Location: Plant Physiology and Genetics Research
2023 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 is the final report for project 2020-21000-013-000D, entitled, “Molecular Genetic and Proximal Sensing Analyses of Abiotic Stress Response and Oil Production Pathways in Cotton, Oilseeds, and Other Industrial and Biofuel Crops” which expired in March 2023, and was replaced by new project 2020-21000-014-000D. For additional information, please review the annual report for this new project.
Substantial results have been achieved over the 5 years of the project. Objective 1 focused on characterizing the molecular and physiological mechanisms governing crop response to heat and drought stresses. In support of Sub-objective 1A, researchers in Maricopa, Arizona, investigated the variation in cuticular wax and cutin monomer composition in cotton, which was conducted using five varieties of Pima cotton and six varieties of upland cotton. Results showed that upland cotton produces more total wax than Pima varieties, whereas Pima cotton produces more total cutin than upland varieties. In both species, alcohols were the most abundant wax compounds and dihydroxy monobasic acids were the most abundant cutin monomers. To support Sub-objective 1B, a growth chamber experiment was conducted to study the mechanisms that rapeseed harbors to cope with drought and high temperatures through physiological changes and gene regulation. Cuticular leaf wax accumulation analyses showed that abiotic stresses affected the variation in wax classes accumulated, where fatty acid waxes increase under both stresses, with differential increase among its constituents (C29 and C30), while primary alcohols increase under drought and decreased under high temperatures and combination of drought and high temperature. Transcriptome analyses showed differential gene expression levels in response to drought, high temperature and the combination of the two stressors. The expressed genes are involved in metabolic pathways related to stress responses and tolerance, such as fatty acid metabolism and elongation, cutin and wax biosynthesis, starch and sucrose metabolism, flavonoid biosynthesis, passive transmembrane transporter activity, and chaperone and heat shock protein binding. The study explored the genetic mechanisms involved in abiotic stress tolerance in brassica napus, and the possibility to use those candidate genes/ molecular markers in breeding for stress tolerance.
In additional support of Objective 1, and in collaboration with ARS researchers at Maricopa, Arizona, a field study investigating the impacts of cotton irrigation timing on fiber yield was conducted. Reduced soil moisture was achieved through varied irrigation amounts based on recommendations from an agroecosystem model. Data showed that reduced irrigation from first square to peak bloom reduced the number of green bolls in the lower middle quarter of mainstem nodes, where most bolls are located. Reproductive development and growth were most sensitive to reduced soil moisture treatments and irrigation rates from squaring to peak bloom, whereas the period from peak bloom to 90% open boll was unaffected by irrigation rates.
Objective 2 focused on developing and validating field-based, high-throughput phenotyping (HTP) strategies for rapid assessment of crop responses to abiotic stresses specially drought and high temperatures. In support Sub-objective 2A, ARS researchers in Maricopa, Arizona, conducted experiments with collaborators from the University of Arizona, and the Donald Danforth Plant Science Center to validate the field scanalyzer located at Maricopa, Arizona, and operated by the University of Arizona. It is the largest high-throughput phenotype (HTP) field robot in the world and is equipped with a chlorophyll fluorescence imaging system, the Photosystem II (PSII) PSII. Validation included development of the field setting and a data processing pipeline to extract measurements needed to determine photosynthetic efficiency, a critical factor for improving crop yields in hot and dry environments. This pipeline has so far been used to process over 2.5 terabytes (TB) of data allowing researchers to investigate phenotypes from over 50,000 data points to improve sorghum and lettuce production. This work has allowed researchers to develop novel germplasm adaptable to hot and dry environments for sustainable crop production. Additionally, an HTP cart mounted with a light induced fluorescence transient (LIFT) system was developed. The LIFT system captures chlorophyll fluorescence, a phenotype that indicates how well plants are utilizing light energy from the sun for photosynthate production. The LIFT system employs a method to collect chlorophyll fluorescence that allows for rapid measurements, making it amenable to HTP.
