2011 Annual Report
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.
Our analysis of knockouts of several maize Squamosa Promotor binding Protein Like (SPLs) targeted by mir156 has revealed how overexpression of Cg1 is able to modify plant development. The first targets analyzed all belonged to the same clade. Knock-out of one of these genes, tasselsheath4 (tsh4), was found to be important for cell allocation during leaf initiation (Chuck et al., 2010). tsh4 is expressed at the sites of leaf initiation in the inflorescence where it ultimately functions as a repressor. In tsh4 mutants, leaf initiation is de-repressed, resulting in ectopic leaves that grow at the expense of axillary meristems. Thus, the reason why Cg1 mutants make so many leaves is because overexpression of miR156 represses leaf repressors such as tsh4. Three other closely related tsh4-like genes may perform similar functions. A pair of genes in the same clade, TC305612 (a.ka. zmsbp5) and TC282500 (a.k.a. zmsbp7), are duplicate factors orthologous to the rice gene first defined by the mutations called WEALTHY FARMERS’ PANICLE (WFP) and IDEAL PLANT ARCHITECTURE (IPA). WFP and IPA are dominant mutations that reduce tillering, and increase inflorescence branching as well as seed yield. The rice mutations are caused by overexpression of the OsSPL14 transcription factor. This phenotype can be mimicked by microRNA binding site mutations in OsSPL14 that release the gene from negative regulation by miR156. In order to understand the functions of the maize orthologs of OsSPL14, we isolated transposon insertions into zmsbp5 and zmsbp7, several of which completely knock out transcription. While single mutants of zmsbp5 and zmsbp7 show no phenotype, double mutants cause increased tillering and unbranched tassels, i.e., the opposite of the gain of function phenotypes seen in rice. Furthermore, triple mutant combinations with tsh4 enhance these defects, resulting in plants that more closely resemble the maize Cg1 mutation. These results show that loss of function of members of the tsh4 clade of SPL genes may be responsible for both the enhanced tillering, and the excess leaf initiation defects of the Cg1 mutant.