2010 Annual Report
1a.Objectives (from AD-416)
Objective 1: To identify additional components of the CLAVATA meristem signal transduction pathway.
Objective 2: To characterize the role of microRNAs and their target genes in regulating Arabidopsis shoot apical meristem activity.
Objective 3: To determine the function of the Arabidopsis BOP1 and BOP2 genes in regulating shoot apical meristem activity and leaf development.
Objective 4: To develop and test hypotheses to determine how knowledge of plant architectural genes in Arabidopsis can be applied to crop plants.
1b.Approach (from AD-416)
Identify, isolate and characterize genes that regulate plant architecture in model systems and agriculturally important crops by.
1)using a sensitized genetic screen to uncover novel components of the Arabidopsis thaliana CLAVATA stem cell signaling pathway;.
2)determining the role of a small regulatory RNA and its five target genes in regulating stem cell maintenance during development;.
3)analyzing the function of the BOP1 and BOP2 genes in regulating stem cell activity and leaf formation; and.
4)developing and testing hypotheses on how knowledge of plant architectural genes in Arabidopsis can be applied to crop plants through the identification and functional analysis of orthologous genes. REPLACES 5335-21000-016-00D (4/06).
The first objective of the research project is to identify additional components of the CLAVATA (CLV) signaling pathway. To accomplish this we are focusing on characterizing enhancers and suppressors of a mutation in ULTRAPETALA1 (ULT1), which affects Arabidopsis stem cell accumulation through a pathway overlapping that of CLV3. We have identified an enhancer of ult1 called uen1 and have initiated characterization of the ult1 uen1 and uen1 mutant meristem phenotypes. We have also crossed the uen1 allele to clv1, clv2 and clv3 alleles and analyzed the double mutant shoot and floral meristem phenotypes.
The second objective of the research project is to characterize the role of microRNAs (miRNAs) and their target genes in regulating shoot apical meristem activity. To accomplish this we have analyzed the phenotype of a suppressor of the jba-1D miRNA mutant phenotype. We have also compared the suppressor phenotype to the loss-of-function phenotype of the receptor-like kinase ERECTA, and sequenced the ERECTA gene from the suppressor line to identify any causative mutations.
Towards accomplishing Objective 3 we have performed BLADE-ON-PETIOLE1 (BOP1) and BOP2 yeast two hybrid assays to identify putative interacting proteins. In addition, we have used molecular genetic techniques to identify two members of the CLV3 family of small signaling molecules, CLE5 and CLE6, as targets of positive regulation by BOP1. We have determined the expression patterns of CLE5 and CLE6 during vegetative and reproductive growth. We have generated transgenic plants carrying artificial microRNA constructs that down-regulate CLE5 and CLE6 transcription, and are currently analyzing their phenotypes.
We collaborated with the Temesek Life Sciences Laboratory in Singapore to determine the functions of CLV3-related proteins CLE8 and CLE18. We made good progress in showing that CLE8 plays a role in Arabidopsis embryo and endosperm development, which are important developmental traits that affect seed size and germination efficiency in plants.
The fourth objective of the research project is to develop and test hypotheses to determine how knowledge of plant architectural genes in Arabidopsis can be applied to crop plants. To accomplish this we have identified putative orthologs of BOP1 interacting proteins in the maize EST and rice genomic sequence databases. In addition we have cloned BOP1 and BOP2-related genes from maize and are analyzing their expression patterns during development. We also obtained a maize BOP2 insertion line and are performing phenotypic analysis of the mutant plants.
Functional Analysis of BOP-Mediated Organ Formation: This work addresses the characterization of genes controlling Arabidopsis organ identity that can be translated to crop species. ARS scientists in the Plant Gene Expression Center in Albany, CA demonstrated that the BOP1 and BOP2 regulatory proteins control leaf formation by suppressing KNOX1 and YABBY protein activity at the base of leaves. We showed that reducing the expression of multiple KNOX and YABBY genes progressively attenuated the phenotype, uncovering a genetic pathway consisting of several protein families that control leaf patterning. This accomplishment may lead to the manipulation of a similar pathway in crop plants for increasing biomass, benefiting domestic farmers as well as biotechnological efforts to achieve energy independence.
Characterization of Small Regulatory RNA in Fertilization: This work addresses the function of small regulatory RNA molecules during Arabidopsis development for future application to crop plants. ARS scientists in the Plant Gene Expression Center in Albany, CA reported in the peer-reviewed journal Genes & Development that the small regulatory RNA KOKOPELLI (KPL) is essential for the process of double fertilization in Arabidopsis. In collaboration with other ARS scientists from the same location, we reported that KPL is specifically expressed in sperm and controls sperm function during fertilization by affecting the expression of a gene that codes for a putative component of the protein degradation pathway. This accomplishment identifies a key controlling factor of double fertilization in plants, a process of enormous agricultural value to domestic farmers.
Identification of Function for ULT1 as Chromatin Regulatory Protein: This work addresses the question of how the critical meristem cell fate specification gene ULTRAPETALA1 (ULT1) functions in Arabidopsis for future application to crop plants. ARS scientists in the Plant Gene Expression Center in Albany, CA determined that ULT1 acts as a part of an epigenetic regulatory complex that counteracts the repressive activity of a well-known chromatin-associated repression complex. We also showed that ULT1 induces the expression of the master floral regulatory gene AG by directly associating with DNA sequences, leading to changes in its methylation status. This accomplishment identifies a novel mechanism that mediates epigenetic switches controlling a key developmental trait, providing a foundation for improving this trait in crop plants.
Carles, C.C., Fletcher, J.C. 2009. The SAND domain protein ULTRAPETALA1 acts as a trithorax group factor to regulate cell fate in plants. Genes and Development. 23(23)2723-2728.
Carles, C.C., Fletcher, J.C. 2010. Missing links between histones and RNA Pol II arising from SAND?. Epigenetics. 5(5):381-385.
Jun, J., Ha, C., Fletcher, J.C. 2010. BLADE-ON-PETIOLE1 coordinates organ determinacy and axial polarity in Arabidopsis by directly activating ASYMMETRIC LEAVES2. The Plant Cell. 22:62-76.
Meng, L., Ruth, K.C., Fletcher, J.C., Feldman, L. 2010. The Roles of Different CLE Domains in Arabidopsis CLE Polypeptide Activity and Functional Specificity. Molecular Plant. Published online doi:10.1093/mp/ssq021.