Location: Plant Gene Expression Center2010 Annual Report
1a. Objectives (from AD-416)
1. Determine the miR156 target genes in tomato and the Corngrass1 (Cg1) microRNA targets in maize, in particular, the direct targets that mediate their role in phase transition. 2. Characterize the role of miR172 and its targets in tomato phase change. 3. Identify downstream targets of select SPL and AP2 target genes in maize. 4. Identify the common and unique factors that are altered upon changes in phase transition. 5. Determine the relationships between the various molecular circuits directing phase change. 6. Determine the effects of regulated manipulation of phase change genes on plant architecture and if applicable, productivity.
1b. Approach (from AD-416)
Reverse genetics will be used to identify mutations in SPL target genes in maize and tomato, and characterization of their phenotypes will determine their functions. In parallel, several miR156 insensitive SPLs will be examined in tomato for their potential promotion of phase change. Experiments will be conducted to identify a role for miR172 in phase change in tomato. These will include modification of miR172 to better match its targets and to be more efficiently processed by the RISC complex, down regulation of miR172 by miR-mimic or by a new strategy involving synthetic microRNAs designed to bind to and inactivate endogenous microRNAs. Preliminary microarray analyses of the Cg1 and ts4 mutants revealed that several GA biosynthesis genes were down regulated in Cg1, while several GA signaling genes were upregulated in ts4. Further microarray experiments with individual SPL and AP2 knockouts will be done to generate a list of selected candidate targets. The promoters of these genes will be searched for the presence of SPL and AP2 DNA binding sites, which will be determined by binding site selection assays. Tomato genes altered in 35S:miR156 plants will be determined by either microarrays or high throughput library pyrosequencing. The sets of the tomato/corn "phase targets" will be used to determine the extent of general vs. species specific responsive genes. These will be subjected to further comparison with an Arabidopsis set, to provide independent assay for general phase change genes. In maize, the focus will be on miR156, miR172 and GA circuits as all were associated with one another, and genetic material required for analyses of epistatic relations are available. In tomato, the miR156, GA and the FT circuit interrelationships will be determined. In both species, existing genetic materials will be utilized, facilitating completion of complex genetic combinations in a reasonable time frame. In addition, effects of GA will also be determined by applications of exogenous GAs and GA biosynthesis inhibitors. This way, both transient long-term GA effects will be thoroughly examined in an otherwise complex genetic backgrounds with altered phase transitions. Since several key regulators of phase change are miRNA targets, timed expression of either the miR156, miR172 and miR-Mimic (MIM) 156 will be employed to specifically regulate these factors. A collection of driver lines developed first in Arabidopsis will be transferred to tomato, and select promoters that maintain their parent will be examined in maize too. The use of these promoters in a transactivation system will facilitate their exploitation for localized manipulation of GA biosynthesis and response through artificial miRNAs. Manipulations with mild morphological consequences will be considered for field trials, depending on potential benefit of manipulated lines. Documents Reimbursable with Weizmann Institute of Science (BARD). Log 37391.
3. Progress Report
The Cg1 mutant phenotype is caused by overexpression of a unique tandem microRNA related to miR156. Of the seven SPL genes that we believe are responsible for the Cg1 phenotype and are downregulated by miR156, we now have independent loss of function mutations for six. We isolated five of these mutations (TC280160, DN213921, TC282500, TC305612 and TC305894) using reverse genetics via a Mutator transposon insertion library, while one, (tga), was isolated by another group. The sequence of the last functionally uncharacterized gene, DR5994, been submitted to the maize TILLING group at Purdue University for directed EMS mutagenesis, and to the TUSC group at Pioneer Hi-Bred for further transposon mutagenesis. A knockout mutant for this gene is expected by the end of the year. We have an antibody for both TSH4 (a target of miR156) and IDS1 (a target of miR172). Preparations are underway to carry out chromatin immunoprecipitation, thereby identifying some of the downstream targets of these microRNAs. 302 A 1 2004