Location: Plant Gene Expression Center
2024 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, titled, “Developmental and Environmental Control Mechanisms to Enhance Plant Productivity”, which started in March 2023.
Under Sub-objective 1A, progress was made in performing genetic engineering of yield traits in pennycress. CRISPR-Cas9 binary vectors carrying single guideRNAs (gRNAs) targeting either the TaCLAVATA2, TaCLAVATA3 or TaCLE16 coding sequences were transformed into wild-type pennycress plants using Agrobacterium-mediated vacuum infiltration. Transformed seeds are being selected on antibiotic medium and sequenced for mutations in the corresponding pennycress gene.
In support of Sub-objective 1B, advances were made in characterizing and stacking mutations in the pennycress CLAVATA (CLV) pathway. Genomic DNA was extracted from two large-scale mapping populations, one for the 158 mutant and the other for the 246 mutant, and whole genome sequencing was performed. The resulting data were analyzed, and a single nucleotide polymorphism (SNP) database was constructed for the pennycress reference genome and used to identify the causal SNPs associated with the 158 and 246 mutations.
For Sub-objective 1C, progress was made in characterizing the functional role of UNUSUAL FLORAL ORGANS (UFO) in stem cell maintenance. Arabidopsis wildtype, ufo, clv3, and ufo clv3 mutant embryo and vegetative meristems were imaged using confocal microscopy and their diameter and height measured. Molecular markers for three distinct meristem domains were introduced into wild-type and ufo mutant plants for analysis of region-specific meristem regulation by UFO. ufo mutant plants were crossed to blade-on-petiole1 (bop1), ultrapetala1 (ult1) and pressed flower (prs) mutant plants to identify additional components of the UFO genetic regulatory pathway.
Under Sub-objective 2A, progress was made in identification of sorghum floral activator genes and determination of their function. To prepare for whole genome resequencing for identification of the locus responsible for the dominant late flowering phenotype of the sorghum ARS313 mutant line, progeny of 72 second backcross second filial (BC2F2) individuals were grown in the field. Of these lines, 20 showed uniform late flowering, indicating these lines were homozygous for the mutation. Samples of these plants were taken to prepare genomic DNA for mutation mapping. A comparable experiment for the ARS223 dominant late flowering experiment yielded less than five lines homozygous for the late flowering phenotype. Since this is an insufficient number of lines for mutant identification, a new BCF2 population will be created.
For Sub-objective 2B, progress was made in elucidating the genetic and biochemical pathways controlling flowering-related growth in sorghum. Growth trials analyzing two mutant lines for the sorghum gigantea (gi) gene showed that the activity of the gi gene is required for internode elongation that promotes growing point extension, which immediately precedes the vegetative to floral transition. The gi mutants also displayed delayed vegetative to floral transition, consistent with previous work demonstrating delayed flowering time in these mutants. These results indicate that the gi gene plays an important role in the growth phase of flowering as well as the transition to floral development.
For Sub-objective 3A, target mimicry constructs were made to knock-down the amount of miR156, and multiple independent events have been isolated in citrus. Additional transformations are ongoing in poplar and citrus.
In support of Sub-objective 3B, progress was made in evaluating RNA-sequencing libraries and tissue stages for identifying which MIR156 genes are most important for maturation. RNA-sequencing was performed on RNA samples from the first true leaves or early apices of Acacia crassicarpa, carrizo citrange and walnut. Additionally, samples and RNA have been obtained for apple and key lime, and seeds are being germinated for peach and Populus species. Walnut was used to develop the bioinformatics pipeline for these datasets, and complete gene models were successfully created for six of the eight predicted MIR156 genes.
For 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. A genetic element known to enhance transcript mobility has also been cloned and combined with MIR156 transcripts and is being tested for increased mobility in Arabidopsis.
Accomplishments
Review Publications
Hong, L., Fletcher, J.C. 2023. Stem cells: Engines of plant growth and development. International Journal of Molecular Sciences. 24(19). Article 14889. https://doi.org/10.3390/ijms241914889.
Monfared, M.M., Fletcher, J.C. 2014. Genetic and phenotypic analysis of shoot apical and floral meristem development. In: Riechmann, J.L., Ferrandiz, C., editors. Flower Development: Methods and Protocols. 2nd edition. New York, NY: Humana. p. 163-198. https://doi.org/10.1007/978-1-0716-3299-4_7.
Hagelthorn, L., Fletcher, J.C. 2023. The CLAVATA3/ESR-related peptide family in the biofuel crop pennycress. Frontiers in Plant Science. 14. Article 1240342. https://doi.org/10.3389/fpls.2023.1240342.
De Riseis, S., Chen, J., Xin, Z., Harmon, F.G. 2023. Sorghum bicolor INDETERMINATE1 is a conserved primary regulator of flowering. Frontiers in Plant Science. 14. Article 1304822. https://doi.org/10.3389/fpls.2023.1304822.
Massaro, I., Poethig, R.S., Sinha, N., Leichty, A.R. 2023. Chromosome-level genome of the transformable northern wattle, Acacia crassicarpa. G3, Genes/Genomes/Genetics. 14(3). Article jkad284. https://doi.org/10.1093/g3journal/jkad284.