Global gene expression analysis of the OsDIS1 overexpression plants under normal and drought stress conditions
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ABSTRACT: To identify genes involved in the OsDIS1-mediated drought-responsive pathway, we performed microarray analysis of the OsDIS1 overexpression and wild-type plants under both normal and drought stress conditions using the Agilent rice Genechip. Seven-day-old plants of the OsDIS1 overexpression line 9-4-2 as well as the wild-type plants were used in the drought treatment. OsLEA3 was used as a positive control for the drought treatment. Genes with more than two-fold changes in the overexpression plants compared with the wild-type plants were selected. The expression pattern of some differentially expressed genes was further confirmed by real-time PCR. The OsDIS1 overexpression 9-4-2 plants and the wild-type plants were cultured on 1/2 MS medium plus 3% sucrose for seven days. About half of the plants were sampled as the untreated control for RNA isolation, and the rest were transferred with 1/2 MS medium onto filter papers to induce drought stress. When the leaves of the OsDIS1 overexpression plants began to show drought stress phenotypes, we collected leaves for RNA isolation. OsLEA3 was used as a positive control for the drought treatment.
Project description:bHLH122 could be induced by salt, osmotic and drought stress except ABA, the transgentic Arabidopsis were more tolerant to abiotic stresses, such as drought and salt. What's more, in the overexpression plants, the endogenesis ABA contents were higher than WT. We found there existed an interaction between bHLH122 and CYP707A3 by virtue of EMSA and ChIP assays. We wanted to learn more about the molecular mechanism of bHLH122 and to explore what had changed in the over-expression plant through Genechips. Two independent overexpression bHLH122 lines and WT Arabidopsis were chosen to RNA extraction and hybridization on Affymetrix microarrays. The plants were sowed on MS medium after 96 h stratification,then transferred into soil after one weeks. After 2 weeks, collected the shoot above the soil and extracted RNA, digested by DNaseM-bM-^EM- and hybrid on Affymetrix microarrays.
Project description:Plant basic helix-loop-helix (bHLH) transcription factors are involved in physiological and developmental processes, and also play essential roles in abiotic stresses. However, their exact roles in abiotic stress are still need to be elucidated, and most of bHLHs have not been functionally characterized. In the present study, we characterized the functional role of AtbHLH112 in response to abiotic stresses. AtbHLH112 is a nuclear-localized protein, and its nuclear-localization is induced by salt, drought and ABA. Besides binding to E-box motif, AtbHLH112 is found to bind to a novel motif with the sequence M-bM-^@M-^\GG[GT]CC[GT][GA][TA]CM-bM-^@M-^] (GCG-box), and the binding affinity is induced by salt and ABA. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by upregulating the expression of P5CS genes and decreasing the expression of P5CDH and PRODH genes to increase proline levels, and via enhancing the expression of POD and SOD genes to improve ROS scavenging ability. All data together suggested that AtbHLH112 regulates the expression of genes through binding to GCG-box and E-box to mediate the physiological stress responses, including proline biosynthesis and ROS scavenging pathways to enhance stress tolerance. Differentially expression genes of AtbHLH112-overexpression plants, mutant (SALK_033618C) plants and wild type of Columbia Arabidopsis thaliana were measured under salt stressed and normal condition for 3 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
Project description:Plant basic helix-loop-helix (bHLH) proteins play essential roles in physiological and developmental processes and are also involved in abiotic stresses. However, their exact roles in abiotic stress are still not fully understood, and most of them have not been functionally characterised. In the present study, we characterised the functional role of AtbHLH112 in response to abiotic stress. A WRKY gene, AtWRKY66, can regulate the expression of the AtbHLH112 via binding to W-box motifs present in its promoter. AtbHLH112 is a nuclear-localised protein, and its nuclear localisation is increased upon exposure to NaCl, mannitol and ABA. In addition to binding to the G-box motif, AtbHLH112 is found to bind to a novel motif M-bM-^@M-^\GGGCCGGTCM-bM-^@M-^] (named the GCG-box) to regulate gene expression. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with tolerance to salt and drought. AtbHLH112 can confer stress tolerance via enhanced expression of POD and SOD genes to improve ROS scavenging ability and via upregulated expression of P5CS genes and decreased expression of P5CDH and PRODH genes to improve proline levels. Our data suggested that AtbHLH112 regulates the expression of genes via binding to the G-box and the GCG-box to improve stress-related pathways, such as ROS scavenging and proline biosynthesis. Differentially expression genes of AtbHLH112-overexpression plants and mutant (SALK_033618C) plants of Arabidopsis thaliana were measured under salt stressed and normal condition for 3 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
Project description:To understand the molecular mechanism of drought stress resistance mediated by OsABA8ox3 gene, we checked the genome-wide expression profile changes in the OsABA8ox3 RNAi and WT seedlings using the Affymetrix GeneChip under the normal condition and drought stress. A total of 1436 genes showed greater than 2-fold higher expression levels in both WT and RNAi-9 seedlings after drought stress, and most of them had higher up-regulated folds in RNAi-9 seedlings than that of WT. Gene expressions in the OsABA8ox3 RNAi and WT seedlings under the normal and drought stress conditions.
