Location: Plant Physiology and Genetics Research
2021 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
In support of Objective 1, a field experiment consisting of a soybean population with over 200 entries was planted in 2020 in Maricopa, Arizona, and Columbia, Missouri. Both locations collected red|green|blue (RGB) imagery from an unmanned aerial system (UAS) of the population including eight collections in Maricopa and three collections in Columbia. Additionally, thermal images from a UAS were collected in Maricopa and thermal images from a handheld thermal imager were collected in Columbia. Extracted traits from the UAS imagery, including canopy temperature, canopy cover, and a calculated leaf area index, will be used in a genome-wide association study to identify molecular markers useful for plant breeding. This trial was planted again in 2021 at both locations and measurements via UAS will be repeated. A more detailed physiological assessment was initiated in 2021 at the Maricopa location using 10 soybean entries selected to represent the 200-entry population diversity. From these 10 entries, bi-weekly measurements for radiation use efficiency and its components will be collected. Radiation use efficiency is a quantitative approach to determine how much plant biomass (leaves, stems etc.) is produced given the amount of light intercepted. This data set will enable a better understanding of how radiation use efficiency and its components respond to high heat in soybeans.
In support of Sub-objective 1A, we continued to develop a field-based, high-throughput plant phenotyping method (FB-HTPP) to measure leaf chlorophyll content in cotton. Chlorophyll are the primary light harvesting pigments and are responsible for driving photosynthesis for production of photosynthates. Our previous work indicated that leaf chlorophyll content in high heat and water deficit conditions was an important factor for maintaining cotton fiber yield. Presumably, chlorophyll stabilization enables the plants to maintain production of photosynthates for growth and development, even under adverse growing conditions. However, the current method for collecting leaf chlorophyll content is very time consuming which has limited our ability to identify the physiological mechanisms underpinning stable chlorophyll content, and the subsequent effects on growth and development. In 2020 we completed a two-year trial to develop a model that utilizes hyperspectral reflectance measurements to estimate leaf chlorophyll content. The model was found to be 80% accurate. In 2021, a cotton mapping population was planted that varies for leaf chlorophyll content and stability in adverse environmental conditions. The FB-HTPP method for leaf chlorophyll content is being used to measure the population along with leaf area and chlorophyll fluorescence, at developmental stages throughout the growing season. With this information genetic regions of the cotton genome associated with chlorophyll stabilization will be able to be identified, which will further efforts to understand the physiological mechanisms underpinning this important adaptive phenotype.
In support of Sub-objective 1B, two Brassica napus genotypes were planted in a replicated growth chamber experiment to study the effects of drought, high temperature, and drought X high temperature on brassica gene expression. The transcriptome analysis showed differential gene expression levels in response to the imposed abiotic stresses. The expressed genes included genes controlling physiological/functional processes, transcription factors such as WRKY genes, and stress-induced proteins such as the HSP70 gene. Gene Ontology analyses annotated expressed genes into biological processes such as response to abiotic stimulus, passive transmembrane transporter activity, and chaperone and heat shock protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated expressed genes to biological/metabolic pathways that could be involved in stress responses and tolerance, such as fatty acid metabolism and elongation, cutin and wax biosynthesis, starch and sucrose metabolism, and flavonoid biosynthesis.
For Sub-objective 2A, a field-based high-throughput plant phenotyping (FB-HTPP) cart mounted with a light induced fluorescence transient (LIFT) system was evaluated in 2020. 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 FB-HTPP. Chlorophyll fluorescence was collected from cotton, sorghum, and soybean plants in 2020 along with validation measurements of chlorophyll fluorescence. Other physiological measurements were also taken including photosynthesis and leaf pigment concentrations (chlorophyll), to further validate the LIFT system’s measurement of chlorophyll fluorescence with other closely associated physiological processes. In 2021 the LIFT system’s applicability to screen large populations will be tested in the cotton and soybean populations mentioned previously. Also, in support of Sub-objective 2A, a cost-effective HTPP system was developed for rapid mapping and analytics using open-source hardware (Arduino and Raspberry Pi) and software (Python). A hub motored robotic HTPP cart was developed to carry a portable HTPP sensor package developed in 2019 that has adjustable width and height capabilities. The cart was assembled and successfully operated on the road by a remote controller in 2020.
