Location: Plant Gene Expression Center2009 Annual Report
1a. Objectives (from AD-416)
We propose to identify genes that regulate cell walls in maize leaves in order to improve saccharification in grass species.
1b. Approach (from AD-416)
We cloned the Corngrass gene in maize that keeps the plant in a juvenile state. We determined that it is a microRNA that regulates SPL transcription factors. These transcription factors are thus responsible for the shift from juvenile to adult leaves and for the production of prop roots in maize. We will determine the genes that are regulated by each SPL gene using reverse genetics and a combination of biochemistry and histology. Documents Reimbursable with DOE. Log 34608.
3. Progress Report
CONTROL OF MAIZE LEAF DIFFERENTIATION AND BIOMASS ACCUMULATION BY THE SPL TRANSCRIPTION FACTOR FAMILY We originally identified a minimum of seven SPL transcription factors that could be responsible for the diverse phenotypes found in Cg1 mutants. Loss-of-function mutations have been isolated in four of the seven, and the remaining three have been submitted to the maize TILLING group at Purdue University for EMS mutagenesis, and to the TUSC group at Pioneer Hi-Bred for transposon mutagenesis. Three loss-of-function mutations were isolated into TC305894. In light of the mutant phenotype, this gene was renamed tasselsheath4 (tsh4). The tsh4-ref allele contains an amino acid change within the DNA binding domain, while the mum1 and m1 alleles contain Mutator and Dissociation transposon insertions, respectively. All three mutants have similar phenotypes. tsh4 tassels have sheath-like bracts replacing the branches of the tassel, while tsh4 ears have fewer kernels and have large bract leaves subtending the base of the ear. An anti-TSH4 antibody localized the protein to a region subtending the lateral meristems of the inflorescence. We hypothesize that tsh4 functions to repress lateral organ development in the area, and when the gene is mutated, bract leaves become de-repressed and grow at the expense of the meristem. Thus, SPL genes appear to be necessary for lateral organ repression, and loss of these genes in the Cg1 mutant may explain the excess number of leaves found in the mutant. The TC282500 and TC305612 are duplicate factors that are highly expressed in vegetative shoots. Several Mutator transposon insertions into exons were isolated in each gene. Homozygous insertions for each gene displayed no obvious phenotype due to the high likelihood that the genes are functionally redundant. Double mutants, however, displayed several aspects of the Cg1 mutant phenotype, including the initiation of extra tillers, extra leaves, and juvenile leaf morphology. Thus, both genes are good candidates to knock out in other biofuel crops as a means of increasing biomass. This idea is currently being tested in switchgrass using artificial microRNAs that may knock down both genes simultaneously. In collaboration with Christian Tobias at the WRRC in Albany, we have generated switchgrass transformants over-expressing the maize Cg1 gene driven behind the ubiquitin promoter. These plants essentially mimic the maize Cg1 mutant phenotype; the plants only produce juvenile leaves with juvenile cell morphology, and produce excess biomass resulting from profuse tillering. One unique aspect of the switchgrass transformants is that they fail to flower, in contrast to Cg1 mutants in maize. This may prove to be useful in the field, since flowering in a biofuel crop may cause reallocation of resources towards reproductive structures, and could cause loss of fermentable substrates. Cell wall analysis is underway with JBEI. The ADODR monitors progress through email, phone conversations, meetings and the publication of findins in peer reviewed journals. NP 302 / 1 A 2004