MRNA changes following induction of GmWRI1 activity in Arabidopsis leaf mesophyll protoplasts
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ABSTRACT:
PROVIDER: PRJNA1064056 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27533219.fastq.gz | Fastqsanger.gz | |||
| SRR27533220.fastq.gz | Fastqsanger.gz | |||
| SRR27533221.fastq.gz | Fastqsanger.gz | |||
| SRR27533222.fastq.gz | Fastqsanger.gz | |||
| SRR27533223.fastq.gz | Fastqsanger.gz |
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Project description:The goal of this experiment was to investigate the early transcript changes (6h) induced by hypoxia treatment in mesophyll protoplasts. A single pair (control & hypoxia) of GeneChips® was used to confirm that hypoxia treatment altered the expression of an overlapping set of genes controlled by KIN10 (At3g01090) in Arabidopsis mesophyll protoplasts. Keywords: KIN10, KIN11, darkness, hypoxia, starvation, stress, sugar signalling, Arabidopsis, SnRK1
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Project description:The goal of this experiment was to investigate the early transcript changes (6h) induced by hypoxia treatment in mesophyll protoplasts. A single pair (control & hypoxia) of GeneChips® was used to confirm that hypoxia treatment altered the expression of an overlapping set of genes controlled by KIN10 (At3g01090) in Arabidopsis mesophyll protoplasts. Experiment Overall Design: For the hypoxic treatment protoplasts were submerged in mannitol buffer and the hypoxia condition was confirmed by the activation of the well-established early marker genes ADH (alcohol dehydrogenase, At1g77120) and PDC1 (pyruvate decarboxylase-1, At4g33070) based on RT-PCR and GeneChip® analyses. Results show a clear overlap in genes induced under hypoxic conditions and those induced by KIN10 expression in protoplasts. The overlap of the gene expression profiles was confirmed by triplicated real-time quantitative RT-PCR analyses of selected marker genes.
2008-06-16 | E-GEOD-8248 | biostudies-arrayexpress
Project description:The goal of this experiment was to explore the extent of KIN10 (At3g01090) transcriptional regulation and identify its early target genes in Arabidopsis mesophyll protoplasts. Results suggest that KIN10 targets a remarkably broad array of genes that orchestrate transcription networks, promote catabolism and autophagy, and suppress anabolism and ribosome biogenesis. The transient expression condition ruled out secondary or long-term effects of metabolism and growth, and circumvented experimental limitations caused by redundancy and embryonic lethality observed in mammals and plants. Experiment Overall Design: Analysis of the transcript changes induced by transient KIN10 (At3g01090) expression (6h) in Arabidopsis mesophyll protoplasts using protoplasts similarly transfected with control plasmid DNA for comparison. To maximize the significance of the duplicated experimental data for defining differentially expressed genes, the biologically independent experiments were carefully performed by two individuals using different soil, plants, growth chambers, DNAs and microarray facilities for hybridization and scanning to cover potential technical and biological variations. For each pair (control & KIN10) of large-scale protoplast transfection experiments, about 3 million protoplasts were isolated from the fifth and sixth leaves of 36 randomly picked plants and pooled. Experiment Overall Design: RNA samples isolated from control and KIN10-transfected protoplasts were subjected to quality controls, including RNA quality and quantity inspection by gel electrophoresis and staining as well as consistent gene expression changes by duplicated real-time quantitative RT-PCR analyses of selected marker genes.
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Project description:The bundle sheath cells (BSCs) layer – a presumed control point for radial transport of water and solutes between the vasculature and the leaf mesophyll cells (MCs) – is still largely understudied. Using isolated protoplasts, we found that 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, suggesting unique functionality of membrane transport in the BSCs, supported also by functional assays (electrophysiology and fluorescence imaging). A tight control of long distance transport is required for the optimization of the hydro-mineral homeostasis of organs in plants under fluctuating ambient conditions. The mechanism of ion selectivity and absorption from the xylem to the leaf is still poorly understood. The bundle sheath (BS), tightly enwrapping the leaf vasculature, has been suggested as a part of this mechanism, acting as a selective barrier which regulates the radial transport of water and solutes from the xylem to leaf cells. This suggestion relies on the anatomy, as well as on the recent physiological transport assays of the BS and its cells (BSCs). We hypothesized that the unique transport functionality of the BSCs is manifested in its transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) of Arabidopsis thaliana leaves. Indeed, in conformation of our hypothesis, 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, with the proton-pump AHA2 as a prominent example. Moreover, fluorescence imaging using the potentiometric dye (di-8 ANEPPS) revealed a more negative membrane potential of the BSCs protoplasts compared to those of MCs, and electrophysiological assays (patch-clamp) showed that the major, AKT2-like, membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Combined, these differences are compatible with an expected possibility of simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs in otherwise similar conditions.
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