Research Project: Developmental and Environmental Signaling Pathways Regulating Plant Architecture

Location: Plant Gene Expression Center

2022 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. Genetic analysis of clv3 cle16 cle17 wus and of clv3 cle16 cle17 stm signaling mutant plants was performed and shoot meristem termination, leaf number and secondary branch number were measured. The expression pattern of the WUS stem cell regulatory gene in wild-type, clv3 and clv3 cle16 cle17 vegetative, reproductive and floral meristems was determined by in situ hybridization. Under Objective 2, research continued to determine how meristem maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. Flowering time, leaf number and rosette diameter were measured in clv2 and clv3 flc meristem maintenance mutant plants as well as clv3 p35S:FLC transgenic lines grown under long day environmental conditions. Flowering time, leaf number and rosette diameter were also measured in ham123 meristem maintenance triple mutant plants grown under long day and short day conditions. The early flowering flc floral repressor mutant allele was crossed to late flowering ham123 plants and F2 seeds collected for genetic epistasis analysis. 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. Two different pennycress clv double mutant combinations were grown and yield traits such as flowering time, flower number, floral organ number and harvest index were measured. RNA was extracted from wild-type pennycress leaf, shoot apex and flower tissues to examine CLE signaling gene expression levels. The putative pennycress ortholog of the Arabidopsis CLE16 gene was cloned and sequenced from wild-type and clv mutant genomic DNA.

Accomplishments
1. A group of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling peptides regulates shoot regeneration. The ability of plants to regenerate complete shoot systems from small fragments of tissues or cells is critical for most tissue culture techniques used in plant breeding, which enable the propagation of a variety of crops including trees as well as many endangered plant species. Shoot regeneration requires the formation of new stem cell reservoirs to provide cells for the shoot, flower and fruit development needed for plant survival. However, it can be a challenging technique for growers and breeders to perform and only a few molecular factors that control the process are known. ARS researchers in Albany, California, showed that a set of seven related CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling peptides act together to control the number of shoots that can be formed by Arabidopsis plants in tissue culture. The study also identified several receptors and a regeneration-promoting transcription factor that function downstream of the CLE peptides in this process. This research identifies a novel set of key regulatory factors that can be targeted in crop species to improve breeding programs by enhancing shoot regeneration and plant survival.

Review Publications
Kang, J., Wang, X., Ishida, T., Grienenberger, E., Zheng, Q., Wang, J., Zhang, Y., Chen, W., Chen, M., Song, X., Wu, C., Hu, Z., Jia, L., Li, C., Liu, C., Fletcher, J.C., Sawa, S., Wang, G. 2022. A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana. New Phytologist. https://doi.org/10.1111/nph.18291.
Wang, G., Zhang, Y., Li, C., Wang, X., Fletcher, J.C. 2022. Signaling peptides direct the art of rebirth. Trends in Plant Science. 27(6):516-519. https://doi.org/10.1016/j.tplants.2022.03.009.