Location: Plant Physiology and Genetics Research
2020 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 Sub-objective 1A, a field trial consisting of eight upland cotton entries was planted with two different planting dates and four irrigation treatments in 2016 and 2017. Measurements for each entry included bi-weekly sampling for leaf chlorophyll content, phenology or growth stage notes, pollen sterility assessments, and weekly collections with a high-throughput phenotyping (HTP) terrestrial platform to capture plant height, canopy temperature, leaf area index, and normalized difference vegetative index. The data were processed, and analysis showed cotton lines that maintained consistent leaf chlorophyll content performed better under the adverse environmental conditions. The manual method of collecting leaf chlorophyll content was very labor intensive and time consuming which limited our ability to explore the physiological mechanisms providing stable leaf chlorophyll content under high heat or low soil moisture conditions. To alleviate this problem, we planted another field trial, first in 2019 and again in 2020, with two planting dates and three irrigation treatments to develop an HTP method to measure leaf chlorophyll content. In this experiment six spectral reflectance sensors and imagers deployed by terrestrial platforms or unmanned aerial systems are collecting information about leaf color to compare to the manual measurements of leaf chlorophyll content. The goal is to develop an accurate leaf chlorophyll content index. With this information we will be able to rapidly screen large populations of upland cotton lines, or other important crops, over time to better understand how some lines are able to stabilize leaf chlorophyll content and therefore maintain photosynthetic efficiency under stressful conditions.
In support of Sub-objective 1B, two Brassica napus (rapeseed) genotypes with significant differences in wax content were selected for a growth chamber experiment to study the effects of heat and drought stresses on wax accumulation and other physiological traits. Genotypes were planted in three replicates each in a split-split plot design with two levels of temperature as the main-plot and two levels of irrigation as the subplot. Analysis of variance showed significant effects of Brassica genotypes and treatments on leaf wax accumulation. Even though total leaf wax accumulation was not affected by genotype nor treatments, accumulations of leaf wax classes and constituents were affected. Primary alcohols and aldehydes increased in response to high temperature, while free fatty acids, wax esters and alkanes decreased. Responses to high temperature also varied within each wax class. Drought treatments did not produce significant changes in leaf wax contents. A second experiment to confirm the results is ongoing.
In support of Sub-objective 2A, a portable HTP system was developed to deliver multi-modal phenotypic metrics using a single-board computer. To accommodate the portable HTP system, a low-cost cart was designed to minimize the frame weight but still rigid enough to carry the portable HTP system. Hub motors and a remote controller were designed and have been assembled and progressed with an industry partner. A second HTP platform, a row-bot that traverses in-between planted rows to measure light interception by the crop canopy was reconfigured. Light interception is an important component of radiation use efficiency, a determination of how well plants are using light energy from the sun to conduct photosynthesis. To test the platform capabilities, bi-weekly measurements in cotton, grain sorghum, and soybean fields were conducted and measurements compared to a manual light-bar meter to determine accuracy. This platform is part of a larger effort to develop a field-based HTP method to measure radiation use efficiency. As part of this effort, a photosystem two (PSII) fluorescence camera, mounted to the University of Arizona, robotic field scanalyzer was assessed. In 2019 a calibration panel was developed for the PSII camera and was used to set and verify the camera settings for image capture at the Maricopa, Arizona location. An experiment was then conducted to validate the imaging system’s ability to quantify reduced efficiency of the light dependent photosynthesis reactions over seven days. The maximum photochemical efficiency was highly correlated with an established manual handheld system indicating the PSII camera can be used for physiological and genetic studies to assess temporal changes in plant photosynthesis in response to high heat and low soil moisture conditions.
In support of Sub-objective 2B, an open source processing pipeline was developed for the PSII imaging system that extracts desired phenotypes and associates them to the appropriate experimental plot. Researchers at Maricopa, Arizona, worked with University of Arizona CyVerse researchers in Tucson, Arizona, to maximize the efficiency of the processing pipeline by using high-performance computing resources available at CyVerse. Using the pipeline on the high-performance computing system reduced the image processing time significantly. The processing pipeline was later used by researchers at Tucson, Arizona, and Davis, California, for analysis on grain sorghum and lettuce experiments. In continuation from last year, the processing pipelines and database for the terrestrial carts remain ongoing with good progress. Beta-users found the web-based user interface highly effective and efficient; within minutes of uploading data from a field collection from one of the terrestrial platforms, users can visualize the data for quality assessment. However, upcoming changes in our information technology infrastructure have prompted a shift to a cloud-based user interface that will work on the USDA SciNET resources. When completed, the pipeline and database will undergo another round of testing with beta-users to seek guidance for improvements although we intend for much of the functionality to remain the same. An image analytic software for plant genetic improvement and management was also developed for the portable HTP system. The software supports high throughput analysis of point data and images collected from plants in the field with a user-friendly graphical interface. Components within the software perform various image processing steps such as segmentation and calibration before performing conversions that enable geographic information system interface to visualize plot metrics.
In support of Sub-objective 3A, two Camelina sativa (camelina) genotypes, one that produced large seeds and one that produced small seeds, were selected for a growth chamber experiment to study the effects of heat and drought on fatty acid accumulation and ratio change during seed growth. Genotypes were planted in three replicates each in a split-split plot design with two levels of temperature as main plot and two levels of irrigation as subplot. Developing seeds were collected at 2, 4, and 6 weeks after anthesis for fatty acids composition, data analysis is ongoing.
