2009 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.
The first objective of the research project is to identify additional components of the CLAVATA (CLV) signaling pathway. To accomplish this we screened mutagenized clv3 families for modifier phenotypes, but we discontinued screening this population because it was an ineffective mutagenesis. Instead, we are focusing on identifying enhancers and suppressors of a mutation in ULTRAPETALA1 (ULT1), which affects Arabidopsis stem cell accumulation through a pathway overlapping that of CLV3. We identified three independent enhancers and one suppressor of the ult1 cellular identity phenotypes. We have cloned one enhancer and determined that it encodes the KAN1 gene, which was previously known to play a role in specifying leaf polarity, and are preparing to clone two other enhancer loci and one suppressor locus using a recently reported new deep sequencing approach.
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 expression patterns of the miR166 family members whose patterns have not yet been reported in the literature. We are currently in the process of determining the molecular mechanism through which a miR166 target transcription factor regulates the stem cell promoting factor WUSCHEL in the developing shoot apical meristem.
The third objective of the research project is to determine the function of the BLADE-ON-PETIOLE1 (BOP1) and BOP2 genes in regulating shoot apical meristem activity and leaf formation. To accomplish this we have used yeast two hybrid assays to demonstrate that BOP1 can form homodimers and also heterdimers with the BOP2 protein, and that mutant bop1-1 protein can dimerize with wild-type BOP1 and BOP2 protein to attenuate their activity. We have also performed a genetic screen to identify additional components of the BOP organ cell fate specification pathway, and are in the process of mapping several candidate enhancer and suppressor loci.
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 most recently have identified and cloned putative BOP1 and BOP2-related genes from maize, and obtained a maize BOP2 insertion line for phenotypic analysis. We have also consulted with the Maize GDB group regarding maize BOP family members.
Expression Analysis of CLE Signaling Gene Family. This work addresses the identification of genes that control Arabidopsis plant architecture that can be used in crop species. ARS scientists in the Plant Gene Expression Center in Albany, CA determined the expression patterns of 24 members of the CLE family of putative signaling molecules throughout Arabidopsis development. We found that most Arabidopsis tissues express multiple CLE genes during the life cycle, and detected dynamic CLE gene expression patterns during developmental processes such as lateral root initiation, stamen maturation and fruit formation. This accomplishment links novel intercellular signaling pathways with key development traits, providing a foundation for improving these traits in crop plants.
Identification of Role for BOP1 in Leaf Polarity. This work addresses the question of how the critical leaf pattern gene Blade-on-Petiole(BOP1) functions in Arabidopsis for future application to crop plants. ARS scientists in the Plant Gene Expression Center in Albany, CA determined that BOP1 induces the expression of the key leaf pattern gene asymmetric leaves(AS2) by directly associating with sequences in its promoter. We also showed that BOP1 and BOP2 are required for establishing proximal-distal leaf polarity by inducing AS2 in a proximal domain, and that disruption of this pathway causes the formation of ectopic blade tissue at the base of mature leaves. This accomplishment may lead to manipulation of such pathways in crop plants for increasing biomass, benefiting domestic farmers as well as biotechnological efforts to achieve energy independence.
Leasure, C.D., Fiume, E., Fletcher, J.C. 2009. The Essential Gene EMB1611 Maintains Shoot Apical Meristem Function During Arabidopsis Development. Plant Journal. 57(4):579-92. Epub 2008 Nov 11.