Transcription profiling by array of Arabidopsis thaliana seedlings (aerial parts) to study the photoperiodic control of starch mobilisation
ABSTRACT: In order to identify genes specifically involved in the photoperiodic control of the mobilisation programme for starch reserve in Arabidopsis thaliana transcription profiling was performed on the following genotypes Columbia wild type (Col-0), CO overexpressor (35S::CO), CO muntant (co-10), GBSSI mutant (gbs-1), aps1 mutant (aps1) and Columbia with sucrose. The different Arabidopsis thaliana genotype seedlings were cultivated in long day conditions (16 h day/ 8 h dark) at 22 C in controlled environment cabinets for two weeks. Samples were collected at ZT 4, immediately frozen in liquid nitrogen and processed.
Project description:Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the main events participating in the healing of a wound, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process, but allow exploring many unanswered features of the healing response; e.g., which are the signal(s) responsible for initiating tissue remodeling? How is the sealing of the epithelia achieved? Or which are the inhibitory cues that cancel the healing machinery upon completion? Answering these and other questions demands in first place the identification and functional analysis of wound-specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method to culture imaginal discs that allows live imaging and biochemical analysis and is healing-permissive. Employing this approach, we performed a comparative genome-wide profiling between those Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. This lets us identify a set of potential wound-specific genes. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in a healing assay. This non-saturated analysis defines a relevant set of new genes whose changes in expression levels are functionally significant for proper tissue repair. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound response. We developed a healing-permissive in vitro culture system for fly imaginal discs: we used one-channel microarrays for comparing healing-engaged cells (showing activation of the JNK signaling cascade) to cells not participating in healing (silent JNK activity) in wounded wing imaginal discs in culture. Employing this method, we aimed detecting the relevant genes involved in disc healing through microarray analysis. We compared cells actively involved in healing to those not involved and identified a whole set of upregulated or downregulated genes. They were annotated, clustered by expression profiles, chromosomal locations and presumptive functions. Most importantly, we functionally tested them genetically in a healing assay.
Project description:Nitric oxide regulates plant development and responses to stress. However, the mechanisms underlying its regulatory role are still poorly known, and the impact of endogenous NO on the genome-wide transcriptome of plants has not been studied. For that purpose, we compared the transcriptomes of NO-deficient nia1nia2, noa1-2 and nia1nia2noa1-2 mutant versus wild type Arabidopsis thaliana plants. A core comprising 66 NO-responsive genes with similar expression in all NO-deficient genotypes was identified. Among them, 46 were down- and 20 up-regulated in NO-deficient plants, and thus positively and negatively regulated by endogenous NO, respectively. Accordingly with changes in its transcriptome, the NO-deficient nia1nia2noa1-2 mutant accumulated anthocyanins and indolic glucosinolates, displayed abnormal iron homeostasis in shoots and roots, and also showed altered root sensitivity to hormones such as ABA, ET, CYK and IAA. Together the presented data suggest NO functions essentially as a modulator of hormone action. Compared analysis of the transcriptomes of 15-day old seedlings from 3 different nitric oxide (NO)-deficient mutant genotypes versus wild type background Col-0 (3 independent biological replicates per genotype). NO-deficient mutant seedlings in Col-0 background were the double nia1nia2 mutant in nitrate reductases (NR/NIA) 1 and 2 (abbreviated as nia); the noa1-2 mutant allele in Nitric Oxide Associated 1 (AtNOA1) (abbreviated as noa); and, the triple nia1nia2noa1-2 (abreviated as nino).
