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ARS Home Pacific West Area Albany, California Plant Gene Expression Center Research





- Fletcher, Jennifer
- Harmon, Frank
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Developmental and Environmental Control Mechanisms to Enhance Plant Productivity **Location:
                            Plant Gene Expression Center** 2023 Annual Report **Objectives**
                                

Objective 1: Perform genome editing of CLAVATA (CLV) pathway genes to improve yield traits in pennycress. Characterize and stack mutations in CLV pathway and other yield-regulating genes to further enhance pennycress productivity. Characterize functional role of UNUSUAL FLORAL ORGANS (UFO) in Arabidopsis stem cell maintenance to increase understanding of yield regulation in brassicas and grasses.

Sub-objective 1.A: Perform genetic engineering of yield traits in pennycress.

Sub-objective 1.B: Characterize and stack mutations in the pennycress CLV pathway. 

Sub-objective 1.C: Characterize the functional role of UFO in stem cell maintenance. 

Objective 2: Identify sorghum floral activator genes and their function. Elucidate genetic and biochemical pathways controlling flowering-related growth in sorghum. Identify how reduced day:night temperature differentials change sorghum growth and development. 

Sub-objective 2.A: Identify sorghum floral activator genes and their function. 

Sub-objective 2.B: Elucidate genetic and biochemical pathways controlling flowering-related growth in sorghum. 

Sub-objective 2.C: Identify how reduced day:night temperature differentials change sorghum growth and development. 

Objective 3: Develop genetic and molecular strategies to accelerate crop breeding including the control of juvenility and rejuvenation; characterize the role of miR156 in the regulation of the juvenile phase and rejuvenation in woody crops.

Sub-objective 3.A: Create miR156 target mimicry lines to determine the contribution to reproductive competence in woody crops. 

Sub-objective 3.B: Characterize the function of individual MIR156 genes in woody plants. 

Sub-objective 3.C: Develop graft-based methods for manipulating juvenility and maturation. **Approach**
                                

Objective 1
Hypothesis: Genetic manipulation of CLV gene function will lead to pennycress yield increases; identifying stem cell maintenance factors and combining stem cell mutations can further increase pennycress yield; UFO regulates microRNA gene expression to control shoot stem cell activity.
  
Experimental Approaches/Procedures: Target pennycress CLV3 and CLV1 for mutagenesis using genome editing. Transform constructs, identify mutants by genotyping, perform RT-qPCR to quantify expression, measure yield traits. Conduct molecular mapping-by-sequencing of pennycress 158 and 246 mutants. Generate Taclv2 158 and Taclv2 246 mutants, measure harvest index. Analyze ufo and ufo clv3 mutants by microscopy, identify genetic pathway, quantify miRNA expression by RT-qPCR.
  
Contingencies: If we are unable to identify conserved CLV3 promoter sequences, gRNAs spanning the promoter will be generated. If molecular mapping fails to identify a mutation in the 158/246 lines, a larger gene interval will be sequenced. If RT-qPCR does not yield results, RNA-seq will be conducted.
 
Objective 2
Hypothesis: Novel late flowering sorghum mutants alter the function of floral activator genes; Sorghum GIGANTEA regulates catabolism of gibberellin phytohormone as part of flowering-related growth; identify how reduced day:night temperature differentials change sorghum growth and development.
  
Experimental Approaches/Procedures: Mapping and mutant analysis to identify causal mutants for late flowering sorghum mutants. Determine gene expression changes to floral regulatory networks in these mutant lines. Gene expression analysis and hormone treatments to identify factors promoting stem growth in sorghum and other grasses. Measure progression of growth and development of sorghum plants exposed to high and low day:night temperature differentials.
  
Contingencies: Late flowering will be screened in the greenhouse if longer growing seasons are needed. Testing growth promotion by phytohormone auxin will be the alternative to gibberellin. More narrow day/night temperature differentials will be tested if no changes in sorghum phenology are initially observed.
 
Objective 3
Hypothesis/Goals: Reduction of miR156 levels will accelerate maturity; identify the most important MIR156 genes for reproductive competence and juvenile traits to aid in improvement strategies utilizing miR156; grafting mature scions onto rootstocks overexpressing miR156 and TFL/ATC will rejuvenate new growth and enhance propagation traits.

Experimental Approaches/Procedures: Measure time to first-flowering and other molecular phenotypes in transgenic plants with elevated and reduced levels of miR156. Use deep sequencing by RNA-seq to generate transcript models for MIR156 genes across a broad phylogenetic sampling of woody crops. Heterograft mature scions with transgenic rootstocks overexpressing miR156/TFL/ATC.

