Expression Analysis of the Gene At1g55430, an Important Gene for Normal Development of Leaves and Roots of Arabidopsis thaliana, by Promoter GFP Fusion and Whole Mount in Situ Hybridization
Rocha, G.C.G1; Arongaus, A.B1; Meyerowitz, E.M 2; Margis Pinheiro, M. 1; Engler, J.D.3; and Alves-Ferreira, M1.
1- Genetic Department, Biology Institute, UFRJ, RJ; 2- California Institute of Technology, Pasadena, USA; 3 - UMR Interact Plantes Microorganismes & Sante Vege, INRA, France.
The developmental pathways that regulate morphogenesis in Arabidopsis thaliana can be accessed by analysis of knockout mutants. An Arabidopsis mutant, called aquitã (aqt), was produced by a T-DNA tagging system which allows also to monitor gene expression of the tagged gene by GFP. In this insertion line, the T-DNA is placed 200bp downstream from the start codon of the gene At1g55430, which encodes a protein containing three putative domains: DAG-PE-bind, DC1 and a PHD finger domain. aqt presents a dwarf phenotype, apical dominance loss, increase in leaves number, slight curling of leaf margins, leaves and cotlyledons with a reduced number of minor veins and often had free vein endings, increase in length and diameter of the main root and reduced number of lateral roots. RT-PCR and real time PCR analysis showed that AQT is predominant expressed in roots of wild type plants, but also presents a low expression in flowers and siliques.
As a next step toward the understanding of At1g55430 protein role in Arabidopsis, we conducted a detailed expression analysis of the T-DNA line by fluorescent microscopy and laser confocal microscopy. In addition, whole mount in situ hybridization experiments were performed in aqt and wt plants to confirm the expression pattern observed by GFP expression. The results of in situ hybridization perfectly matched GFP expression, what validated the use of T-DNA GFP line to monitor At1g55430 expression. At1g55430 showed a transient expression during vascular development of leaves, flower pedicel and roots and expression in the shoot apical meristem and stipules. A strong expression was also observed in tricoblast, epidermal cells of root elongation zone and in apical meristem of lateral roots. Furthermore, a complex expression pattern was observed during embryo development. Since the expression sites of At1g55430 mostly coincide with high auxin concentration, future experiments will establish a relation between the absence of AQT and auxin distribution in Arabidopsis.
Supported by CBAB/CNPq, PIBIC/CBAB. M. Alves-Ferreira is recipient of research fellowship from CNPq (307219/2004-6).
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