Performing Department
(N/A)
Non Technical Summary
In many crop species, vernalization accelerates flowering. Gaining a molecular understanding of genes potentially involved in vernalization has important agricultural implications. We are interested in characterizing gene function in the model plant Arabidopsis thaliana. Specifically we aim to characterize a family of genes (the REM genes) in Arabidopsis that contains 44 members. The REM genes are found only in plants; there are no family members in animals, fungi, or bacteria. An unusual feature is that many of the REM genes are clustered in the genome; nine REM genes are clustered on chromosome 4, five REM genes on chromosome 2, and three REM genes on chromosome 5. Several lines of evidence suggest that the REM genes play a role in plant growth and development. The one REM gene for which a loss-of-function phenotype has been described is VRN1/REM39. VRN1/REM39 is involved in maintaining the response to vernalization, an extended cold treatment that accelerates the
transition to flowering. Understanding the molecular basis of vernalization and how vernalization affects flowering timing has important practical implications for US agriculture. Increasing our understanding of the molecular basis of vernalization will make it possible, for example, to engineering early flowering of vernalization-sensitive crop plants in the absence of vernalization.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this proposal is to molecularly and genetically characterize members of a gene family in Arabidopsis called the REproductive Meristem (REM) gene family. There are 44 REM genes in the Arabidopsis genome. At present, the function of only one of the REM genes has been determined at present; specifically, a gene called VERNALIZATION1 (VRN1) / REM39 has been demonstrated to be necessary for a proper response to vernalization. The REM genes are found only in plants; there are no family members in animals, fungi, or bacteria. The REM genes are a subclass of the B3-domain transcription factor family. An unusual feature is that many of the REM genes are clustered in the genome; nine REM genes are clustered on chromosome 4, five REM genes on chromosome 2, and three REM genes on chromosome 5. We will focus our studies on the REM genes in the gene cluster on chromosome 4 as well as on REM19 (the gene most similar to VRN1/REM39). We plan to 1) determine the spatial and
temporal RNA expression profiles, 2) construct and analyze gain-of-function overexpression lines, 3) construct and analyze deletions of the REM gene cluster on chromosome 4, 4) phenotypically characterize T-DNA loss-of-function mutants, 5) test whether rem19 is defective in the vernalization response, and 6) determine the protein expression pattern and subcellular localization of REM3.
Project Methods
To determine the spatial and temporal expression patterns of the REM genes, we will use in situ hybridization, Northern analysis, RT-PCR, and construction of promoter-reporter fusion transgenic lines. Transgenic overexpression lines will be constructed by fusing either cDNA or genomic clones to the 35S promoter of cauliflower mosaic virus. Deletions in the chromosome 4 REM gene cluster will be constructed using gamma-irradiation to mutagenize pollen from an enhancer trap line in one of the chromosome 4 REM genes that exhibits a shoot apical meristem-specific expression pattern. Mutagenized pollen will be crossed to a male sterile mutant and putative deletions will be identified in M1 plants by loss of the shoot meristem enhancer trap staining pattern. The goal is to identify deletions that remove multiple REM genes in the chromosome 4 cluster. Presently, many T-DNA insertions in REM genes are publicly available. We will obtain these T-DNA insertion lines and examine
them for growth and developmental phenotypes. Specifically, we will test rem19 mutants for defects in response to vernalization. We have generated antisera to REM3. We plan to use this antisera to determine the spatial and temporal expression pattern of the REM3 protein by immunohistochemistry using seedling and inflorescence sections. Since the REM genes that have been examined to date are nuclear, we assume that REM 3 will be present in the nucleus as well. REM3 subcellular localization will be determined by observing co-localization of REM3 protein with known nuclear markers.