Research Project: Developmental and Environmental Signaling Pathways Regulating Plant Architecture

Location: Plant Gene Expression Center

2018 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
This report documents progress for project 2030-21000-048-00D, which began in March 2018 and continues research from project 2030-21000-041-00D, "Signaling Pathways Regulating Plant Architecture." Towards accomplishing Sub-objective 1A, images and measurements of clv3 cle16 vegetative and floral meristems were generated using light and confocal microscopy. For Sub-objective 2A, the flowering time of wild-type Arabidopsis and clv3 mutant plants under several different day lengths was measured, and several replicates of target gene expression were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). In support of Sub-objective 3A, seeds were planted and initial crosses were made between three different pennycress mutant lines.

Accomplishments