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United States Department of Agriculture

Agricultural Research Service

Title: Functional Analysis of Regulatory Elements in the Gene Promoter for An Abscission-Specific Cellulas from Bean (Phaseolus Vulgaris) and Characterization of Candidate Transcription Factors

item Whitelaw, Catherine -
item Lyssenkoa, Nicholas -
item Natha, Pravendra -
item Tucker, Mark

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 5, 2002
Publication Date: N/A

Interpretive Summary: Premature shedding of flowers, fruits and pods reduces the potential yield of many crops. Moreover, mechanical harvesting of fruit is impeded by the non-uniform ripening and loosening of the fruit. The purpose of this project is to identify and characterize DNA fragments and DNA- binding proteins that are responsible for regulating the accumulation of protein during leaf, flower and fruit abscission (organ separation). We identified a short DNA-fragment (18 bp) that can confer abscission- specific expression to a non-specific gene promoter. In addition, we identified several candidate proteins which may bind to this DNA fragment. Our goal is to use this information to genetically manipulate organ separation to benefit agricultural productivity (e.g., increase yield and reduced harvesting costs). The results reporter in this manuscript will be useful to other academic & industry researchers working on abscission.

Technical Abstract: Site-directed mutagenesis was used to identify both positive and negative cis-acting elements that control hormonal and abscission- specific expression of the bean abscission cellulase (BAC) promoter. One of the 15 different mutations created in a 2.9 kb BAC promoter reduced reporter gene expression by 60%. The native sequence for this 10 bp mutation includes a TGA-type basic leucine zipper (bZIP) motif. Ligation of an 18 bp BAC element (Z-BAC) that includes the bZIP motif to a minimal 50 35S CaMV promoter enhanced abscission-specific expression 13-fold over that of the minimal promoter alone. The Z-BAC element was used in a yeast one-hybrid screen to identify putative DNA-binding proteins in a bean leaf abscission zone (AZ) cDNA library. Both NAC domain and homeodomain leucine-zipper (HD-Zip) transcription factors were identified in this screen. In addition, PCR was used to identify three TGA-type bZIP transcription factors in the AZ cDNA library. RNA blot analysis was completed for these genes. Electrophoretic mobility shift assays (EMSA) indicated binding of the TGA-type bZIP factors to the Z-BAC element; however, under our conditions, neither the NAC nor HD-Zip factors bound. Overexpression of the PvNAC1 transcription factor suppressed transient BAC promoter expression by 84%. The results indicate a complex interaction of negative and positive regulating transcription factors that control BAC gene expression.

Last Modified: 4/22/2015
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