In support of Sub-objective 2A, ARS researchers developed improved methods to determine plant height and seed size in cotton. Plant height is an important characteristic of growth that is a useful indicator of plant stress resulting from water and nutrient deficit. Accurate plant height assessment throughout the growing season can improve breeding and selection decisions. While this trait is relatively simple, it can be difficult to measure accurately in crops with complex canopy architectures, like cotton. ARS researchers and university collaborators at Maricopa, Arizona, have developed and validated two high-throughput phenotyping methods for determining plant height in cotton. One method utilizes terrestrial platforms and inexpensive ultrasonic transducers, and the other utilizes images captured with a small hobby drone. Another novel method was developed to rapidly assess cottonseed size. Cottonseed is an important by-product of fiber production and supports the cattle and food industries. Due to large increases in lint yield, cottonseed size has diminished, resulting in a call by the National Cotton Council for breeders to focus on cottonseed size improvement. ARS researchers at Maricopa, Arizona, along with collaborators from Cotton Incorporated and the Regional Breeders Testing Network developed an imaging method that is inexpensive and provided a custom processing pipeline to facilitate high-throughput cottonseed phenotyping. This method has allowed novel research into cottonseed size and the effects on seed germination and plant stand establishment in the field. This technology provides a valuable new method for plant researchers to develop germplasm that meets grower and industry needs.
In support of Sub-objective 2B, ARS researchers in Maricopa, Arizona, used the open-source coding language, Python, to develop processing pipelines for each terrestrial phenotyping platform. The pipelines process data by calculating the geospatial position of each data point captured using geospatial satellite positions, which enable users to associate captured data to each experimental plot for statistical analysis. The georeferenced data are then automatically transferred into a database for storage, curations, and subsequent analysis. Image Mapping and Analytics for Phenotyping (IMAP) software was developed and enhanced with an algorithm for georeferencing data, a graphical user interface for spectral calibration, and a batch process of gridding. The IMAP was tested with Unmanned Aircraft Systems (UAS) derived imagery and successfully delivered plot-level metrics.
Objective 3 focused on characterizing the molecular mechanisms of oil accumulation in agriculturally important plants under abiotic stress conditions, including high temperature and drought. In support of Sub-objective 3A, two Camelina genotypes, with large or small seeds, were planted in a replicated growth chamber experiment to study the effects of high temperatures and drought stresses on fatty acid accumulation. Developing seeds were collected at two, four, and six weeks after flowering and fatty acids were extracted and analyzed using gas chromatography. Data analyses indicated that accumulation of certain fatty acids is correlated with seed development. Some fatty acids were synthesized and accumulated during early seed development, while other fatty acids were synthesized and accumulated at higher rates during late seed development. Our data indicated that drought, high temperature, and drought and high temperature combined have significant effects on fatty acid accumulation and abundance. Transcriptomic analyses showed differential expression of fatty acid biosynthesis genes in response to the abiotic stresses and seed development stage. The study explored the genetic mechanisms involved in abiotic stress tolerance in camelina, and feasibility to use those candidate genes/molecular markers to genetically increase oil content and fatty acids under abiotic stress conditions.
In support of Sub-objectives 3B and 3C, and in collaboration with scientists from the University of Guelph, University of Texas, and University of Goettingen, researchers in Maricopa, Arizona, examined the role of two proteins involved in lipid accumulation. Seipin, which plays a critical role in the formation of lipid-storage organelles “lipid droplets” (LDs), was shown to physically interact with a protein called Vesicle Associated Protein 27 (VAP27). Both proteins were critically important for normal LD production in plant cells. The results showed that VAPs are important for LD formation and describe the mechanism for function through physical association with the Seipin protein. In the same collaborative group, the Early Responsive to Dehydration 7 (ERD7) protein was shown to localize specifically to LDs in drought-stressed plant leaves. These findings describe a new gene family in plants and establish clear linkages between a known stress-related protein, ERD7, and the proliferation of LDs during the plant stress response. These observations open new avenues of research for further exploring the functional mechanisms of stress adaptation in plants.
Accomplishments
