Unknown,Transcriptomics,Genomics,Proteomics

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Identification of early target genes of the DELLA protein GAI in etiolated seedlings

ABSTRACT: The hormones gibberellins (GA) control many aspects of plant growth and development thruough the whole life cycle of the plant. For instance, GA participate in the establishment of the skotomorphogenic developmental program that is triggered when seeds germinate in darkness, i.e. under the soil. Under these conditions seedlings appear etiolated and developmental features usually triggered by light are kept repressed. The GA signaling elements GAI and RGA have a major, partially redundant role in this process. These proteins belong to the DELLA family of transcriptional regulators and are destabilized by GA, acting as negative regulators of the pathway. In order to understand the molecular basis of the regulation of the skotomorphogenesis by GA, we sought to identify early target genes of the activity of GAI in etiolated seedlings. For that purpose we analyzed rapid, global changes in gene expression in response to a transient accummulation of a dominant version of GAI, gai-1, which is resistant to GA-induced destabilization. Experiments were performed using wild type Col-0 and the transgenic line HS:gai-1 that expresses the dominant version gai-1 under the control of the promoter of the HSP18.2 gene, which is highly and rapidly induced at 37ºC. Three biological repeats were performed, and wild type and transgenic samples were labelled with Cy3 and Cy5, respectively. For each point in the time course (0, 1, 2 and 4h after a 30 minute treatment at 37ºC), a sample from the transgenic line was compared to the corresponding wild type control.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: David Alabadi

PROVIDER: E-GEOD-24253 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The proper transition between the skotomorphogenic and photomorphogenic developmental programs is key for seedling survival and it is therefore tightly regulated. Besides light, which is the major cue that triggers this transition, several hormone pathways participate in this control, mainly antagonizing the light effect. Two of these hormones are gibberellins (GA) and brassinosteroids (BR). Both hormones promote the skotomorphogenesis and prevent photomorphogenesis, as evidenced by the partially de-etiolated phenotype caused by GA or BR deficiency, or by a blockage in the respective signaling pathways. We have previously shown that BR mediate GA activity in the control of this transition. For instance, treatment of dark-grown, GA-deficient seedlings with BRs was able to partially restore morphological phenotypes such as hypocotyl growth, and importantly to restore completely the molecular phenotype of CAB2 and RbcS gene expression. On the contrary, GA-treatment did not alter the same phenotypes in the BR deficient det2 mutant. Thus, the aim of this study was to investigate to what extent BRs mediate GA activity in dark-grown seedlings, and for that purpose we have the examined global changes in gene expression caused by treatment with GA and BRs in BR- and GA-deficient seedlings, respectively. We performed two sets of experiments, each one including three different samples. The first experiment included dark-grown, WT Col-0 mock-treated seedlings, or seedlings treated with either the GA biosynthesis inhibitor paclobutrazol (PAC) or with PAC+epibrassinolide (EBR). In this experiment, PAC samples were labelled with Cy3 and compared to WT and PAC+EBR samples, which were labelled with Cy5. Two biological repeast were performed. The second experiment included dark-grown, WT Col-0 seedlings and the det2-1 mutant, which was either mock-treated or treated with GA3. Two biological replicates were performed, in the first one the WT and det2-1+GA3 were labelled with Cy3 and the det2-1 sample with Cy5. The reverse labelling was used in the second replicate.

Project description:Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes including respiration, photosynthesis and oxidative stress protection. In many eukaryotic organisms, including yeast and mammals, copper and iron homeostases are highly interconnected; however such interdependence is not well established in higher plants. Here we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis thaliana. COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We have characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 expression could play a dual role under Fe deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation maybe aimed to minimize further iron consume. On the other hand, global expression analyses of copt2-1 mutants versus wild type Arabidopsis plants indicate that low phosphate responses are increased in copt2-1 plants. In this sense, COPT2 function under Fe deficiency counteracts low phosphate responses. These results open up new biotechnological approaches to fight iron deficiency in crops. Four biological replicates of Arabidopsis seedlings were generated for 2 genotypes, Col-0 and copt2-1 mutant; and 3 growth condictions; first one an iron(Fe) and copper(Cu) sufficient medium (+Fe + Cu), second one an Fe deficient and Cu sufficient medium (-Fe+Cu) and third one an Fe and Cu deficient medium (-Fe-Cu). For each growth one comparasion was made, copt2-1 mutant versus Col-0; in each comparasion four biological replicates were made, two replicas were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 sample, while the other two replicas were reversed-labeled.

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Identification of early target genes of the DELLA protein GAI in etiolated seedlings

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