Location: Plant Gene Expression Center2012 Annual Report
1a. Objectives (from AD-416):
1: To map and characterize leaf mutants in maize. [NP 301, C4, PS 4A] 2: To positionally clone genes that regulate plant architecture in maize. [NP 301, C4, PS 4A] 3: To determine the function of genes through transgenic tests. [NP 301, C4, PS 4A]
1b. Approach (from AD-416):
Maize is an important crop as well as a model system for other cereals such as sorghum, barley, rice and wheat. The large number of genetic mutants, in combination with the ease of obtaining and mapping additional mutants, makes maize an excellent system for determining the function of genes. We propose to identify genes that regulate maize leaf development and shoot architecture. We will carry out mutagenesis with defined inbreds using the chemical mutagen EMS. Mutants will be characterized genetically and histologically. The genes will be mapped to position and cloned. The functions will be determined by expression analysis and further genetics. We already have two mutants at different stages of analysis. The dominant Liguleless narrow mutant has been mapped to position and beginning characterization is under way. The dominant Wavy auricle in blade1 (Wab1) mutant has been localized to a BAC contig. Once we clone Wab1, we will determine the function of the wild-type gene product. In order to follow expression of the genes we clone, we have developed a vector for gene fusions. We are presently testing this vector with the liguleless1 gene.
3. Progress Report:
Objective 1. We mapped liguleless narrow (lgn) to chromosome 9. We carried out high throughput sequencing and found a mutation in a kinase gene. A second allele was identified through transposon tagging. Lgn mutants are background and environmentally sensitive. In one inbred, the plants die in Indiana and Davis, but in another inbred, they survive to produce progeny. The lethality in Indiana and Davis is not seen in Berkeley where the temperatures are lower. Lgn was crossed to recombinant inbred lines and a major modifier was identified on chromosome 1. The modifier regulates the levels of lgn RNA levels, suggesting an explanation for the striking background effects. We also analyzed PIN and LG1 expression in Lgn mutants and have that data under review. Last year we reported the identification of the gene for the candy leaf mutation, which increases glucans in cell walls. The gene encodes a lichenase. This year we identified a second allele in collaboration with our research partners at Pioneer. We also discovered interesting differences in expression levels of the candy leaf gene between inbreds as well as during development. Objective 2. We mapped Wavy auricle in blade (Wab) to a region containing two genes. One of the genes encodes a TCP transcription factor, which is upregulated in the dominant Wab mutants. We carried out an EMS screen to identify a revertant and found one in 5000 that lost the Wab phenotype but still carried the parental polymorphisms. We sequenced the TCP gene in the revertant and found an arginine to tryptophan conversion in the coding region, helping us prove we have identified the correct gene. Fas maps to a region containing two genes, a YABBY and a MADS box transcription. Revertants were identified by an EMS screen and are being sequenced now. Objective 3. Auxin is an important hormone for plant growth and development. If auxin transport is inhibited, plants fail to initiate organs and multiple defects may occur. We made transgenic Brachypodium plants that express three different PIN1 auxin transporters. Each construct carries the regulatory regions with the coding regions fused to fluorescent protein reporters. The constructs reveal that in the grasses, auxin tranport is subdivided. One auxin transporter moves auxin in the epidermis to the site of leaf initiation. Another transporter moves auxin from the site of leaf initiation to developing vasculature and the third pumps auxin from leaf to leaf down the stem. Antibodies were made in maize that corroborate the different patterns. We made artificial miRNA lines to knock down the levels of PIN1 expression in Brachypodium. We have obtained transposon insertion lines into three different PIN1 genes in maize. These Brachypodium and maize lines will be analyzed in the next year. We obtained lines that have LG1 fused to a fluorescent protein reporter from collaborators in Wyoming. Unfortunately, these lines do not compliment the mutant and do not express the FP protein. Some of them showed a liguleless mutant phenotype, suggesting that the transgene suppressed the endogenous gene. An antibody has not been made that will be used instead for LG1 analysis.
1. Targets of meristem gene which is a KNOTTED1 master regulator of stem cells. Plant growth and development is regulated by meristems, stem cell populations at the growing tips of plants. In collaboration with scientists at Ohio State, ARS scientists identified the genes that are bound and regulated by the meristem gene, KNOTTED1 (KN1) in maize. KN1 regulates other transcription factors and hormone pathways. The auxin hormone pathway is tightly regulated by KN1. In collaboration with UC Berkeley scientists, they examined the genes regulated by the rice ortholog to KN1, OSH1. Many KN1/OSH1 targets are conserved, providing useful information for improving growth and development of these cereal crops.
Bolduc, N., Yilmaz, A., Mejia-Guerra, M.K., Morohashi, K., O'Connor, D., Grotewold, E., Hake, S.C. 2012. Unraveling the KNOTTED1 regulatory network in maize meristems. Genes and Development. 26:1685-1690.