Contingencies: Use RNA-seq and qRT-PCR if transgenic plants fail to produce visible maturation related phenotypes. Explore different types and depths of RNA-seq libraries for ranking MIR156 importance. If miR156/TFL/ATC transcripts show mobility, fuse tRNA-like sequences to their 3’ ends. **Progress Report**
                                

This report documents progress for project 2030-21210-001-000D, Developmental and Environmental Control Mechanisms to Enhance Plant Productivity, which started in March 2023 and continues research from projects 2030-21000-048-000D, Developmental and Environmental Signaling Pathways Regulating Plant Architecture; 2030-21000-049-000D, Conserved Genes and Signaling Networks that Control Environmental Responses of C4 Grain Crops; 2030-21000-051-000D, Dissecting the Mechanisms of Phytochrome Photoperception, Signaling and Gene Regulation; and 2030-21000-054-000D, Developing Tools to Accelerate Genetic Improvement for Woody Horticultural Crops.

Under Sub-objective 1A, progress was made in performing genetic engineering of yield traits in pennycress. Conserved non-coding sequences within the TaCLAVATA3 (TaCLV3) promoter sequence were aligned to those of closely related Brassica species and three highly conserved WUSCHEL transcription factor binding sites were identified. One guide RNA (gRNA) targeting each binding site was designed, along with a fourth gRNA targeting the downstream untranslated region corresponding to the clv3-3 mutation. The four TaCLV3 gRNAs were cloned into Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 entry vectors and Gateway-assembled into a single pennycress binary vector. In parallel, single gRNAs targeting the TaCLV1 and TaCLV2 coding sequences were separately designed, cloned and assembled into the binary vector. The resulting constructs are being transformed into wild-type pennycress plants using Agrobacterium-mediated vacuum infiltration.

Under Sub-objective 1B, advances were made in characterizing and stacking mutations in the pennycress CLV pathway. Two large-scale mapping populations of 250 plants, one population from a homozygous 158 plant twice backcrossed to the MN106 reference line and the second from a homozygous 246 plant twice backcrossed to MN106, are currently being grown and will be scored for homozygous plants from which to extract DNA for whole genome sequencing.

Under Sub-objective 1C, progress was made in characterizing the functional role of UNUSUAL FLORAL ORGANS (UFO) in stem cell maintenance. Arabidopsis wild-type, ufo, clv3, and ufo clv3 mutant inflorescence and floral meristems were imaged using confocal microscopy and their diameter and height measured. In addition, average flower number in wild-type, ufo, clv3, and ufo clv3 mutants was quantified using scanning electron microscopy and total floral organ number was quantified using light microscopy.

Under Sub-objective 2A, progress was made in constructing mapping populations for late flowering mutants ARS223 and ARS313. Large field grow outs of each line will identify BC2F3 late flowering individuals for subsequent testing of homozygosity. This advancement of mutant generations also will develop material for gene expression analysis by RNAseq.  
 
Under Sub-objective 2B, preliminary experiments have been done to identify the appropriate time window from plant sowing for dissection of plants to accurately measure differences in when the vegetative to reproductive transition occurs. Sampling every 3 weeks shows that normal plants achieve reproductive growth ahead of gigantea mutants. Future fine scale sampling will confirm these observations and identify the ideal stages to sample for gene expression analysis by RNA-sequencing (RNAseq).

Under Sub-objective 3A, transformation was established in the lab for Citrus and Populus. Target mimicry constructs were made to knock-down the amount of microRNA156 (miR156), and transformations with these constructs have begun in Citrus.

Under Sub-objective 3B, progress was made in evaluating which RNA-seq libraries and stage of tissue to use for identifying which MIR156 genes are most important for maturation. mRNA-sequencing and 3’-Digital Gene Expression sequencing was performed on RNA samples from the first true leaves of Acacia crassicarpa, and used for constructing gene models and rank order abundance of all 21 MIR156 genes. Seeds have been germinated for many of the remaining proposed species for comparative analysis, and libraries will be constructed and sequenced at depths determined by the initial pilot experiment in Acacia crassicarpa.

Under Sub-objective 3C, an in vitro micrografting system was established for Populus. miR156 over-expressing/wildtype heterografts have been initiated using this system. Full-length transcripts and coding sequences from the ATC gene have been successfully cloned from Arabidopsis thaliana and integrated into the Golden Gate system. **Accomplishments**


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