A detailed analysis of plant gene regulation is necessary to understand both the molecular basis of plant development and how plants respond to external and internal changes in their environment. This proposal analyzes the regulation of ocs-element enhancers, a group of closely related DNA sequences which have been exploited by two distinct groups of plant pathogens, Agrobacterium and the Caulimoviruses, to express genes in plants. We have found that ocs-element sequences respond to the plant hormone auxin in the root tip of transgenic Arabidopsis plants. A number of plant genes whose expression is induced by auxin also contain ocs- element like sequences in their promoters. Auxin is important for many aspects of plant development including roles in cell division, cell elongation and cell differentiation, all processes highly pertinent to the root tip region. We have cloned the genes for two, closely related, ocs- element binding proteins (OBF) in Arabidopsis, that are bZIP proteins and good candidates for the ocs-element transcription factor. Through a detailed characterization of the Arabidopsis OBF proteins we hope to determine their function(s) and if they mediate the auxin responsiveness of the ocs-element sequence.
The specific aims of this proposal are: 1) Determine if the expression of the OBF proteins changes in response to auxin, in different auxin mutant backgrounds and/or during development of Arabidopsis plants. These studies would analyze RNA levels and include in situ hybridization studies with gene specific probes unique to each of the OBF proteins. We will also analyze if the expression of the ocs- element/GUS fusions is altered in any of the auxin mutant backgrounds. 2) Analyze the role of the Arabidopsis OBF proteins using reverse genetic approaches to enhance, eliminate or alter the developmental timing and tissue specificity of their expression. These approaches will use antisense RNA, overexpression of the Arabidopsis OBF proteins and expression of modified forms of these proteins that act either as dominant repressors or constitutive/inducible activators. A primary goal of these experiments will be to determine if any of the reverse genetic approaches affect the auxin inducibility of the ocs-element sequence in the transgenic Arabidopsis plants containing the ocs-element/GUS reporter gene and if so, are there changes in other -phenotypes controlled by auxin such as root gravitropism. We will also analyze if any of the reverse genetic constructs alter the expression of endogenous genes known to be responsive to auxin. Through the studies described in this proposal we hope to contribute to an understanding of the control of gene expression in plants and whether structurally related transcription factors interact during plant development.
| Kang, H G; Singh, K B (2000) Characterization of salicylic acid-responsive, arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects. Plant J 21:329-39 |
| Chen, W; Singh, K B (1999) The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element. Plant J 19:667-77 |
| Kang, H G; Fang, Y; Singh, K B (1999) A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induces defense-related genes. Plant J 20:127-33 |
| Buttner, M; Singh, K B (1997) Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. Proc Natl Acad Sci U S A 94:5961-6 |
| Chen, W; Chao, G; Singh, K B (1996) The promoter of a H2O2-inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBP1-binding sites. Plant J 10:955-66 |
