2011 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.
Tissue was grown from switchgrass, maize and Brachypodium that either was wild-type or over-expressed the Corngrass microRNA, miR156. The tissue was harvested in order to avoid the shoot apex and thus consisted of leaves and stems. RNA was prepared and sequenced by collaborators. We have found ~ 1000 genes that are differentially expressed in each species. The genes that are expressed in the Corngrass tissue represent juvenile tissue, while the wild-type tissue represents adult tissue. Our goal is to determine a grass juvenile transcriptome. Toward that end, we are collaborating with a UC Berkeley graduate student to determine which of the genes are orthologs.
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.