2O2 in erf6 plants. Interestingly, the knockout mdar3 mutant plants did not show the reduced growth phenotype of erf6 plants. This indicates the possibility that, in addition to MDAR3, other ROS-related genes may have compromised expression in erf6 plants. Moreover, under high light, erf6 mutant plants showed high levels of anthocyanin pigments. This was accompanied with high expression levels of the anthocyanin biosynthesis gene chalcone synthase (CHS), inferring that erf6 plants accumulate anthocyanin as an antioxidant to cope with the high levels of ROS in mutant plants. In addition, this high level of anthocyanin may be contributing to the reduced growth phenotype of erf6 plants owing to growth regulating properties of flavonoids. Collectively, the results presented in this PhD project suggest that ERF6 is one of the genes that establish the specific responses to ROS produced by different stresses. ERF6 TF is required to maintain ROS under control by upregulating genes required for ROS-scavenging. Overall, these results provide new insights into better understanding of the ROS signalling in plants and may help with the development of new stress protection strategies in plants.

Evidence for surface perception of abscisic acid by rice suspension cells as assayed by Em gene expression

Abiotic stresses are major limiting factors for growth, development, and productivity of crop plants. Here, we report on OsSKIPa, a rice homolog of human Ski-interacting protein (SKIP) that can complement the lethal defect of the knockout mutant of SKIP homolog in yeast and positively modulate cell viability and stress tolerance of rice. Suppression of OsSKIPa in rice resulted in growth arrest and reduced cell viability. The expression OsSKIPa is induced by various abiotic stresses and phytohormone treatments. Transgenic rice overexpressing OsSKIPa exhibited significantly improved growth performance in the medium containing stress agents (abscisic acid, salt, or mannitol) and drought resistance at both the seedling and reproductive stages. The OsSKIPa-overexpressing rice showed significantly increased reactive oxygen species-scavenging ability and transcript levels of many stress-related genes, including SNAC1 and rice homologs of CBF2, PP2C, and RD22, under drought stress conditions. More than 30 OsSKIPa-interacting proteins were identified, but most of these proteins have no matches with the reported SKIP-interacting proteins in animals and yeast. Together, these data suggest that OsSKIPa has evolved a specific function in positive modulation of stress resistance through transcriptional regulation of diverse stress-related genes in rice.

The plant hormone abscisic acid (ABA) is involved in regulating a number of major processes such as seed dormancy, seedling development, and biotic and abiotic stress responses. The function and effect of ABA on pathogens are still unclear, but the roles of ABA in seed germination and abiotic stress responses have been well characterized. Abiotic stresses elevate ABA levels and activate ABA signaling; thus, inducing a variety of responses, including the expression of stress-related genes and stomatal closure. The past decade has witnessed many significant advances in our understanding of ABA signal transduction due to application of a combination of approaches including genetics, biochemistry, electrophysiology, and chemical genetics. A number of proteins associated with the ABA signal transduction pathway such as PYR/PYL/RCAR family of START proteins, have been identified. These ABA receptors bind to ABA and positively regulate ABA signaling via inactivation of PP2C phosphatase activity, which inhibits SnRK2-type kinases by direct interaction and dephosphorylation. Additionally, SnRK2-type kinases and PP2Cs interact with one another and with other components of ABA signaling and function as positive and negative ABA regulators, respectively. In this review, we focus on ABA function to abiotic stresses and highlight each component in relation to ABA and its interactions.

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.

BackgroundDue to global warming and gradual climate change, plants are subjected to a wide range of environmental stresses, adversely affecting plant growth and production worldwide. Plants have developed various mechanisms to overpower these abiotic stresses, including salt stress, drought, and high light intensity. Apart from their own defense strategies, plants can get help from the beneficial endophytic bacteria inside host plants and assist them in enduring severe growth conditions. A previously isolated plant endophytic bacteria, Burkholderia seminalis 869T2, from vetiver grass can produce auxin, synthesize siderophore, and solubilize phosphate. The B. seminalis 869T2 can colonize inside host plants and increase the growth of bananas, Arabidopsis, and several leafy vegetables.ResultsWe further demonstrated that different growth parameters of Arabidopsis and pak choi plants were significantly increased after inoculating the B. seminalis 869T2 under normal, salt, and drought stress conditions compared to the mock-inoculated plants. Both transcriptome analysis and quantitative real-time PCR results showed that expression levels of genes related to phytohormone signal transduction pathways, including auxin, gibberellin, cytokinin, and abscisic acid were altered in Arabidopsis plants after inoculated with the strain 869T2 under salt stress, in comparison to the mock-inoculated control with salt treatments. Furthermore, the accumulation levels of hydrogen peroxide (H2O2), electrolyte leakage (EL), and malondialdehyde (MDA) were lower in the 869T2-inoculated Arabidopsis and pak choi plants than in control plants under salt and drought stresses.ConclusionsThe plant endophytic bacterium strain B. seminalis 869T2 may affect various phytohormone responses and reduce oxidative stress damage to increase salt and drought stress tolerances of host plants.