Project description:Chemical hybridization agent (CHA)-induced male sterility is becoming one of the most useful tools for the crop heterosis in seed production. We had previously discovered monosulfuron ester sodium (Mes), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, could induce rapeseed (Brassica napus L.) male sterility. To investigate the mechanism of Mes inducing male sterility, a cytological analysis and comparative transcriptome analysis were performed between Mes-treated plants leaves and different developmental stage anthers and those of the Mock-treated plants. Cytological analysis exhibited that Mes destroyed plastids ultrastructure and depressed the materials accumulation during anther development and maturation process. Comparative transcriptome analysis identified a total of 1501 transcripts differentially expressed in the leaves and different developmental stage anthers. Subcellular localization analysis, functional analysis and pathway analysis were carried out to obtain the clues that plastid was the seriously damaged organelle, and many genes involved in carbon and lipid metabolism and cellular transport differentially expressed. Detailed expression pattern analysis of genes related with these functions were conducted and verified by RT-PCR. In addition, several transcription factors, protein kinases and hormone related genes were identified. Carbohydrate content analysis confirmed carbon metabolism were influenced by Mes. Taken together, we proposed a putative action mode that Mes inhibited activity of acetolactate synthase, which localized in plastid in plants, and then disturbed the normal supply of carbon and lipid metabolite for anther development, finally displayed male sterility. This results have important significance for uncovering the metabolic gene regulation during anther development and may provide more potential targets for developing new male sterility inducing CHAs in rapeseed breeding. Rapeseed (Brassica napus L.) plants were foliar sprayed with 0.1μg mL-1 Monosulfuron ester sodium (Mes) solution containing 50ppm DMF and 5ppm Tween 80 for about 15 ml per plant. Young leaves (C2Y1, C2Y2, C2Y3; T2Y1, T2Y2, T2Y3) of the main inflorescences and developing anthers (including small buds (C2S1, C2S2, C2S3; T2S1, T2S2, T2S3), middle anthers (C2M1, C2M2, C2M3; T2M1, T2M2, T2M3), and large anthers (C2L1, C2L2, C2L3; T2L1, T2L2, T2L3)) from Mes-treated and Mock-treated plants were collected for each experiment. Each tissue has three independent biological replicates.
Project description:Environmental stresses influence the growth of plants and the productivity of crops. Salinity is one of the most important abiotic stresses for agricultural crops. PCD is induced by various biotic and abiotic stresses in algae and higher plants, including high salinity treatment. OsPDCD5, an ortholog to mammalian-programmed cell death 5, is up-regulated under low temperature and NaCl treatments. We found that the transgenic rice which constitutively expressed anti-OsPDCD5 increased salt stress tolerance in unique ways. By using the Rice Genome Microarray, we identi?ed target genes that were regulated in transgenic rice plants by anti-OsPDCD5. Leaf tissues of 2-week-old transgenic and nontransgenic seedlings (10 plants each) before 200mM NaCl treatment, 20mins and 3 hours after 200mM NaCl treatment, respectively, were selected.