In support of Sub-objective 2B, progress continues to be made on the processing pipelines and database for terrestrial platforms. In 2020 a small team of students worked remotely to improve the various pipelines’ user interface and database functionality. A referenced journal article was published detailing the pipeline for a tractor-based platform and database and provided the open-source code in a GitHub repository. However, as the upcoming changes to the USDA information technology infrastructure have not been finalized, no further progress has been made on a shift to a cloud-based user interface that will work on the USDA SciNET resources. In preparation for the shift, HTPP datasets have been published through the National Agricultural Libraries, Ag Data Commons to facilitate testing. The Image Mapping and Analytics for Phenotyping (IMAP) software, developed in 2019, was further enhanced with an algorithm for georeferencing data, a graphical user interface for spectral calibration, and a batch process of gridding. IMAP was tested with UAS derived imagery and successfully delivered plot-level metrics. The technology transfer of the IMAP software is in process to share with universities and other government institutes by publishing in Ag Data Commons.
In support of Sub-objective 3A, two Camelina sativa genotypes, with large or small seeds, were planted in a replicated growth chamber experiment to study the effects of heat and drought stresses on fatty acid accumulation. Developing seeds were collected at two, four, five, and six weeks after flowering and fatty acids were extracted and analyzed using gas chromatography. Preliminary analyses of data indicated that production 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 X high temperature have significant effects on fatty acid accumulation and abundance.
In support of Sub-objectives 3B and 3C, we continued to study the molecular mechanisms of oil production in plants and the connections between lipid storage and plant stress responses. Prior studies showed that a protein called SEIPIN played a critical role in the formation of lipid-storage organelles called “lipid droplets” (LDs), which emerge from the surface of the endoplasmic reticulum. How SEIPIN functions in this process is not well understood. In collaboration with scientists at Maricopa, Arizona, the University of Guelph, University of Texas, and University of Goettingen, SEIPIN was shown to physically interact with a protein called VAP27 (Vesicle Associated Protein 27). Both proteins were critically important for normal LD production in plant cells. VAP-type proteins have well-established roles in the formation of membrane contact sites and lipid transfer between organelles. Our results are the first to show that VAPs are important for LD formation, and describe the mechanism for function through physical association with the SEIPIN protein. These results provide foundational information for increasing oil production in oilseed crops.
In addition to lipid storage in plant seeds, LDs are also known to proliferate in green tissues during plant stress response. The role of LDs in this process are not well understood, due in part to the few proteins known to associate with LDs during stress adaptation. In the same collaborative group described above, a protein called ERD7 (Early Responsive to Dehydration 7) was shown to localize specifically to LDs in drought-stressed plant leaves. ERD7 is known to be strongly upregulated at the gene expression level in response to drought and other abiotic stresses, but its subcellular location was previously unknown. ERD7 was further shown to be a member of a small gene family in plants that contain two distinct types of proteins, with one group targeting to LDs, and another to mitochondria. 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 plant stress response. These observations open new avenues of research for further exploring the functional mechanisms of stress adaptation in plants with an eye towards improving drought stress tolerance in crops.
Accomplishments
1. Field-based high-throughput plant phenotyping for chlorophyll fluorescence. Photosynthetic efficiency is an important phenotype for improving crop yields in a hot dry environment. The LEMNA-TEC field scanalyzer located at Maricopa, Arizona, the largest field robot in the world, and operated by the University of Arizona, is equipped with a chlorophyll fluorescence imaging system, the Photosystem II (PSII). ARS researchers at Maricopa, Arizona, along with collaborators from the University of Arizona, and the Donald Danforth Plant Science Center, validated the PSII system for a field setting and developed a data processing pipeline to extract measurements needed to determine photosynthetic efficiency. The validation of this system and development of the processing pipeline has enabled field trials that capture the temporal dynamics of chlorophyll fluorescence for plants grown in a hot dry environment. This system provides a valuable new tool for plant researchers to develop novel germplasm adaptable to a hot dry environment for sustainable crop production.