In support of Sub-objectives 3B and 3C, we continued to study the molecular mechanisms of oil production in plants. Our recent studies have shed significant light on the life cycle of lipid droplets (LDs), the subcellular organelles that store lipids, including synthesis of LDs at the surface of the endoplasmic reticulum (ER), dynamics and function of LDs in the cytoplasm, and degradation of stored lipids as a source of energy. Our prior studies indicated that three proteins, including ER-localized SEIPIN, which in humans is associated with a neutral lipid disorder called Berardinelli-Seip congenital lipodystrophy, lipid droplet-associated LDAPs, and a third protein called LDIP (LDAP-Interacting Protein) played key roles in formation of LDs at the surface of the ER in plants. However, it was unclear how these proteins worked together to produce the droplets. By reconstituting various combinations of the proteins in SEIPIN-deficient yeast cells, we showed that LDIP and SEIPIN function together at the earliest stages of LD formation, and that LDAP modulates the number of LDs produced.
Once LDs are formed, they accumulate in the cytoplasm and are involved in various cellular processes. The size of LDs is an important determinant of metabolic accessibility to the stored lipids: smaller droplets allow for easier access to the lipid interior, while large droplets are more resistant to lipid degradation and turnover. Therefore, one strategy for increasing oil content in plants is to increase LD size. To investigate whether this was possible, scientists at the ARS lab in Maricopa, Arizona, collaborated with scientists at the University of Guelph and University of North Texas to express a protein called FSP27 in plants. FSP27 plays a key role in regulating LD size in mammalian cells by promoting the fusion of normal-sized LDs to produce larger-sized LDs. Expression of this protein in transgenic model plants showed that the protein also functioned in a plant cell context, producing larger-sized LDs and a significant increase in lipid content in both leaves and seeds.
When plant cells require additional energy, the lipids stored in LDs are degraded by organelles called peroxisomes. How lipids are transferred from LDs to peroxisomes is not well understood. Evidence from our lab, and the labs of collaborators at the University of Guelph and University of North Texas, has revealed an emerging role for “tethering” proteins in bringing the two organelles into close proximity. This “shortening the distance” between the two organelles allows for more efficient lipid transfer and helps facilitate the process of lipid breakdown. Characterization of this mechanism also provides new targets for increasing oil content in plants, as disruption of this process should decrease efficiency of lipid turnover, resulting in an increase in oil content. Studies to test this hypothesis are ongoing.
Accomplishments
Review Publications
Esnay, N., Dyer, J.M., Mullen, R.T., Chapman, K.D. 2020. Lipid droplet-peroxisome connections in plants. Contact. 3:1-14. https://doi.org/10.1177/2515256420908765.
Lin, Y., Chen, G., Mietkiewska, E., Song, Z., Caldo, K.P., Singer, S.D., Dyer, J.M., Smith, M., McKeon, T.A., Weselake, R.J. 2019. Castor patatin-like phospholipase A IIIß facilitates removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds. Plant Molecular Biology. 101:521-536. https://doi.org/10.1007/s11103-019-00915-w.
Chapman, K.D., Aziz, M., Dyer, J.M., Mullen, R.T. 2019. Mechanisms of lipid droplet biogenesis. Biochemical Journal. 476(13):1929-1942. https://doi.org/10.1042/BCJ20180021.
Price, A.M., Doner, N., Gidda, S.K., Puri, V., James, C., Schami, A., Yurchenko, O., Mullen, R.T., Dyer, J.M., Chapman, K.D. 2019. Mouse Fat-Specific Protein 27 (FSP27) expressed in plant cells localizes to lipid droplets and promotes lipid droplet accumulation and fusion. Biochimie. 169:41-53. https://doi.org/10.1016/j.biochi.2019.08.002.
Kim, J.Y. 2020. Roadmap to high throughput phenotyping for plant breeding. Journal of Biosystems Engineering. 45:43-55. https://doi.org/10.1007/s42853-020-00043-0.
Sturtevant, D., Lu, S., Zhou, Z., Shen, Y., Wang, S., Song, J., Zhong, J., Burks, D.J., Yang, A., Yang, Q., Cannon, A.E., Herrfurth, C., Feussner, I., Borisjuk, L., Munz, E., Verbeck, G.F., Wang, X., Azad, R.K., Singleton, B.B., Dyer, J.M., Chen, L., Chapman, K.D., Guo, L. 2020. The genome of jojoba (Simmondsia chinensis): A taxonomically isolated species that directs wax ester accumulation in its seeds. Science Advances. 6(11). https://doi.org/10.1126/sciadv.aay3240.
Gazave, E., Tassone, E.E., Baswggio, M., Cyder, M., Byrel, K., Oblath, E.A., Lueschow, S.R., Poss, D.J., Hardy, C.D., Wingerson, M., James, D.B., Abdel-Haleem, H.A., Grant, D.M., Hatfield, J.L., Isbell, T., Vigil, M.F., Dyer, J.M., Jenks, M.A., Brown, J., Gore, M.A., Pauli, D. 2020. Genome-wide association study identifies acyl-lipid metabolism candidate genes involved in the genetic control of natural variation for seed fatty acid traits in Brassica napus L. Industrial Crops and Products. 145. https://doi.org/10.1016/j.indcrop.2019.112080.
Thorp, K.R., Thompson, A.L., Bronson, K.F. 2020. Irrigation rate and timing effects on Arizona cotton yield, water productivity, and fiber quality. Agricultural Water Management. 234. https://doi.org/10.1016/j.agwat.2020.106146.