Project description:In the current report, we report that ThbZIP1 is a direct target gene of the ThABF1 transcription factor. There are three ABRE motifs in the promoter of ThbZIP1, Yeast one-hybrid (Y1H) assays showed that a ABF protein, ThABF1, specifically binds to the ABRE motifs. The interaction between ThABF1 and the promoter of ThbZIP1 was further confirmed by transient expression assays in tobacco leaves. Chromatin Immunoprecipitation (ChIP) results suggested that binding of ThABF1 to ABRE motifs in the promoter of ThbZIP1 occurs in vivo in Tamarix hispida to regulate the expression of ThbZIP1. Moreover, ThABF1 and ThbZIP1 share similar expression patterns in response to salt, drought, ABA, methyl viologen (MV) and cold stress. Microarray analyses results showed there were 1,662 and 1,609 genes that were significantly upregulated or downregulated, respectively, under ABA stress conditions. ThbZIP1 regulated the genes via binding to the C-, G- or A-box motifs in their promoter sequences. Based on these data, the results suggested a regulatory network model mediated by ThbZIP1, under abiotic stress conditions, ThABF1 regulates the expression of ThbZIP1, and the activated ThbZIP1 binds to bZIP recognition sequences or other motifs to regulate the expression of genes containing these motifs in their promoters. Differentially expression genes of ThbZIP1-overexpression plants and wild type of Columbia Arabidopsis thaliana were measured under ABA stressed and normal condition for 3 hours, respectively. Two independent experiments were performed at each treatment using different plants for each experiment.
Project description:To better understanding the genetic and physiological changes behind the dormancy process in tree peony, we performed customized cDNA microarray to investigate gene expression profiling in tree peony M-bM-^@M-^XFeng Dan BaiM-bM-^@M-^Y buds during chilling induced dormancy release. Endo-dormant tree peony plants were exposed to 0-4M-BM-0C from 5 November to 30 December 2009 in Qingdao, Shandong, China. Buds were collected after 0 d, 6 d, 12 d, 15 d, 18 d and 24 d chilling endured. DNA microarrays were customized using Agilent eArray 5.0 program, containing spots with 14,957 gene-specific 60-mer oligonucleotides representing 14,957 non abundant ESTs obtained from 454 sequencing normalized cDNA of tree peony buds during chilling duration (TSA, 65,217). Total 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release, and the number of up-regulated (1,611) and that of down-regulated (1,563) was almost same. Expression of differentially-expressed genes associated with GA biosynthesis and signaling, cell growth and development was confirmed by quantitative RT-PCR, which displayed similar trends pattern in expression. Transcript profiling of tree peony was measured during chilling (0-4M-BM-0C) induced dormancy release. Mixed buds, three buds for each individual, were collected after 0, 6, 12, 15, 18 (endo-dormancy release), 24 days (eco-dormancy) chilling requirement fulfilling. Three replications (3 plants/ replication) were harvested between November and December.
Project description:ABA deficient mutant Osaba1-1 exhibits great resistance to Xanthomonas oryzae pv. oryzae (Xoo) infection. To investigate gene expression profile changes at whole genome level between Osaba1-1 and wild-type (Nipponbare) rice during Xoo infection, we employed microarray expression profiling as a discovery platform. Osaba1-1 and wild-type rice plants about 6.5 leaf stage were used in this experiment. For Xoo inoculation, tips (about 3cm) of the fifth and sixth fully expanded leaves were cut off, and then immersed into Xoo (PXO99 strain) inoculum (suspended in sterile distilled water containing 10mM MgCl2, OD≈0.5) immediately for about fifteen seconds. Inoculated rice leaves were collected (approximately 2 cm leaf fragment from the inoculation site) at 0h and 72h post inoculation. Three independent replicate samples were collected at each time point for microarray.