2013 Annual Report
1a.Objectives (from AD-416):
Our overall objective is to understand the molecular genetic control of plant architecture so that it can be manipulated to improve agriculture. We study the molecular mechanisms that regulate the activity of the shoot apical meristem (SAM), the growing tip of the plant. Evidence indicates that cell to cell communication mediated by small secreted signaling molecules is crucial to balance stem cell function with organ formation at the shoot tip, yet very little is known about the expression or function of small signaling molecules in these critical biological processes. We have previously shown that the small signaling gene CLV3 plays a critical role in communicating stem cell fate information in the SAM and developing flower meristems. This project describes a functional genomics approach designed to identify roles in Arabidopsis SAM growth and/or organ formation for four CLV3-related genes, which are members of the CLE (CLAVATA3/ESR-RELATED) family of signaling molecules. We will utilize a combination of genetic and molecular approaches to analyze the expression and regulation of the CLE10, CLE16, CLE17 and CLE27 genes in the SAM and initiating organs, and to determine their functions in regulating stem cell activity and/or organ early organ formation. We will also take advantage of the high degree of molecular and functional conservation between different plant species to develop and test hypotheses regarding how knowledge of architectural genes in Arabidopsis can be applied to crop plants to aid in the improvement of agriculturally important traits.
The specific objectives of this project plan are:
Objective 1. Analyze the expression and regulation of signaling genes in plant shoots.
Objective 2: Determine the function of signaling genes in plant shoot meristem activity.
Objective 3: Determine the function of signaling genes in plant organ and tissue formation.
Objective 4: Computationally or molecularly test the hypothesis that signaling genes have conserved expression patterns between model plants and crop plants.
1b.Approach (from AD-416):
Hypothesis: Additional CLE genes are expressed in specific patterns in the SAM and/or organ primordia during the Arabidopsis life cycle, and these CLE genes will be regulated by known pathways acting in the SAM or organ primordia.
Experimental Design: Analyze CLE10, CLE16, CLE17 and CLE27 gene expression in Arabidopsis SAM tissue. Use in situ hybridization and quantitative RT-PCR to analyze CLE16 and CLE17 expression in plants carrying mutations in major SAM regulatory pathway components. Analyze the expression of all four CLE genes in plants that display defects in leaf organ patterning.
Contingencies: If in situ hybridization fails to detect CLE gene expression, then CLE promoter:GUS reporter construct analysis will be conducted.
Hypothesis: The CLE16 and CLE17 genes function to control aspects of Arabidopsis SAM activity.
Experimental Design: Generate CLE16 and CLE17 loss-of-function lines using insertion mutants and artificial microRNA constructs. Characterize their SAM and lateral organ phenotypes via light and electron microscopy, and histological sectioning. Perform in situ hybridization and GUS staining in the mutants to examine the expression of genes that mark SAM functional domains.
Contingencies: If CLE16/17 loss-of-function phenotypes are not detected in the mutant lines, their over-expression phenotypes will be analyzed using the same methologies.
Hypothesis: The CLE10, CLE16, CLE17 and CLE27 genes have overlapping functions in regulating Arabidopsis organ formation.
Experimental Design: Generate null mutants and/or amiRNA lines for each of the four CLE genes. Cross these lines to produce triple and quadruple mutant combinations. Analyze their leaf morphogenesis events and growth parameters using computational methods. Analyze their leaf patterning defects using electron microscopy, histological sectioning and quantitative measurements. Perform in situ hybridization, RT-qPCR and GUS staining to examine the expression of genes that mark various leaf domains and regulate different aspects of leaf formation.
Contingencies: If phenotypes are not observed in the quadruple mutants, cross them to amiR-CLE5/6 lines to eliminate all CLE gene activity in initiating organs.
Hypothesis: Arabidopsis CLE sequences are conserved in crop species, and crop CLE genes with highly similar expression patterns to those of Arabidopsis CLE genes can be identified via computational methods.
Experimental Design: Perform computational sequence analysis to identify CLE sequences in the rice, maize and soybean genomes, and generate a phylogenetic tree. Use in silico gene expression data to compare the expression patterns of Arabidopsis CLE genes with their rice and grass counterparts. Combine the phylogenetic and expression data to determine which rice and grass CLE genes are most similar and thus most likely to share biological functions.
Contingencies: Crop genes orthologous to Arabidopsis SAM and organ regulatory genes are likely to be of economic value. Existing evidence idicates that CLE genes regulating Arabidopsis meristem architecture have similar functions in crop plants as rice and maize.
This report documents progress for Project Number 5335-21000-041-00D, which started in July 2013 and continues research from Project Number 5335-21000-038-00D, entitled “Functional Genomics of Plant Architecture.” The four project Objectives fall under National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement. Progress on this project focuses on addressing Problem 3A – the need for fundamental knowledge of plant biological and molecular processes. During the first month of this project we have made progress on attaining Objective 1 to determine CLE gene expression in wild-type plants by preparing plant materials for the in situ hybridization experiments. We have made progress on Objective 2 to generate cle16 cle17 double mutants by planting cle16 and cle17 single mutants for the cross. We have also made progress on Objective 4 to identify CLE gene sequences in rice by gathering the accession numbers of all of the OsCLE genes in the sequenced rice genome.