Transcription profiling by array of Arabidopsis thaliana wild type and atglr3.3 mutant seedlings treated with CK buffer containing GSH and Glu
ABSTRACT: Arabidopsis wild type and atglr3.3 mutant seedlings were cultured on 0.7% (w/v) agar medium containing half strength of MS basic salt with 1% sucrose (w/v), 5mM MES, pH 5.7. 10-day-old seedlings were equilibrated in the CK buffer for 24 hours. Then, these seedlings were either treated with the CK buffer or the CK buffer containing 100 ?M GSH and 100 ?M Glu for 1 hour, respectively.
Project description:Overexpression of programmed cell death 5 gene (PDCD5) in tumor cells enhances apoptosis triggered by growth factor or serum deprivation, and overexpression of its homolog, OsPDCD5, induces the early death of transgenic plants. In this work, a system of inducible OsPDCD5 expression using a heat shock promoter was developed to study PCD in rice at different developmental stages.The results showed that in three-leaf aged and older seedlings, OsPDCD5 could independently induce PCD. In altered plants, OsPDCD5 expression caused lesion mimic phenotype, abnormal leaf morphology, nuclear condensation, DNA fragmentation, and H2O2 production. But two-leaf aged and younger seedlings seedlings showed no visibly morphological phenotype after OsPDCD5 expression, suggested that young seedlings possessed some mechanism inhibiting OsPDCD5 induced PCD. The transcripts of 24-day-old transgenic Zhonghua 11 seedlings heat-shocked for 6 h (before the appearance of PCD phenotypes) were compared with untreated seedlings using microarray analysis (3-Leaf Group) to study the genes involved in OsPDCD5 induced PCD. The experiment was also performed using 18-day-old seedlings to discover why OsPDCD5 expression did not induce PCD in very young seedlings (2-Leaf Group). RNA samples were extracted using TRIzol (Invitrogen) as described by the manufacturer. Microarray analyses were carried out using a competitive hybridization method using the Agilent microarray system. All procedures were carried out according to the manufacturer's protocols. Briefly, total RNA from each sample was used to synthesize cRNA and was labeled with cyanine-5 (Cy5)- or cyanine-3 (Cy3)-labeled CTP. The labeled cRNAs (including a heat-shocked and a control sample) were competitively hybridized to the Agilent Rice Oligo Microarrays, and then washed. The hybridized slides were scanned using Agilent DNA Microarray Scanner and data points were extracted using Agilent Feature Extraction software. Two comparisons were made between biologically independent samples.
Project description:Comparative transcriptome analysis was performed to study differentially expressed genes (DEGs) between CK and Cocytodes caerulea Guenée challenged (CH) leaves of ramie.We obtained 40.2 and 62.8 million reads for CH and CK libraries respectively.De novo assembling of these reads generated 26,759 and 29,988 unigenes respectively. After a integrate assembly for all data of these two libraries, a total of 46,533 unigenes with an average length of 845 bp were obtained.A total of 1179 genes were identified as DEGs, 657 and 1230 of them were up- and down- regulated respectively, in response to C.caerulea infestation. Leaf samples of Cocytodes caerulea infested (T2) and un-infested (T1) ramie were RNA-Seq sequenced to compare differented expressed genes.
Project description:Digital gene expression (DGE) analysis was performed to study differentially expressed genes (DEGs) between CK and P. coffeae challenged (CH) roots of ramie. A total of 10.16 and 8.07 million clean reads were detected in the CK and CH libraries, respectively. A total of 137 genes were identified as DEGs, 117 and 20 of them were up- and down- regulated respectively, in response to P. coffeae infection. Roots samples of Pratylenchus coffeae infected(E2) and un-infected (E1) ramie were RNA-Seq sequenced to compare differented expressed genes.
Project description:Purpose: Understanding of MeJA, CK effect on regulation of gene expression patterns in Arabidopsis roots Total mRNA was extracted from 7-day-old Arabidopsis roots grown in MeJA (10uM), CK (50nM) and MeJA/CK-treated conditions. To generate cDNA libraries with TruSeq RNA library kit, 1 μg of total RNA was used. This step consisted of polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100 nt paired-end sequencing by Illumina HiSeq2000. 4,403 genes satisfying |fold change|≥2 in at least one data set were collected.
Project description:Living organisms have to cope with multiple and combined fluctuations in their environment. According to their sessile mode of life, plants are even more subjected to such fluctuations impacting their physiology and development. In particular, nutrient availability is known to tune plant development through modulating hormonal signaling, and conversely, hormonal signals are key to control nutrient related signaling pathways (Krouk et al., 2011a). However, very few is known about molecular mechanisms leading to plant adaptation to such combined signals. Here we deployed an unprecedented combinatorial treatment matrix to reveal plant adaptation in response to nitrate (NO3-), ammonium (NH4+), auxin (IAA), cytokinins (CK) and abscisic acid (ABA) and their exhaustive binary combinations. In order to study the effect of 5 signaling molecules we developed a matrix of treatment including NO3- (1mM or 0.5mM), NH4+ (1mM, or 0.5mM), indol-acetic-acid (IAA: 500 nM), Kinetin (CK: 500 nM), abscisic acid (ABA: 1µM). When present, the overall nitrogen treatment has been maintained to 1mM. As such, when NO3- and NH4+ are present in the same media their concentration was divided by 2. Proper mock controls include KCl, and DMSO. Plants are grown on NH4+-succinate for 12 to 14 days (first visible true leaves). Transferred 24 hours on a refreshed N-free media (reset the background that may be exhausted in several elements), and then transferred toward fresh media containing combinations of signals. Plant roots are harvested after 4 hours of treatment for gene expression analysis. Please note that the treatment details for each sample were provided in the sample 'characteristics' column in 'NO-NH-IA-CK-AB' format followed by the Presence/Absence code used for each hormone/nutrient.
