Unknown,Transcriptomics,Genomics,Proteomics

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Stress response study in Arabidopsis thaliana using DNA millichips

ABSTRACT: Our goal was to create a DNA chip that is as easy, convenient and inexpensive as an agarose gel. For a first generation solution, we describe a low cost, easy to use de novo synthesis oligonucleotide microarray technology that draws on the inherent flexibility of the maskless array synthesizer for in situ synthesis of thousands of photolithographically produced oligonucleotides covalently attached to a microscope slide. The method involves physically subdividing the slide into 1 x 1 mm 'millichips' that are hybridized to fluorescent RNA or DNA of biological origin, in a microfuge tube at an ordinary laboratory benchtop, rather than in dedicated hybridization chambers. Fluorescence intensity is then measured with a standard microxcope rather than sophisticated DNA chip scanners. For proof of principle, we measured changes in the transcriptome of Arabidopsis plants induced by growth in the presence of three major environmental abiotic stresses (temperature, light and water status), in all possible combinations. Validation by comparison with qRT-PCR showed a high correlation coefficient and analysis of variance indicated a high technical reproducibility. These experiments demonstrate that low cost DNA 'millichips' can be made and reliably used at the benchtop in a normal laboratory setting, without assistance of core facilities containing costly specialized instrumentation. An average of three to four technical replicates and six biological replicates for each treatment and control group were performed resulting in a total of 457 individual millichip experiments for this study.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Jamison Wolfer

PROVIDER: E-GEOD-37118 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abscisic acid (ABA) is a key hormone for plant growth, development and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYR/PYL/RCAR branch reduces vegetative growth and seed production, and leads to a severely open stomata and dramatic ABA insensitive phenotype, even though other branches for ABA perception remain functional. Arabidopsis sextuple mutant impaired in 6 PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8, was able to germinate and grow even on 100 mM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role to regulate seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture and transcriptional response to the hormone. Four biological replicates were generated for the 3 genotypes, pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant, snrk2.2/2.3/2.6 triple mutant, and Col-0. All the samples were treated with ABA for 3 hours before harvesting. The four Col-0 samples were mix to create an unique reference Col-0 sample. Two comparasion were made, fisrt one, PYR/PYL sextuple mutant versus Col-0 reference sample and second one, SnRK2 triple mutant versus Col-0 reference sample. In each comparasion four biological replicates were made. Replicas number 1 and 2 were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 reference sample, while the other two replicas,#3 and #4, were reversed-labeled.

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Stress response study in Arabidopsis thaliana using DNA millichips

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