Research Project: Developmental and Environmental Signaling Pathways Regulating Plant Architecture

Location: Plant Gene Expression Center

2019 Annual Report

Objectives
The long-term objective of this project is to determine how developmental and environmental signaling pathways regulate plant architecture by controlling shoot and floral meristem activity. During the next five years we will focus on the following objectives: Objective 1: Identify the mechanisms by which signaling gene pathways combine to control plant shoot meristem cell activity in floral induction and flower development. • Sub-objective 1A: Conduct functional analysis of clv3 cle16 SAM phenotypes. • Sub-objective 1B: Characterize regulation of key downstream target genes. Objective 2: Determine how meristem cell maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. • Sub-objective 2A: Analyze the interaction between the photoperiod pathway and the CLV-WUS pathway. • Sub-objective 2B: Analyze the contribution of FLC and CLE16 to regulation of the floral transition by the CLV-WUS signaling pathway. Objective 3: Translate knowledge of signaling gene functions and floral induction and flower development to specifically enhance yield traits in crop plants. • Sub-objective 3A: Quantify the effect of clv3-like mutations on floral induction and yield in pennycress. • Sub-objective 3B: Translate information on CLE16 function to improve yield traits in pennycress.

Approach
Objective 1. Hypothesis: CLV3 and CLE16 genes function together to control shoot meristem maintenance during plant development. Experimental Approaches: Quantify shoot meristem cell accumulation in clv3 cle16 plants throughout development using confocal microscopy, scanning electron microscopy, and histology. Determine if the CLV3 and CLE16 genetic pathways regulate WUS and HAM gene expression through in situ hybridization and genetic epistasis analysis. Contingencies: If neither WUS nor HAM genes are targets of CLV3 and CLE16 regulation, then expression analysis of cytokinin signaling genes such as CKX3/5 and AHK2/4 will be conducted using RT-qPCR. Objective 2. Hypothesis: CLV-WUS meristem maintenance pathway regulates the floral transition in response to photoperiod cues. Experimental Approaches: Measure shoot meristem size in wild-type plants under different photoperiods using histology and analyze meristem markers using in situ hybridization. Assess contribution of key photoperiod-responsive factor FLC to CLV3- and WUS-regulated floral transition using genetic epistasis analysis. Quantify FLC gene expression levels using RT-qPCR and measure histone methylation levels through ChIP-qPCR. Determine whether CLE16 contributes to CLV-WUS mediated regulation of floral transition using histology and RT-qPCR. Contingencies: If FLC does not fully mediate the effect of CLV-WUS signaling on the floral transition, the contribution of the photoperiod-responsive factor CONSTANS will be tested using RT-qPCR and genetic epistasis analysis. Objective 3. Hypothesis: Knowledge regarding signaling gene functions and floral induction and flower development can be translated from a model plant system to enhance yield traits in the emerging crop species pennycress. Experimental Approaches: Quantify shoot meristem cell accumulation in clv3-like pennycress plants using histology. Measure floral induction in clv3-like pennycress plants grown under laboratory and field conditions, and quantify total yield using harvest index method. Generate loss-of-function mutations in the pennycress CLE16 gene using CRISPR-Cas9 genome editing and quantify total yield in mutant plants using harvest index method. Contingencies: If multiple pennycress genes display homology to CLE16, then they will be targeted for simultaneous disruption using multiplex CRISPR genome editing. Conversely, if the CLE16-like gene is not annotated in the pennycress genome, then it will be amplified from wild-type pennycress genomic DNA using degenerate PCR.

Progress Report
Research continued under Objective 1 to identify the mechanisms through which signaling pathways combine to control plant meristem cell activity. Embryo and vegetative meristem phenotypes of clv3 cle16 and clv3 cle16 cle17 plants were visualized and measured using confocal microscopy. In addition, plate assays were performed to determine the sensitivity of clv3 cle16 and clv3 cle16 cle17 meristems to application of synthetic CLE16 or CLE17 signaling peptides. Finally, genetic crosses were initiated between clv3 cle16 cle17 plants and plants carrying mutations in putative receptor genes such as CLV1, RPK2 and BAM1/2/3 in order to determine which receptor(s) respond to CLE16 or CLE17 signaling peptides in meristems. Research continued under Objective 2 to determine how meristem maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. Vegetative meristem size and leaf formation in wild-type Arabidopsis and clv3 mutants was visualized and measured under three different sets of light conditions. Further, plants carrying mutations in the CLV3 downstream target genes HAM1 through HAM4 were obtained and initial measurements of their flowering time under specific light conditions was performed. Research continued under Objective 3 to translate knowledge of signaling gene functions in floral induction and flower development to specifically enhance yield traits in crop plants. Yield traits such as meristem size, flower number and flower organ number were measured in wild-type and clv mutant pennycress plants. In addition, pairwise crosses between the three clv mutants were carried out and double mutants for several different combinations of clv alleles were generated.

Accomplishments
1. Identification of downstream target genes of meristem maintenance factor ULT1. ARS scientists in Albany, California, addressed the need to identify novel genes that affect plant architecture and yield by analyzing and identifying the global downstream target genes of the Arabidopsis meristem maintenance factor ULTRAPETALA1 (ULT1). Using a method called transcriptomics analysis, the scientists identified many ULT target genes that are involved in development and in regulating gene expression. ULT1 was also discovered to regulate many genes involved in plant defense responses as well as in external stress responses. This finding positions ULT1 as an important gene for controlling how plants alter their growth in response to fluctuations in their environment.

Review Publications
Fletcher, J.C. 2018. The CLV-WUS stem cell signaling pathway: a roadmap to crop yield optimization. Plants. 7(4):87. https://doi.org/10.3390/plants7040087.
DiGennaro, P., Grienenberger, E., Dao, T.Q., Jun, J., Fletcher, J.C. 2018. Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin. Plant Direct. 2(12):1-14. https://doi.org/10.1002/pld3.103.
Gregory, E., Alexander, M., Miller, M.J., Fletcher, J.C. 2018. The signaling peptide-encoding genes CLE16, CLE17 and CLE27 are dispensable for Arabidopsis shoot apical meristem activity. PLoS One. 13(8):e0202595. https://doi.org/10.1371/journal.pone.0202595.