Location: Plant Physiology and Genetics Research
2018 Annual Report
Objectives
Objective 1: Characterize the molecular and physiological mechanisms governing crop response to heat and drought, including interactions, to use the information to identify and verify new genes and molecular markers useful for plant breeding.
Sub-objective 1A: Characterize the physiological and genetic mechanisms governing wax content and composition and heat shock proteins in cotton, under heat and drought conditions.
Sub-objective 1B: Characterize the physiological and genetic mechanisms governing wax content and composition and aquaporins in oilseeds, under heat and drought conditions.
Objective 2: Develop and validate field-based, high-throughput phenotyping strategies for rapid assessment of crop responses to heat and drought, including evaluation and validation of sensors, proximal sensing vehicles, and methods of data capture, storage, analysis, and interpretations.
Sub-objective 2A: Develop and deploy novel sensing platforms, sensor calibration devices, and sensor validation protocols for field-based high-throughput phenotyping.
Sub-objective 2B: Develop a database that can be queried, and a geospatial data processing pipeline for proximal sensing and imaging data collected from terrestrial platforms for field-based high-throughput phenotyping.
Objective 3: Characterize the molecular mechanisms of oil accumulation in agriculturally important plants under various inclement conditions, including heat and drought conditions, to identify and verify new genes and molecular markers to increase oil yields in both food and bioenergy crop plants.
Sub-objective 3A: Characterize the molecular and physiological mechanisms governing seed number, size, and weight for oilseeds and biofuel crops in response to heat and drought stress conditions.
Sub-objective 3B: Characterize the function of lipid droplet-associated proteins (LDAPs) and identify new genes involved in abiotic stress responses and oil production pathways in plants.
Sub-objective 3C: Use transgenic and gene-editing approaches to increase oil content and abiotic stress tolerance in crop plants.
Approach
A variety of experimental approaches including phenomics and associated “big data” management, field studies of cotton and camelina, genomics, and the molecular and biochemical studies of the model plant Arabidopsis, as well as camelina, Brassica napus, and cotton are involved.
Objective 1: To characterize the physiological and genetic mechanisms governing crop response to heat and drought, cotton and Brassica napus plants will be examined for genetic variability of these traits using conventional and high-throughput phenotyping approaches to determine canopy temperature, cuticular wax content and composition, and leaf chlorophyll content. A transcriptomics approach will be used to determine if known genes involved in wax or chlorophyll biosynthesis are underpinning the observed phenotypes, and ribonucleic acid (RNA) sequencing will be conducted with either PacBio or Illumina HiSeq technology.
Objective 2: To develop and validate field-based high-throughput phenotyping (FB-HTP) strategies for assessment of crop responses to heat and drought, novel platforms and sensor arrays, including carts, small robots and imagery, will be tested in cotton fields grown under high heat or drought stress. The FB-HTP collected traits will be assessed for accuracy and consistency using in-field calibration targets and ground truthing measurements. Semi-automated pipelines and databases will be developed to process and manage the data for statistical analysis of crop response to the environmental conditions.
Objective 3: To characterize the molecular and physiological mechanisms governing seed development and lipid-droplet-associated proteins (LDAPs) in biofuel crops, candidate gene-based and transgenic approaches will be used to examine the model system Arabidopsis and camelina. Gene function will be characterized using a combination of forward and reverse genetic approaches, coupled with cellular and biochemical studies of protein activity. Oil production in response to abiotic stress tolerance will be studied by examining the function of LDAPs and other lipid-related proteins in leaves and seeds of plants. Transgenic approaches will be used to increase oil content and abiotic stress tolerance in camelina.
Progress Report
This report documents progress for this new project which started March 2018. Progress was made toward the three Objectives and their Sub-objectives, all of which fall under National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement. Please see the report for the previous project, 2020-21000-012-00D, “Molecular Genetic Analysis of Abiotic Stress Tolerance and Oil Production Pathways in Cotton, Bioenergy and Other Industrial Crops”, for additional information.
Objective 1: The new project focuses on characterizing the molecular and physiological mechanisms governing crop response to heat and drought to identify and verify new genes and molecular markers useful for plant breeding. Sub-objective 1A focuses on characterizing mechanisms governing cuticular wax content and composition and heat shock proteins in cotton. Cuticular waxes are secreted by epidermal cells and help form a protective barrier to prevent water loss in plants. In support of this research the Regional Breeders Testing Network (RBTN) population was planted and assessed for variation in wax content and composition in leaves and fiber under well-watered and water-limited conditions. Initial analysis has identified ten cultivars with sufficient variation to proceed with candidate gene sequencing. The ten cultivars will again be planted in a well-watered and water-limited field trial once candidate gene primers have been developed. Leaf tissue will be collected and sequenced in which transcript abundance differences for the candidate genes between cultivars will be assessed.
Heat shock proteins (HSPs) can be found in many cell types and help to stabilize other proteins and cellular membranes under high heat. A certain family of HSPs, the HSP70 family, has been shown to help stabilize plant chloroplasts and prevent degradation of the photosystem components which are necessary for plant photosynthesis. In the previous five-year project, HSP70 genes that are thought to contribute to heat tolerance were identified in upland cotton. Cultivars were planted at two different planting dates for normal and high heat treatments. The cultivars were assessed for leaf chlorophyll content and rate of photosynthesis to identify cultivars with variation to these traits. Initial analysis identified three cultivars with sufficient variation in chlorophyll content and photosynthesis. The objective for this project was to identify 5 cultivars with and without tolerance so additional cultivars will be assessed in upcoming field trials.