Project description:Many of duplicated genes are enriched in signaling pathways. Recently, gene duplication of kinases has been shown to provide genetic buffering and functional diversification in cellular signaling. Transcription factors (TFs) are also often duplicated. However, how duplication of TFs affects their regulatory structures and functions of target genes has not been explored at the systems level. Here, we examined regulatory and functional roles of duplication of three major ARR TFs (ARR1, 10, and 12) in Arabidopsis cytokinin signaling using wild-type and single, double, and triple deletion mutants of the TFs. Comparative analysis of gene expression profiles obtained from Arabidopsis roots in wild-type and these mutants showed that duplication of ARR TFs systematically extended their transcriptional regulatory structures, leading to enhanced robustness and diversification in functions of target genes, as well as in regulation of cellular networks of target genes. Therefore, our results suggest that duplication of TFs contributes to robustness and diversification in functions of target genes by extending transcriptional regulatory structures. Duplication of TFs can confer an extension of transcriptional regulatory structures for target genes by providing new regulatory relationships between duplicated TFs and new or old target genes. To examine the nature of the extension in the regulatory structure, we performed gene expression profiling of Arabidopsis root tissues obtained from wild-type (WT) and deletion mutants of three type-B ARR1, 10, and 12. To examine how the extended regulatory structures by the duplicated ARR TFs are utilized for the responses to external CK, we generated gene expression profiles of WT Arabidopsis roots treated with mock or exogenous CK for 1 hour. Total RNAs were isolated from two biological replicates at each condition and used to measure gene expression level.
Project description:We were interested in investigating the transcriptome responses to exogenous applications of brassinosteroid hormone when Arabidopsis seedlings are pre-stressed with a reactive oxygen species, hydrogen peroxide. We were interested in seeing which subsets of BR-responsive gene transcripts were most affected and how BR-responsive gene transcripts responded to increasing concentrations of hydrogen peroxide both as a whole and individually. Liquid culture Arabidopsis seedlings are grown under standard conditions. Hydrogen peroxide is added at various concentrations to pre-stress the seedlings. Following this pretreatment, the seedlings are then treated with brassinosteroid (BR) hormone (epi-brassinolide, BL). Following this treatment, seedlings are harvested and total RNA is extracted for genome-wide transcriptome analysis.
Project description:To explore the molecular mechanism of low-K tolerance in sugarcane, we have employed whole genome microarray expression profiling to identify sugarcane genes in response to low-K stress. seeldings were transplanted to low-K hydroponic (containing 0.1 mmol.L-1 K+) and the roots were collected at 0 (CK), 8, 24 and 72 h after exposure to low-K condition. The expressions of genes in sugarcane roots were detected by microarray analysis. Totally 1545 genes at 8 h, 1053 genes at 24 h and 3155 at 72 h differentially expressed under low-K stress, when the 2-fold change was adopted as the threshold for determining differentially expressed genes. Among these genes, a certain amount of transcription factors, transporters, kinases, oxidative stress-related genes and genes in Ca+ and ethylene signaling pathway were detected to differentially express. Seeldings were treated with low-K hydroponic (containing 0.1 mmol.L-1 K+) and after 0 (CK), 8, 24 and 72 h exposure to low -K stress, the roots of sugarcane were collected. Four independent experiments were performed using roots collected at different time points
Project description:Potassium (K+) is one of the most important nutrient ions in plant cells and play crucial roles in many plant physiological and developmental processes. K+ deficiency is the common abiotic stress in natural environment, which inhibits plant growth and reduces production of crops. We used microarrays to analyse the transcriptomic changes in rice roots after suffering K+ starvation at different times. The results of GO analysis showed that these transcriptionally changed genes mainly fell into the categories of metabolic process, membrane, cation binding, kinase activity, transport and so on. The two-week-old hydroponic rice seedlings were transferred to K+-free solution (-K) and K+-replete solution (+K, 1 mM K+) as treatment (LK) and control (CK) conditions respectively. The fresh roots of both control and treated rice seedlings were collected after K+-deficient treatment at indicated times (6 h, 3 d and 5 d). The roots were frozen immediately using liquid nitrogen and stored at -80℃ for further RNA extraction. Every RNA sample was derived from 5 independent seedlings. In sum, three time points were selected and three biological replicates were performed at each time point, totally 18 rice genome arrays were used.
Project description:Columbia (Col) seeds were sown on half-strength Murashige and Skoog (MS) medium, supplemented with 1% sucrose and 0.8% agar and grown vertically in culture room conditions. The 5-d-old homogenous seedlings were washed five times with sterile water and lastly with liquid half strength MS medium without sugar to remove residual exogenous sugar. In order to deplete internal sugars seedlings were grown in sugar free liquid half strength MS medium for 24 h in dark. Thereafter, the seedlings were treated with half-strength MS medium containing 0% G, 0% G + 1 uM BAP, 3% G, and 3% G + 1 uM BAP for 3 h in dark. RNA was extracted and microarray analysis was performed. Please note: G stands for glucose and BAP stands for 6-Benzylaminopurine (cytokinin)