ABSTRACT: Dissecting physiological and transcriptional responses of nitric oxide to drought stress by increasing in vivo nitric oxide content in Arabidopsis
Project description:Nitric oxide (NO) is involved in all major environmental stresses. However, most of these understandings were mainly based on pharmacological study using NO modulator compounds. Recently, our studies together with others provided a new class of plant experimental system with specific in vivo NO release through constitutively overexpressing rat neuronal NO synthase (nNOS) in plants. In this study, we found that the nNOS transgenic Arabidopsis plants displayed lower level of H2O2 content, but higher levels of antioxidant enzyme activities and osmolytes under drought stress conditions. Transcriptomic analysis identified 490 and 20 genes that were differentially expressed in wild type (WT) and nNOS transgenic plants under control and drought stress conditions, respectively. Pathway analysis revealed that many genes involved in photosynthesis, cell, misc, co-factor and vitamin metabolism, major CHO metabolism, OPP and secondary metabolism were largely changed in nNOS vs. WT under control or drought stress conditions. Interestingly, CBF1/2 and 13 zinc finger family proteins, known as important family of transcription regulators in modulating several stress-responsive genes, were differentially expressed by nNOS transgenic effect. Additionally, some genes were commonly regulated by nNOS transgenic and abscisic acid (ABA) effects, indicating new insights to cross-talk between ABA and NO. Taken together, in vivo NO modulated antioxidant enzyme activities, osmolyte level, and the expression of genes involved in several pathways, thereby resulting in enhanced stress tolerance in nNOS transgenic plants. These observations might provide some insights to understand the physiological and molecular mechanisms of NO in response to drought stress in Arabidopsis. Two transgenic Arabidopsis lines (nNOS-2 and nNOS-25) with the nNOS gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, as well as Col-0 (WT), were used in this research. RNA samples from two nNOS transgenic lines were pooled for cRNA labelling and chip hybridization.
Project description:Nitric oxide (NO) is involved in all major environmental stresses. However, most of these understandings were mainly based on pharmacological study using NO modulator compounds. Recently, our studies together with others provided a new class of plant experimental system with specific in vivo NO release through constitutively overexpressing rat neuronal NO synthase (nNOS) in plants. In this study, we found that the nNOS transgenic Arabidopsis plants displayed lower level of H2O2 content, but higher levels of antioxidant enzyme activities and osmolytes under drought stress conditions. Transcriptomic analysis identified 490 and 20 genes that were differentially expressed in wild type (WT) and nNOS transgenic plants under control and drought stress conditions, respectively. Pathway analysis revealed that many genes involved in photosynthesis, cell, misc, co-factor and vitamin metabolism, major CHO metabolism, OPP and secondary metabolism were largely changed in nNOS vs. WT under control or drought stress conditions. Interestingly, CBF1/2 and 13 zinc finger family proteins, known as important family of transcription regulators in modulating several stress-responsive genes, were differentially expressed by nNOS transgenic effect. Additionally, some genes were commonly regulated by nNOS transgenic and abscisic acid (ABA) effects, indicating new insights to cross-talk between ABA and NO. Taken together, in vivo NO modulated antioxidant enzyme activities, osmolyte level, and the expression of genes involved in several pathways, thereby resulting in enhanced stress tolerance in nNOS transgenic plants. These observations might provide some insights to understand the physiological and molecular mechanisms of NO in response to drought stress in Arabidopsis.
Project description:Cadmium treatment induces slow but long lasting nitric oxide production in plant tissues. This NO production can be suppressed using the commonly used Nitric Oxide Synthase inhibitor L-NAME. This inhibitor tends to partially alleviate Cd toxicity. This effect is correlated with a strong diminution of Cd content in roots of plants treated both with Cd and L-NAME compared to roots from plants treated with Cd only. The main goal of this study is the identification of transcriptionnal changes caused by Cd-induced nitric oxide, and that could potentially result in enhanced Cd root accumulation.
