Location: Plant Gene Expression Center
2023 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 is the final report for project 2030-21000-048-000D, Developmental and Environmental Signaling Pathways Regulating Plant Architecture, which has been replaced by new project 2030-21210-001-000D Developmental and Environmental Control Mechanisms to Enhance Plant Productivity. For additional information, see the new project report.
Significant results were generated over the five years of the project. Under Objective 1, insight was gained into the mechanisms through which signaling pathways combine to regulate stem cell activity in plant meristems. Arabidopsis CLV3, CLE16 and CLE17 gene expression was analyzed in wild-type, clv3 and clv3 cle16 cle17 meristem mutant plants and the three genes found to cross-regulate one another. The cle16 and cle17 mutations determined to singly and together enhance the severe clv3 meristem maintenance defect in embryonic, vegetative and reproductive meristems. Genetic analysis of clv3 cle16 cle17 wus and of clv3 cle16 cle17 stm mutants showed that like CLV3, both CLE16 and CLE17 function in the same pathway as and upstream of WUS activity. The expression patterns of the WUS and HAM stem cell regulatory genes were showed to be altered in wild-type, clv3 and clv3 cle16 cle17 vegetative, reproductive and floral meristems. CLE16 and CLE17 synthetic peptides were applied to clv3, clv3 cle16, clv3 clv17, clv3 cle16 cle17 as well as clv1, clv2 and bam1/2/3 mutant meristems to show that CLE16 and CLE17 peptides signal through the BAM1/2/3 receptor kinases.
Under Objective 2, research advances were made in determining how meristem maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. Arabidopsis vegetative meristem size and leaf formation in wild-type and clv3 meristem mutants was visualized and measured under three different sets of light conditions, showing that CLV3 buffers the meristem to variable light environments. CLV3 was determined to slightly delay the timing of the transition from vegetative to reproductive development under long day (LD) light conditions through a mechanism that is likely to involve the key flowering repressor locus FLC. ham1/2/3 meristem maintenance triple mutant plants were grown under LD and short-day (SD) conditions, revealing that the HAM1, HAM2 and HAM3 genes are collectively required for flowering in SD. Finally, genetic analysis of clv3 cle16 double mutant flowering time under LD and SD conditions was conducted and showed that the CLE16 gene plays no role in CLV3-mediated floral repression.
Under Objective 3, advancements were made in translating knowledge of signaling gene functions in floral induction and flower development to specifically enhance yield traits in crop plants. Pennycress clv2 single mutant and two different clv2 double mutant combinations were demonstrated to significantly improve the yield traits of flowering time, flower number, floral organ number and harvest index. A dataset was generated consisting of a full list of 27 pennycress CLAVATA3/ESR-related (CLE) signaling gene symbols, locus IDs, protein lengths, and CLE peptide motif sequences, as well as a multiple sequence alignment, gene phylogeny, and fully annotated genome structure for each pennycress CLE locus. RNA was extracted from various wild-type pennycress tissues and CLE gene expression levels were measured, revealing that most pennycress CLE genes are broadly expressed during development. The putative pennycress ortholog of the Arabidopsis CLE16 gene was cloned from wild-type and clv mutant genomic DNA into a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing vector and transformed into wild-type Spring32 plants.
Accomplishments
1. Analysis of the CLAVATA3/ESR-related (CLE) peptide family in the biofuel crop pennycress. The CLE family of small signaling proteins is responsible for controlling key aspects of plant development. Pennycress is a relative of broccoli, cauliflower and canola that is being grown across the United States in the Midwest during an otherwise fallow period of the typical corn/soybean rotation to improve soil quality and to harvest the seeds for biofuels. Understanding the CLE family offers a major avenue for future domestication efforts of pennycress, as development is closely tied with yield. ARS researchers in Albany, California, identified and analyzed all 27 members of the CLE family in pennycress. The study reported their gene structure, evolutionary relationships, and expression in various plant tissues including seed-specific expression, by combining quantitative gene expression and previously published whole-genome transcriptome analysis to uncover the expression during plant growth of 25 of the 27 CLE family members. The new data will increase the accuracy of the Pennycress Genome Project draft genome, which previously contained correct sequence data for only two thirds of the CLE family members, and accelerate the targeting of tissue-specific CLE genes for genome editing to improve pennycress yield traits. Academic and industry scientists as well as growers and breeders will also find the results useful for identifying and manipulating CLE gene activity to improve pennycress yield, growth and development.
