Location: Plant Gene Expression Center
2020 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 mechanism through which signaling pathways combine to control plant meristem activity. Reproductive and floral meristem phenotypes of clv3 cle16 and clv3 cle16 cle17 plants were visualized using confocal laser scanning microscopy (CLSM) and meristem size was quantified as a measure of stem cell activity. Fruit chamber number was quantified in plants containing combinations of CLV3, CLE14, CLE16, CLE17 and CLE27 mutant alleles as a measure of flower meristem activity. Plate assays were performed to determine the sensitivity of clv1, clv2, and bam1 bam2 bam3 receptor mutant plants to application of synthetic CLV3, CLE16, or CLE17 signaling peptides. In addition, CLE16 and CLE17 expression in wild-type and clv3 vegetative and reproductive meristems was analyzed using in situ hybridization and florescence microscopy of green florescent protein (GFP) marker lines.
Under Objective 2, research continued to determine how meristem maintenance pathways integrate with environmental signaling pathways to regulate plant architecture. The timing of the transition from vegetative to reproductive development under long day environmental conditions was analyzed in wild-type and clv3 meristem mutant plants using confocal laser scanning microscopy. Genetic crosses were performed to introduce a florescent marker line that is specifically expressed in initiating flower meristems into the clv1, clv2, clv3 and wus meristem mutant backgrounds in order to accurately measure the transition to flowering in these lines. Homozygous mutant lines with insertion mutations in the HAIRY MERISTEM (HAM) transcription factor genes that promote meristem activity in combination with WUS were obtained from the Arabidopsis Biological Resource Center (ABRC) and crossed into wild-type plants in order to generate clean backcrossed lines for phenotypic 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. Phenotypic analysis of developmental traits such as total leaf number, plant diameter, floral induction date, and fruit chamber number was performed in wild-type and three clv mutant pennycress backgrounds. Mature embryos, seedlings, reproductive meristems and young flowers were visualized using confocal laser scanning microscopy and meristem size was quantified as a measure of stem cell activity. In addition, two sets of double mutants between the various clv mutant lines were generated and a third was initiated. Finally, genomic DNA was extracted from two of the clv mutant lines and sent for whole-genome sequencing to identify the causative gene(s).
Accomplishments
1. Small signaling peptides govern shoot and floral stem cell maintenance in plants. The ability of plants to produce leaves, stems, flowers and fruits during their lifetime is dependent upon their sustained stem cell activity, which is maintained by intricate networks of cell-to-cell signaling pathways. In Arabidopsis, the small signaling peptide CLV3 regulates shoot and flower stem cell maintenance by modulating the expression of the transcription factor WUSCHEL. However, prior research has shown that additional unknown signaling molecules also contribute to stem cell maintenance and therefore influence yield traits. ARS scientists in Albany, California, characterized two CLV3-related genes, CLE16 and CLE17, that restrict stem cell accumulation in the absence of CLV3 by signaling upstream of WUSCHEL. Both CLE16 and CLE17 contribute independently to stem cell maintenance in clv3 mutants at all stages of development by signaling through a subset of CLV3 receptors. This study reveals a complex functional relationship among related peptide genes and their cognate receptors that buffers stem cell activity at the shoot apex and identifies new target genes for manipulation to enhance yield in agricultural crop species.