Project description:High-throughput sequencing of small RNAs from rice was used to identify distinct miRNAs that are responsive to elicitors from the fungal pathogen Magnaporthe oryzae. [Expression profiling by array] We used microarrays to determine the expression behaviour of target genes for elicitor-regulated miRNAs. [High throughput sequencing] High-throughput sequencing of rice small RNAs was performed in two different tissues, leaves and roots, and two different time point of elicitor treatment, 30' and 2h Amplicons were prepared by 5´and 3´adaptor ligation in which the 5'-adaptor contained a 'barcode' consisting of a 4-nucleotide identifier sequence for each sample. The libraries containing unique barcodes were combined and subjected to pyrosequencing (454 Life SciencesTM, Roche) [Expression profiling by array] Leaves from rice plants were harvested at two time points after the onset of treatment (30' and 2h) with elicitors of Magnaporthe oryzae 18.1 and used for RNA extraction and hybridization on Affymetrix microarrays. Mock inoculations were performed with sterile water for control experiments. Three biological replicates were analyzed. Each sample represented a pool of approximately 150 rice plants. [High throughput sequencing] 8 samples examined: leaves and roots, treated or not with elicitors at two different time points, 30' and 2h (2x2x2)
Project description:Genome-wide transcriptional profiling shows that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression. However, simulation experiments on ground, without space constraints, show weaker effects than space environment. A global and integrative analysis using the “gene expression dynamics inspector” (GEDI) self-organizing maps, reveals a subtle response of the transcriptome using different populations and microgravity and hypergravity simulation devices. These results suggest that, in addition to behavioural responses that can be detected also at the gene expression level, the transcriptome is finely tuned to normal gravity. The alteration of this constant parameter on Earth can have effects on gene expression that depends both on the environmental conditions and the ground based facility used to compensate the gravity vector. Alternative and commons effects of mechanical facilities, like the Random Positioning Machine and a centrifuge, and strong magnetic field ones, like a cryogenically cooled superconductive magnet, are discussed. We compare the effects over the gene expression profile of different gender/age Drosophila imagoes in 3-4 days-long experiments under altered gravity conditions into three GBF ("Ground Based Facilities" for micro/hyper- gravity simulation) using whole genome microarray platforms. Descriptions of different GBFs ("treatments"): LDC means "Large Diameter Centrifuge". Samples can be placed under three conditions: inside LDC (at certain g level), at the LDC rotational control and at external 1g control (outside the LDC). RPM means "Random Positioning Machine". Samples can be placed under two conditions: inside RPM (at nearly 0g, Microgravity level) and at external 1g control (outside the RPM). At the magnet, means INSIDE the Magnetic levitator (another GBF). Samples can be placed under four conditions: inside Magnet 0g* (at microgravity with magnetic field), inside Magnet at 1g* (internal control with magnetic field) or inside the magnet 2g* (at hypergravity with magnetic field) and at external 1g control (outside the magnet)
Project description:Formation of epithelial tissues requires the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. MDCK cell line has proven to be a powerful model to study mammalian polarized epithelia in vitro. MDCK cells plated in extracellular matrix (ECM) form cysts, a spherical structure of polarized cells enclosing a central lumen which resembles epithelial tubular structures. The morphogenetic process requires drastic changes in cell architecture, which are regulated by change in gene expression. We used microarrays to identify genes up-regulated in lumen formation. The identification of up-regulated genes could lead us to characterize novel pathways needed for this process. MDCKII cells were plated in two different conditions: Cells cultured in confluence in plastic dishes, forming polarized monolayers (2D); or cells cultured in plastic dishes covered with Matrigel (ECM) forming three dimensional cysts (3D). Comparison of both transcriptomic profiles would lead us to identify up-regulated genes in the 3D condition, which would be good candidates to be key regulators of novel processes involved in lumen morphogenesis.
Project description:In plants, endogenous and environmental signals such as light control the timing of the transition to flowering . Two phytochrome B-interacting transcription factors, VASCULAR PLANT ONE–ZINC FINGER1 (VOZ1) and VOZ2 redundantly promote flowering in Arabidopsis thaliana. In the voz1 voz2 mutant the expression of FLOWERING LOCUS C (FLC) was up-regulated and expression of FLOWERING LOCUS T (FT) was down-regulated, which was proposed to be the cause of late flowering in voz1 voz2. However, the detailed mechanism by which the VOZ genes promote flowering is not well understood. Here, we show that neither the reduced FT-expression nor the late-flowering phenotype of voz1 voz2 is suppressed in the voz1 voz2 flc triple mutant . Genetic interaction experiments between voz1 voz2 and constans-2 (co-2) mutants reveal that the VOZs and CO work in the same genetic pathway. Using in vitro pull-down, electrophoretic mobility shift assays and bimolecular fluorescence complementation assays, we show that VOZ1 and VOZ2 interact with CO. The voz1 voz2 35S::CO:YFP plants show suppression of the early-flowering phenotype induced by CO-overexpression, showing that CO requires VOZ for induction of flowering. Determination of the VOZ consensus binding site followed by genome-wide sequence analysis failed to identify any VOZ-binding sites near known flowering-time genes. Together, these results indicate that the VOZ genes regulate flowering primarily through the photoperiod pathway, independent of FLC, and suggest that VOZs modulate CO function to promote flowering. Overall design: Agilent Microarray Gene Expression 8X60K-Arabidopsis thaliana (AMADID: 37661) designed by Genotypic Technology Pvt.Ltd.