Genome-wide transcriptome analysis of 35S::amiPPD line
Ontology highlight
ABSTRACT: To obtain more insight into the molecular changes associated with PPD down-regulation, RNA was extracted from the first leaf pair of ami-ppd and wild-type plants at 13 DAS (the time point at which differences in leaf area start to be visible), and subjected to micro-array transcript profiling.
Project description:The expression level of the 31 genes (ERF-1 (AT4G17500), ERF2 (AT5G47220), ERF5 (AT5G47230), ERF6 (AT4G17490), ERF11 (AT1G28370), ERF8 (AT1G53170), ERF9 (AT5G44210), ERF59 (AT1G06160), ERF98 (AT3G23230), MYB51 (AT1G18570), STZ (AT1G27730), WRKY28 (AT4G18170), WRKY33 (AT2G38470), WRKY15 (AT2G23320), ZAT6 (AT5G04340), WRKY48 (AT5G49520), RAP2.6L (AT5G13330), K11J9.4 (AT5G61590), bHLH (AT2G43140), GATA3 (AT4G34680), GA2OX6 (AT1G02400), GA3OX1 (AT1G15550), GA2OX4 (AT1G47990), GA20OX1 (AT4G25420), EXLB3 (AT2G18660), MPK3 (AT3G45640), RAP2.2 (AT3G14230)) was measured upon mild osmotic stress (25mM mannitol) in the third leaf of wild-type plants during the proliferating (9 days after stratification (DAS)), expanding (15 DAS) and mature (22 DAS) developmental stage. The expanding third leaf (15 DAS) was harvested at a high temporal resolution (20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h and 48 h), whereas the proliferating and mature leaf tissues were harvested 24h after transfer to control or 25 mM mannitol-containing medium. A detailed expression pattern over time for each gene was generated with the nCounter Nanostring® technology.
Project description:Leaf senescence is an essential developmental process that involves altered regulation of thousands of genes and changes in many metabolic and signaling pathways resulting in massive physiological and structural changes in the leaf. The regulation of senescence is complex and although several senescence regulatory genes have been identified and characterized there is little information on how these individual regulators function globally in the control of the process. In this paper we use microarray analysis to obtain a high-resolution time course profile of gene expression during development of a single leaf over a three week period from just before full expansion to senescence. The multiple time points enable the use of highly informative clustering tools to reveal distinct time points at which signaling and metabolic pathways change during senescence. Analysis of motif enrichment in co-regulated gene clusters identifies clear groups of transcription factors active at different stages of leaf development and senescence. A novel experimental design strategy (A Mead et al, in preparation), based on the principle of the “loop design”, was developed to enable efficient extraction of information about key sample comparisons using a two-colour hybridisation experimental system. With 88 distinct samples (four biological replicates at each of 22 time points) to be compared, the experimental design included 176 two-colour microarray slides, allowing four technical replicates of each sample to be observed. Half of the slides were devoted to assessment of changes in gene expression between time points, using a simple loop design to link 11 samples from either the 7h time points or the 14h time points across the 11 sampling days, directly comparing samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, 27 DAS with 29 DAS, etc.), and directly comparing the samples collected at 39 DAS with those collected at 19 DAS. Four separate loops were constructed for the 7h time points and for the 14h time points, using the arbitrary biological replicate labelling to identify the samples to be included in each loop. The remaining slides provided assessment of differences between the 7h and 14h samples and between the arbitrarily labelled biological replicates, with some further assessment of changes between sampling days. All direct comparisons (pairs of samples hybridised together on a slide) were between 7h and 14h samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, etc.), including comparisons between samples collected at 39 DAS and at 19 DAS, and different arbitrarily labelled biological replicates. These 88 comparisons formed a single loop connecting all 88 treatments, therefore ensuring that the design was fully connected (allowing each sample to be compared with every other sample).
Project description:Seedlings are grown on a mesh covering MS media without carbon source, and subsequently transferred at 9 DAS to control media without sucrose or medium supplemented with 15 mM sucrose. 3 hours and 24 hours after transfer, seedlings were harvested and the third leaf micro-dissected for RNA extraction.
