Functional Genomics Analysis of the ABI5 bZIP Subfamily

Recipient Organization
TEXAS A&M RESEARCH FOUNDATION
400 HARVEY MITCHELL PKWY S #100
COLLEGE STATION,TX 77845-4321

Performing Department
BIOLOGY

Non Technical Summary
Seed development and germination are of immense agronomic importance yet little is known about the molecular genetic events that control these processes. We do know that the plant hormone abscisic acid (ABA) plays a pivotal role in this process. ABA modulates the accumulation of seed storage components required for germination, and it prevents the seed from germinating precociously. We and others have identified a gene family that encodes regulatory proteins called basic leucine zipper or bZIP proteins; these proteins play a key role in mediating the effect(s) of ABA during seed development and germination. Because one member of this gene family is the ABA INSENSITIVE5 (ABI5) gene, we have chosen to name the subfamily after this gene. The present project will study how the ABI5 bZIP proteins interact with DNA sequences, called cis regulatory sequences, that control the expression of a carrot gene called Dc3. We will try and understand how ABI5 bZIP proteins transduce developmental and hormonal (ABA) signals and how this impacts seed development and germination. This project will provide novel insights into the function of seed-specific bZIP transcription factors that mediate gene regulation in developing embryos and in response to ABA, and will also contribute to the understanding of more global aspects of the molecular basis of plant development. Results from this project may afford the opportunity to manipulate the storage process to enhance the quantity and quality of seed protein and lipids and to more precisely control seed germination.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	1040	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;

Goals / Objectives
We have identified a small family of novel basic leucine-zipper (bZIP) proteins that includes ABA INSENSITIVE5 (ABI5). Because these proteins bind to the promoter region of the lea class gene Dc3, we named them the Arabidopsis Dc3 promoter binding factors (AtDPBFs). At least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these (AtDPBF-1) is identical to ABI5. Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization and transcriptional activity. The overall objective of this project is a detailed functional analysis of AtDPBF1-5 of the ABI5 subfamily of bZIP transcription factors that contribute to the regulation of gene expression during seed development and in response to ABA. Specific objectives include: 1. Isolation of additional genes encoding proteins that interact with AtDPBFs. 2. Exploration of the molecular aspects of ABA signaling and Dc3 expression by determining the specificity of interaction of AtDPBFs with the Dc3 promoter and synthetic binding sites. Specific questions to be asked are: (a) What are the optimal AtDPBF binding site(s). (b) Do AtDPBF heterodimers have the same binding characteristics and function as homodimers. (c) Do AtDPBFs bind cooperatively to E motifs. (d) Are AtDPBFs global seed-specification/ABA-response factors. 3. Analysis of the expression of selected genes involved in ABA signaling and seed development. This project will provide novel insights into the function of seed-specific bZIP transcription factors that mediate gene regulation in developing embryos and in response to ABA, and will also contribute to the understanding of more global aspects of the molecular basis of plant development.

Project Methods
Objective 1. We will attempt to clone additional genes encoding proteins that interact with AtDPBFs employing the yeast two-hybrid system. Various portions of AtDPBF cDNAs will be fused to coding sequences of GAL4 DNA-binding domains and the hybrid constructs will be co-transformed into yeast with the GAL4 activation domain-cDNA library. Objective 2. We will perform random binding site selection to determine the optimal binding sites for AtDPBFs. We will bind recombinant AtDPBFs to a pool of oligos containing fifteen random nucleotides flanked by two nonidentical primer sequences. Binding will be assessed by EMSA and DNAse footprinting. Similar methods will be used to assess the role of heterodimer formation on DNA binding specificity and to assess whether AtDPBFs bind cooperatively. Objective 3. We will use low density DNA microarrays to survey the expression patterns of approximately 100 genes we have identified that are involved in ABA signaling or seed development. We will examine expression in developing seeds and seedlings as a function of treatment with ABA and a limited set of other plant growth regulators and physiological conditions. In addition and if time allows, we will examine expression in the following genetic backgrounds, abi3, abi5, lec1 and fus3.

Progress 09/01/01 to 08/31/04

Outputs
During the previous funding period, the overall objective of this project was a functional analysis of AtDPBF1-5, members of the Arabidopsis ABI5 subfamily of bZIP transcription factors that contribute to the regulation of gene expression during seed development and in response to ABA. We used quantitative PCR (qPCR) to study the expression of the ABI5 bZIP transcription factor genes in developing seeds and tissues from mature plants. One important observation is that most of the ABI5 bZIP transcription factor genes are expressed at some point during seed development, and with the exception of AREB1, these genes are expressed at significantly lower levels in vegetative tissues grown under optimal, non-stress conditions. We showed that the ABI5 promoter contains a seed-specification element, and we demonstrated that ABI5 bZIP transcription factors interact with this seed specification element. We isolated T-DNA mutants in ABI5, EEL and AtDPBF2, and studied the effect of these mutants on germination and gene expression. All goals of the project were accomplished.

Impacts
This project enhanced our understanding of the role of the ABI5 bZIP transcription factor gene family in seed development. This will enable further insights into the molecular mechanisms involved in seed maturation and dormancy. Seed maturation is arguably one of the most agronomically important processes with enormous implications for the health and prosperity of U.S. citizens and worldwide.

Publications

Kim, S. Y., Ma, J., Perret, P., Li, Z. and Thomas, T. L. (2002) Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities. Plant Physiol. 130, 688-697.
Chung, H.J., Fu, H. Y. and Thomas, T.L. 2004. ABA inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo regulated expression of the carrot Dc3 gene. Planta, Sep 2004; 10.1007/s00425-004-1366-7.
Damaj, M., Hur, J., Chuang, H., Perret, P., Ma, J., Kim, S.Y., Thomas, T.L. 2005. The ABI5 bZIP transcription factor gene family: structure, expression and function. Submitted.