Project description:Water availability is the biggest single limitation on plant productivity worldwide. In Arabidopsis, adjustments to drought stress, involving changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that integrate these complex responses we hypothesised that we needed to identify genes that govern early responses to drought. To this end, we produced a high-resolution time series transcriptomics dataset, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to the onset of drought conditions. 1825 differentially expressed genes (DEGs) were identified which showed no significant enrichment in gene ontology terms associated with dehydration responses and abscisic acid (ABA) regulation, confirming that the gene expression time series had targeted events prior to severe drought stress. Initial changes in gene expression coincided with a drop in carbon assimilation, not the later increase in foliar ABA content. Thus the early physiological and gene expression responses to drought were not driven by changes in leaf ABA content. In order to identify gene regulatory networks (GRNs) linked to early events, we used Bayesian network modelling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE 22 as key hub gene in a TF GRN. AGL22 is involved in the transition from vegetative state to flowering. Loss of AGL22 expression affected flowering time and drying rate providing a link between early changes in metabolism and the subsequent initiation of developmental responses to stress that govern plant productivity.
Project description:Calorie restriction is known to extend lifespan among organisms by a debating mechanism underlying nitric oxide-driven mitochondrial biogenesis. We report here that nitric oxide generators including artemisinin, sodium nitroprusside, and L-arginine mimics calorie restriction and resembles hydrogen peroxide to initiate the nitric oxide signaling cascades and to elicit the global antioxidative responses in mice. The large quantities of antioxidant enzymes are correlated with the low levels of reactive oxygen species, which allow the down-regulation of tumor suppressors and accessory DNA repair partners, eventually leading to the compromise of telomere shortening. Accompanying with the up-regulation of kinases, acetylases, and biomarkers, mitochondrial biogenesis occurs with the elevation of adenosine triphosphate levels upon exposure of mouse skeletal muscles to the mimetics of calorie restriction. In conclusion, calorie restriction-triggered nitric oxide provides antioxidative protection and alleviates telomere attrition via mitochondrial biogenesis, thereby maintaining chromosomal stability and integrity, which are the hallmarks of longevity. Six samples were analyzed, in which a control sample was included.
Project description:Abscisic acid (ABA)-, stress-, and ripening-induced (ASR) proteins are involved in abiotic stress responses. However, the exact molecular mechanism underlying their function remains unclear. Notably, the direct targets of ASRs that confer drought stress tolerance have not yet been identified.In this study, we report that MaASR expression was induced by drought stress and MaASR overexpression in Arabidopsis strongly enhanced drought stress tolerance. Physiological analyses indicated that transgenic lines had higher survival rates, germination rates and proline content, and lower water loss rates (WLR) and malondialdehyde (MDA) content. MaASR-overexpressing lines also showed smaller leaves and reduced sensitivity to ABA. Further, microarray and chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis revealed that MaASR participates in regulating photosynthesis, respiration, carbohydrate and phytohormone metabolism and signal transduction to confer plants with enhanced drought stress tolerance. Direct interactions of MaASR with promoters for the hexose transporter and Rho GTPase-activating protein (RhoGAP) genes were confirmed by electrophoresis mobility shift array (EMSA) analysis. Our results indicate that MaASR acts as a crucial regulator of photosynthesis, respiration, carbohydrate and phytohormone metabolism and signal transduction to mediate drought stress tolerance.
Project description:microRNA (miRNA), recently identified, non-coding, small RNA, are emerging as key regulators in homeostasis of the immune system. Therefore, aberrant expression of miRNA may be linked to immune dysfunction, such as in chronic inflammation and autoimmunity. In this study, we investigated the potential role of miRNA in estrogen-mediated regulation of innate immune responses, as indicated by upregulation of LPS-induced IFNg, inducible nitric oxide synthase (iNOS), and nitric oxide in splenic lymphocytes from estrogen-treated mice. We found that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in freshly-isolated splenic lymphocytes from estrogen-treated mice compared to placebo controls. Increasing the activity of miR-146a significantly inhibited LPS-induced IFNg and iNOS expression in mouse splenic lymphocytes. Further, miRNA microarray and Real-time RT-PCR analysis revealed that estrogen selectively upregulates/downregulates the expression of miRNA in mouse splenic lymphocytes. miR-223, which is highly upregulated by estrogen, regulates LPS-induced IFNg, but not iNOS or nitric oxide in splenic lymphocytes. Inhibition of miR-223 activity decreased LPS-induced IFNg in splenic lymphocytes from estrogen-treated mice. Our data are the first demonstrating selective regulation of miRNA expression in immune cells by estrogen and are indicative of an important role of miRNA in estrogen-mediated immune regulation. Keywords: microRNAs, estrogen, splenic lymphocytes, TLR4 signaling, mouse Total RNAs were isolated from freshly-isolated splenic lymphocytes from 2 pairs of placebo-and estrogen-treated mice