Research Project: Developmental and Environmental Signaling Pathways Regulating Plant Architecture

Location: Plant Gene Expression Center

2021 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
Under Objective 1, research continued to identify the mechanisms through which signaling pathways combine to regulate stem cell activity in plant meristems. Floral organ number was measured for two alleles each of the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED16 (CLE16), CLE17 and CLAVATA3 (CLV3) stem cell signaling genes, as well as for all of the double mutant combinations. The expression patterns of the CLV3, CLE16 and CLE17 genes were determined in wild-type, clv3 single mutant and clv3 cle16 cle17 triple mutant inflorescence and floral meristems by in situ hybridization. The effect of CLE16, CLE17 and CLV3 peptide treatment on the size of CLE receptor quadruple mutant meristems was measured using confocal microscopy. Under Objective 2, research continued to determine how meristem maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. Leaf production and secondary branch outgrowth under long day and short day environmental conditions was measured in two different loss of function alleles of the meristem maintenance genes CLV3. Leaf production was also measured in loss of function alleles of the ULTRAPETALA1 (ULT1) and ULT2 meristem maintenance genes under constant light, long day and short day conditions. Homozygous mutant lines with insertion mutations in each of three different HAIRY MERISTEM (HAM) meristem regulatory genes were crossed to wild-type plants and clean lines lacking secondary mutations were obtained by genotyping. The clean lines were crossed to one another to generate all the various higher order mutant combinations for phenotypic analysis. In addition, a separate set of ham alleles, including a null allele of a fourth HAM gene called HAM4, was obtained and initial crosses to wild-type were performed. Under Objective 3, research continued to translate knowledge of signaling gene functions in floral induction and flower development to specifically enhance yield traits in crop plants. Double mutant combinations between the three pennycress clv mutant lines were generated and initial measurements of fruit chamber number were made. Whole genome sequencing data from two of the mutant lines was analyzed to identify mutations in the causative genes. The pennycress CLAVATA1 (CLV1) and CLV3 homologous genes were cloned and sequenced from the wild-type and mutant lines. In addition, a multiple sequence alignment and phylogeny of all pennycress CLV3-related CLE genes was generated from the draft genome. These datasets were used to identify a putative pennycress ortholog of the Arabidopsis CLE16 and CLE17 genes to utilize as a target of genome editing to enhance fruit and seed yield.

Accomplishments
1. Identification of genes involved in regulating plant reproductive development. Stamens and carpels are male and female reproductive organs crucial for plant breeding, fruit formation and seed dispersal; yet the molecular mechanisms that control their development are poorly understood. ARS scientists in Albany, California, performed a transcriptomics study to identify genome-wide ULTRAPETALA1 (ULT1) and KANADI1 (KAN1) target genes during reproductive development in Arabidopsis. Several hundred genes were discovered to be regulated by ULT1, KAN1 or both factors together. Many of the novel downstream target genes of ULT1 and/or KAN1 are expressed in the developing carpels, including a unique subset restricted to the stigmatic tissue, which receives the pollen from the stamens. Also identified were a number of downstream genes expressed and/or functioning in stamens, revealing for the first time that stamen, as well as carpel development, is regulated by ULT1 and KAN1. This study identifies a wealth of new genes that can be targeted by plant researchers and breeders to enhance fruit and seed yield in various crop species.

Review Publications
Fletcher, J.C. 2020. Recent advances in Arabidopsis CLE peptide signaling. Trends in Plant Science. 25(10):1005-1016. https://doi.org/10.1016/j.tplants.2020.04.014.