Sub-objective 1B focuses on characterizing the physiological and genetic mechanisms governing wax content and composition and aquaporins in oilseeds, under heat and drought conditions. Aquaporins are membrane bound proteins that facilitate water, small solute, and gas transport between plant cells. Aquaporin gene families are different within and among plant species and have been shown to have differential responses to abiotic stresses like heat and drought. The hypothesis being tested is that these aquaporin genes would be good candidates for breeding under stress conditions. To test the hypothesis and study the interaction among aquaporin genes and other secondary traits related to heat and drought stress tolerance, a B. napus diversity panel was screened for wax content and found that genotypes varied in wax content and composition. Future experiments will be conducted on four B. napus genotypes selected from the diversity panel showing high and low wax content. The replicated experiment will be conducted under controlled conditions in growth chambers, with high and normal temperature and with water deficient and normal water conditions. During the experiment, morphological and physiological data related to stress tolerance will be collected.
Objective 2: The emphasis is development and validation of field-based, high-throughput phenotyping strategies for rapid assessment of crop responses to heat and drought, including evaluation and validation of sensors, proximal sensing vehicles, and methods of data capture, storage, analysis, and interpretations. Sub-objective 1A focuses on the development and deployment of novel sensing platforms for field-based high-throughput phenotyping. In support of this work, two platforms, named “Professor” and “Deadpool” were refurbished and published to better fit phenotyping needs. Two new platforms, named “Wolverine” and “Cyclops”, have also been developed and are in initial field testing phase. The Wolverine platform mimics the previously developed high-throughput phenotyping tractor in terms of capabilities but at a reduced cost compared to the tractor. This fits the needs of research groups adopting terrestrial high-throughput phenotyping strategies with limited budgets. The Cyclops platform is being developed to phenotype canopy and below canopy traits difficult to capture with proximal nadir sensors or imagers, e.g. branching angle and fruit load in cotton. Initial trials for both platforms have found they are sufficiently rugged and reliable to collect data in closed-canopy cotton during an Arizona summer where average temperatures reach 106 degrees Fahrenheit or 41 degrees Celsius; however, some minor modifications are still required. Sub-objective 2B centers on the development of a database that can be queried, and a geospatial data processing pipeline for proximal sensing and imaging data collected from terrestrial platforms for field-based high-throughput phenotyping. In support of this work two databases and processing workflows have been developed and are in initial alpha-testing for the Avenger high-throughput phenotyping tractor and new Wolverine platform. The database and workflow are being tested for efficiency, ease of use, and overall effective-ness. The alpha-testing is being done in-house but will progress to use by collaborators for beta-testing soon.
Objective 3: Molecular mechanisms of oil accumulation in plants and the identification of new genes and molecular markers that can be used to increase oil content is examined. In the previous 5-year project, a new class of proteins called Lipid Droplet-Associated Proteins (LDAPs) that coat the surface of lipid droplets in non-seed cell types were identified. Lipid droplets are the subcellular organelles that store oil in plant cells, and the LDAPs are involved in both the formation and stability of these organelles in the aqueous environment of the cytoplasm. The LDAPs were also used to identify interacting protein partners, one of which was a previously unknown protein termed LDIP (for LDAP-Interacting Protein). Notably, disruption of the LDIP gene in plants increased lipid droplet size and oil content in both leaves and seeds, indicating that this protein plays a key role in negatively regulating oil content in plants. Current studies aim to elucidate the molecular mechanisms of LDIP activity. Results showed that the LDIP protein localized specifically to the surface of lipid droplets in plant cells, where it physically interacted with the LDAPs. To test the functional significance of this interaction, experiments were developed to ascertain whether LDIP might serve as an anchor for LDAP targeting to the lipid droplet surface, since LDIP is a hydrophobic protein while LDAP is hydrophilic. Several different experiments, however, revealed that the opposite was true. LDAPs were required instead for LDIP association with lipid droplets. To characterize the role of LDIP in lipid droplet formation, tests were performed to analyze the lipid composition of wild-type and LDIP mutant plants. The results showed distinct changes in the ratios of certain types of polar lipids, suggesting that LDIP might play a role in determining the polar lipid composition of the lipid droplet outer membrane. Current experiments aim to further dissect the mechanisms by which LDIP influences polar lipid composition and determine how these changes modulate lipid droplet size and oil content in plants.
Accomplishments
Review Publications
Thompson, A.L., Thorp, K.R., Andrade-Sanchez, P., Conley, M.M., Heun, J.T., Dyer, J.M., White, J.W. 2018. Deploying a proximal sensing cart to identify drought-adaptive traits in upland cotton for high-throughput phenotyping. Frontiers in Plant Science. 9:507. https://doi.org/10.3389/fpls.2018.00507.
Tomasi, P., Dyer, J.M., Jenks, M.A., Abdel-Haleem, H.A. 2017. Phenotypic variations in leaf cuticular wax classes and constituents in a spring Camelina sativa diversity panel. Industrial Crops and Products. 112:247-251.