We studied the link between abscisic acid (ABA), auxin, MAPK (mitogen-activated protein kinase) signaling, and the cell cycle in cadmium (Cd)-stressed rice (Oryza sativa L. cv. Zhonghua No. 11) roots. ABA can partially compensate for root growth inhibition and counteract over-accumulation of auxin caused by Cd. GUS staining of rice plants harboring DR5-GUS showed that ABA affects auxin distribution in Cd-stressed roots. Detection using DRB (5,6-dichlorobenzimidazole 1-β-d-ribofuranoside, an RNA synthesis inhibitor), MG132 (a protein degradation inhibitor), BFA (brefeldin A, a protein transport inhibitor), and TIBA (2,3,5-triiodobenzoic acid, a polar auxin transport inhibitor) revealed that ABA regulates the distribution of auxin via transcription, protein degradation, and transport pathways under Cd stress. Several genes related to ABA and MAPK, key components of the auxin signaling pathway, and the cell cycle were differentially regulated by Cd plus ABA vs. Cd plus tungstate (TS) (a ABA biosynthesis inhibitor) at 7 days or 11 days of treatment in roots, indicating that ABA levels affect the transcription of these genes, and that these genes are differentially regulated by ABA in rice seedlings at different developmental stages. Furthermore, the expression of some of these genes differed between Cd + ABA/TS-treated plants and plants treated with TS or ABA alone, suggesting that ABA signaling serves specific functions in the regulation of gene expression under Cd stress. Overall, these results suggest that ABA coordinates auxin and MAPK signaling and the cell cycle in response to Cd stress. The ABA signal transduction pathways in Cd-stressed rice plants are discussed.

Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Rice (Oryza sativa L.) is one of the most important food crops in the world. In plants, jasmonic acid (JA) plays essential roles in response to biotic and abiotic stresses. As one of the largest transcription factors (TFs), basic region/leucine zipper motif (bZIP) TFs play pivotal roles through the whole life of plant growth. However, the relationship between JA and bZIP TFs were rarely reported, especially in rice. In this study, we found two rice homologues of Arabidopsis VIP1 (VirE2-interacting protein 1), OsbZIP81, and OsbZIP84. OsbZIP81 has at least two alternative transcripts, OsbZIP81.1 and OsbZIP81.2. OsbZIP81.1 and OsbZIP84 are typical bZIP TFs, while OsbZIP81.2 is not. OsbZIP81.1 can directly bind OsPIOX and activate its expression. In OsbZIP81.1 overexpression transgenic rice plant, JA (Jasmonic Acid) and SA (Salicylic acid) were up-regulated, while ABA (Abscisic acid) was down-regulated. Moreover, Agrobacterium, Methyl Jasmonic Acid (MeJA), and PEG6000 can largely induce OsbZIP81. Based on ChIP-Seq and Random DNA Binding Selection Assay (RDSA), we identified a novel cis-element OVRE (Oryza VIP1 response element). Combining ChIP-Seq and RNA-Seq, we obtained 1332 targeted genes that were categorized in biotic and abiotic responses, including α-linolenic acid metabolism and fatty acid degradation. Together, these results suggest that OsbZIP81 may positively regulate JA levels by directly targeting the genes in JA signaling and metabolism pathway in rice.

The phytohormone abscisic acid (ABA) is essential in plant responding to biotic and abiotic stresses. Although ABA signaling model is well established in Arabidopsis, ABA receptor PYL family and clade A PP2C subfamily are not yet characterized in monocot model plant Brachypodium distachyon. In this study, we identified 12 PYLs and 8 clade A PP2Cs from B. distachyon genome and successfully cloned 12 PYLs and 7 clade A PP2Cs. Bioinformatic and expression analyses showed that most of the identified genes respond to several signal molecules and abiotic stresses. Protein–protein interaction analysis revealed that many BdPYLs and BdPP2CAs participate in the classic ABA-PYL-PP2C-SnRK2 signaling pathway. A clade A PP2C, designated BdPP2CA6, interacted with BdPYL11 in the absence of ABA and localized in nucleus. Most clade A PP2C members from Arabidopsis showed negatively regulation in ABA signaling pathway, whereas BdPP2CA6-overexpression transgenic Arabidopsis showed ABA hypersensitive phenotype, resulting in enhanced stomatal closure and salinity tolerance. Our results indicate that BdPP2CA6 positively regulates ABA and stress signal pathway in transgenic Arabidopsis plant seedlings.