Project description:An indica rice cultivar IET8585 (Ajaya), resists diverse races of the Xanthomonas oryzae pv oryzae (Xoo) pathogen attack, and is often cultivated as bacterial leaf blight (blb) resistant check in India. Earlier we reported a recessive blb resistance gene mapped to the long arm of chromosome 5 in IET8585. To further understand the mechanism of recessive and durable resistance response, two indica rice genotypes namely, i) IET8585 (Ajaya), a disease resistant indica veriety from India and ii) IR24, a bacterial leaf blight disease susceptible genotype were selected for this study. We used the 22K rice Oligoarray from Agilent technologies to study the transcript profile in the leaves of the two contrasting rice genotypes under inoculated and un-inoculated conditions during seedling stage. Experiment Overall Design: We used Agilent rice gene chips (G4138A) to investigate the transcript level changes in rice leaf tissues during bacterial pathogen infection. We used two contrasting rice genotypes (IET8585 (Ajaya) blb resistant IR24 blb susceptible) differing in bacterial disease response. Plants were grown growth chambers and inoculated with bacterial pathogen on 18th DAS. Leaf sampling was done in both un-inoculated and inoculated plants at 3 time points. Two replications of microarray experiments were carried out by hybridizing the resistant samples against the susceptible samples.
Project description:An indica rice cultivar FR13A, is widely grown as submergence tolerant variety and can withstand submergence up to two weeks. The tolerance is governed by a major QTL on chromosome 9 and represented as sub1. Recently the gene for sub1 has been mapped and cloned. However, the trait is governed by several QTLs and not by a single gene. To understand the mechanism of submergence tolerance we selected, two indica rice genotypes namely, I) FR13A, a tolerant indica variety and ii) IR24, a susceptible genotype for this study. We used the 22K rice Oligoarray from Agilent technologies to study the transcript profile in the leaves of the two contrasting rice genotypes under constitutive and submerged conditions at seedling stage. SUBMITTER_CITATION: Combining In Silico Mapping and Arraying: an Approach to Identifying Common Candidate Genes for Submergence Tolerance and Resistance to Bacterial Leaf Blight in Rice. Mol. Cells 2007 24:394-408. Experiment Overall Design: We used Agilent rice gene chips (G4138A) to investigate the transcript level changes in rice leaf tissues during submergence treatment. We used two contrasting rice genotypes (FR13A tolerant and IR24 susceptible) differing in submergence response. Plants were grown in growth chambers and treated by submerging the plants in transparent polythene bags on14th DAS. Leaf sampling was done in both constitutive and treated plants at 3 time points. Two replications of microarray experiments were carried out by hybridizing the RNA from tolerant samples against the susceptible lines.
Project description:RNA-seq data of Arabidopsis thaliana accessions exposed to mild drought or control treatments. The sampled tissue is the third leaf at the last day of proliferation (cell division phase).
Project description:We performed a transcriptome profiling on the leaf 3 microdissected from plants with altered cytokinin levels 3h after activation at 9 DAS, when this leaf is normally fully composed of proliferating cells to get an insight into dynamics of molecular network underlying cell responses to CK excess and deficiency in proliferating cells. Seeds of Arabidopsis thaliana transgenic lines (CaMV35S>GR>ipt and CaMV35S>GR>HvCKX2) and corresponding wild-types (Col-0) were sown on nylon meshes placed on half-strength Murashige and Skoog (MS) medium containing 0.5% (w/v) MES and 0.8% (w/v) agar. After 2 days of stratification, plants were removed to growing chambers with 16-h day/8-h night, 21 C and 19 C, respectively, under continuous light (110 mol m-2 s-1). DEX-inducible lines and wild-types were activated at 9 DAS by transfer of nylon meshes to half-strength MS medium supplemented with either 2.5 M DEX dissolved in 5x10-4% (v/v) DMSO or just 5x10-4% (v/v) DMSO (mock). The leaf 3 was dissected from i > 250 individual seedlings per variant on a cooling plate under a stereomicroscope with precision needle and removed into the liquid nitrogen. For RNA-seq 2 biological replicates of WT, ipt and HvCKX2 activated by DEX were used. The RNA was extracted according the same method described for qRT-PCR. RNA sequencing was done by GATC Biotech AG (Konstanz, Germany).