2. Measuring cottonseed size for cotton improvement. Cotton production in the United States is a multi-billion-dollar industry that reaches far beyond textile and fabric production. Cottonseed is an important by-product of cotton fiber production and supports the cattle and food industries. Over the last 20 years 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 using inexpensive, off-the-shelf imaging equipment and a custom processing pipeline to facilitate high-throughput cottonseed phenotyping. The development of this method has enabled 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.
Review Publications
Tomasi, P., Herritt, M.T., Jenks, M., Thompson, A.L. 2021. Quantification of leaf wax and cutin monomer composition in Pima (Gossypium barbadense) and upland (G. hirsutum L.) cotton. Industrial Crops and Products. 169. Article 113670. https://doi.org/10.1016/j.indcrop.2021.113670.
Herritt, M.T., Mockler, T.C., Pauli, W.D., Thompson, A.L. 2020. Chlorophyll fluorescence imaging captures photochemical efficiency of grain sorghum (Sorghum bicolor) in a field setting. Plant Methods. 16. Article 109. https://doi.org/10.1186/s13007-020-00650-0.
Greer, M.S., Cai, Y., Gidda, S.K., Esnay, N., Kretzschmar, F.K., Seay, D., Mclinchie, E., Ischebeck, T., Mullen, R.T., Dyer, J.M., Chapman, K.D. 2020. SEIPIN isoforms interact with the membrane-tethering protein VAP27-1 for lipid droplet formation. The Plant Cell. 32(9):2932-2950. https://doi.org/10.1105/tpc.19.00771.
Ischebeck, T., Mullen, R.T., Dyer, J.M., Chapman, K.D. 2020. Lipid droplets in plants and algae: distribution, formation, turnover and function. Seminars in Cell and Developmental Biology. 108:82-93. https://doi.org/10.1016/j.semcdb.2020.02.014.
Xu, Y., Caldo, K.P., Singer, S.D., Mietkiewska, E., Greer, M.S., Tian, B., Dyer, J.M., Smith, M., Zhou, X., Qiu, X., Weselake, R.J., Chen, G. 2020. Physaria fendleri and Ricinus communis lecithin: cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine. The Plant Journal. 105(1):182-196. https://doi.org/10.1111/tpj.15050.
Thompson, A.L., Thorp, K.R., Conley, M.M., Roybal, M.D., Moller Jr, D.C., Long, J.C. 2020. A data workflow to support plant breeding decisions from a terrestrial field-based high-throughput plant phenotyping system. Plant Methods. 16. Article 97. https://doi.org/10.1186/s13007-020-00639-9.
Herritt, M.T., Jones, D., Thompson, A.L. 2020. Upland cotton (Gossypium hirsutum L.) fuzzy seed counting by image analysis. Journal of Cotton Science. 24:112-120.
Doner, N.M., Seay, D., Mehling, M.E., Sun, S., Gidda, S.K., Schmitt, K., Braus, G.H., Ischebeck, T., Chapman, K.D., Dyer, J.M., Mullen, R.T. 2021. Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 localizes to lipid droplets via its senescence domain. Frontiers in Plant Science. 12. Article 658961. https://doi.org/10.3389/fpls.2021.658961.
Herritt, M.T., Long, J.C., Roybal, M.D., Moller Jr, D.C., Mockler, T.C., Pauli, D., Thompson, A.L. 2021. FLIP:FLuorescence imaging pipeline for field-based chlorophyll fluorescence images. SoftwareX. 14. Article 100685. https://doi.org/10.1016/j.softx.2021.100685.