The phytohormone abscisic acid (ABA) plays a crucial role in numerous aspects of plant growth and environmental stress responses. Endogenous ABA levels are regulated by a balance between its biosynthetic and catabolic activities. This balance may occur at multiple levels and includes the expression of genes involved in these processes. ABA UDP-glucosyltransferase (UGT), the major player in the ABA conjugation pathway, has been shown to have a marginal effect on free ABA levels. However, recent studies provide new insight into the importance of the ABA conjugation pathway in contributing to the control of ABA homeostasis. Gain-of-function and loss-of-function mutant analyses have revealed that UGT71B6, an ABA UGT, and its 2 closely related homologs, UGT71B7 and UGT71B8, play a crucial role in ABA homeostasis and in adaptation to various abiotic stresses.

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

The effect of abscisic acid (ABA) on growth, abiotic stress tolerance, and physiology of tomato seedlings was investigated. To examine the effect of ABA concentration on growth and abiotic stresses, six ABA concentrations (0, 10, 50, 100, 150, or 200 mg·L-1) were applied by foliar spraying once a day for 10 days. The effect of ABA application number was also studied by using different timing at one ABA concentration (100 mg·L-1) once a day for 1, 3, 5, 7, and 9 days. The effect of ABA on physiology of tomato seedlings was examined by using two concentrations (50 and 100 mg·L-1) as compared to the control (non-ABA). Foliar application of ABA decreased the growth characteristics of tomato seedlings in a concentration-dependent manner; however, no statically significant difference was observed between the 50 and 100 mg·L-1 treatments. Furthermore, although growth parameters decreased statistically with increasing number of ABA treatments, there was no difference between the 3 and 5 application treatments. Application of ABA enhanced stress tolerance (cold and drought) of tomato seedlings by delaying the starting time of wilting point in drought conditions and reducing the relative ion leakage and chilling injury index in low temperature in all treatments. The transpiration rate decreased significantly, while stomatal diffusive resistance increased significantly with increasing ABA concentration. The relative water content decreased significantly during the period without irrigation. However, relative water content increased with increasing ABA concentration. The ABA enhanced drought tolerance of tomato seedlings by delaying the start time of wilting point from day 3 in the control to day 5 and 7 in the 50 and 100 mg·L-1 treatments, respectively. Integrating this result data, we can determine the ABA’s ability to maintain of seedling quality at low temperature and water deficit condition.

Since plants cannot move to avoid stress, they have sophisticated acclimation mechanisms against a variety of abiotic stresses. The phytohormone abscisic acid (ABA) plays essential roles in abiotic stress tolerances in land plants. Therefore, it is interesting to address the evolutionary origins of ABA metabolism and its signaling pathways in land plants. Here, we focused on 48 ABA-related Arabidopsis thaliana genes with 11 protein functions, and generated 11 orthologous clusters of ABA-related genes from A. thaliana, Arabidopsis lyrata, Populus trichocarpa, Oryza sativa, Selaginella moellendorffii, and Physcomitrella patens. Phylogenetic analyses suggested that the common ancestor of these six species possessed most of the key protein functions of ABA-related genes. In two species (A. thaliana and O. sativa), duplicate genes related to ABA signaling pathways contribute to the expression variation in different organs or stress responses. In particular, there is significant expansion of gene families related to ABA in evolutionary periods associated with morphological divergence. Taken together, these results suggest that expansion of the gene families related to ABA signaling pathways may have contributed to the sophisticated stress tolerance mechanisms of higher land plants.

Rice (Oryza sativa L.) is susceptible to various abiotic stresses, such as salt, cold, and drought. Therefore, there is an urgent need to explore the relevant genes that enhance tolerance to these stresses. In this study, we identified a gene, OsNCED4 (9-cis-epoxycarotenoid dioxygenase 4), which regulates tolerance to multiple abiotic stresses. OsNCED4 encodes a chloroplast-localized abscisic acid (ABA) biosynthetic enzyme. The expression of OsNCED4 gene was significantly induced by 150 mM NaCl and cold stress. Disruption of OsNCED4 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated mutagenesis resulted in significant sensitivity to NaCl and cold stress. The salt and cold sensitivity of osnced4 mutant was due to the reduction of ABA content and the excessive accumulation of reactive oxygen species (ROS) under stress. Moreover, OsNCED4 also regulates drought stress tolerance of rice seedlings. Taken together, these results indicate that OsNCED4 is a new regulator for multiple abiotic stress tolerance in rice, and provided a potential target gene for enhancing multiple stress tolerance in the future.