Project description:A network model has been generated that describes the immediate gene expression cascade surrounding three similar but distinct NAC transcription factors that have roles to play in leaf senescence and in many stress responses in Arabidopsis. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1 hybrid analysis and modeling using gene expression time course data has been used to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modeling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions.. Gene expression measurements in mutants of ANAC019 and ANAC055 at different times during leaf senescence have shown a distinctly different role for each of these genes. Leaf 7 from the anac019 (genotype 9C11) and anac055 (genotype IM4) mutants and their WT controls, Col-5 and Col-0 respectively, was tagged with cotton 18 days after sowing (DAS) and harvested from five randomly selected plants (biological replicates), 8 h into the light period, at 23, 29, 31, 33 and 35 DAS (full senescence). Variance in expression between lines at timepoints was analysed using a local adaptation of the MAANOVA package as previously described (Breeze et al., Plant Cell 2011 Mar;23(3):873-94. PMID: 21447789) using a loop design including dye swaps. Genotype DM (double mutant cross between IM4 and 9C11) was also included in the experiment, and was part of the loop design. Results for DM are included here in so far as they played a role in the ANOVA definition of values, but are not discussed in the paper.
Project description:Arabidopsis thaliana shows hybrid vigour (heterosis) in progeny of crosses between Col and C24 accessions (1). Hybrid vigour was evident as early as the mature seeds and in the seedlings 3 days after sowing (DAS). At 3 DAS genes encoding chloroplast-located proteins were significantly overrepresented (187) among the 724 genes which have greater than mid parent values of expression in the hybrid. Many of these genes are involved in chlorophyll biosynthesis and photosynthesis. The rate of photosynthesis was constant per unit leaf area in parents and hybrids. Larger cell sizes in the hybrids were associated with more chloroplasts per cell, more total chlorophyll and more photosynthesis. The increased transcription of the chloroplast-targeted genes was restricted to the 3 to 7 DAS period. At 10 DAS only 118 genes had expression levels different from the expected mid parent value in the hybrid and only 12 of these genes were differentially expressed at 3 DAS. The early increase in activity of genes involved in photosynthesis and the associated phenomena of increase in cell size and number through development, leading to larger leaf areas of all leaves in the hybrid, suggest a central role for increased photosynthesis in the production of the heterotic biomass. In support of this correlation we found that an inhibitor of photosynthesis eliminated heterosis and higher light intensities enhanced both photosynthesis and heterosis. In hybrids with low level heterosis (Ler x Col) chloroplast-targeted genes were not upregulated and leaf areas were only marginally increased.
Project description:Constituting the final growth phase during the lifecycle of maize (Zea Mays L.), leaf senescence plays an important biological role in grain yield in crops. We undertook proteomic and physiological analyses in inbred line Yu816 in order to unravel the underlying mechanisms of leaf senescence induced by preventing pollination. A total of 6,941 proteins were identified by Isobaric tags for Relative and Absolute Quantitation (iTRAQ) analysis. Proteomic analyses between pollinated (POL) and non-pollinated (NONPOL) plants indicated that 973 different proteins accumulated in NONPOL plants. The accumulated proteins were classified into various groups, including response to stimuli, cellular processes, cell death and metabolic processes using functional analysis. Furthermore, in accordance with the changes in these different accumulated proteins, analysis of changes in leaf total soluble sugars and starch content showed that the prevention of pollination can disturb endogenousplant hormone and sugar metabolism and lead to ROS bursts, protein degradation and photosystem breakdown, eventually resulting in leaf senescence. This represents the first attempt at global proteome profiling in response to induced leaf senescence by preventing pollination in maize, and provides a better understanding of the molecular mechanisms involved in induced leaf senescence.Constituting the final growth phase during the lifecycle of maize (Zea Mays L.), leaf senescence plays an important biological role in grain yield in crops. We undertook proteomic and physiological analyses in inbred line Yu816 in order to unravel the underlying mechanisms of leaf senescence induced by preventing pollination. A total of 6,941 proteins were identified by Isobaric tags for Relative and Absolute Quantitation (iTRAQ) analysis. Proteomic analyses between pollinated (POL) and non-pollinated (NONPOL) plants indicated that 973 different proteins accumulated in NONPOL plants. The accumulated proteins were classified into various groups, including response to stimuli, cellular processes, cell death and metabolic processes using functional analysis. Furthermore, in accordance with the changes in these different accumulated proteins, analysis of changes in leaf total soluble sugars and starch content showed that the prevention of pollination can disturb endogenousplant hormone and sugar metabolism and lead to ROS bursts, protein degradation and photosystem breakdown, eventually resulting in leaf senescence. This represents the first attempt at global proteome profiling in response to induced leaf senescence by preventing pollination in maize, and provides a better understanding of the molecular mechanisms involved in induced leaf senescence.