Research Project: Integrated Approaches to Improve Fruit and Vegetable Nutritional Quality with Improved Phenolics Contents

Location: Food Quality Laboratory

2024 Annual Report

Objectives
Objective 1: Identify, characterize and manipulate key regulatory genes for antioxidant biosynthesis in pre- and post- harvest produce to optimize product quality and nutritive value. [NP 306, C1, PS1A] Sub-objective 1A: Analyze global gene expression profiles in response to treatments and identify candidate genes and signaling pathways that regulate fruit ripening and biosynthesis of sugars, acids and phenylpropanoids. Sub-objective 1B: Produce transgenic plants/fruits with increased or reduced expression of selected candidate genes, and determine their functional significance in fruit ripening and nutritive quality. Objective 2: Identify pre-harvest parameters and develop commercially relevant treatments that enhance microgreen productivity, quality and nutritive value for urban and space farming. [NP 306, C1, PS1B] Sub-objective 2A: Evaluate the effect of preharvest treatments on microgreen productivity, quality and nutritive value in controlled environment settings. Sub-objective 2B: Conduct global gene expression analysis of microgreens in response to abiotic stresses encountered in space or under microgravity.

Approach
For first objective, strawberry fruit at early and late stage of fruit development will be treated with BZT and AMD, two compounds showing impact in controlling fruit color and firmness, etc. Global gene expression will be studied to identify candidate genes related to fruit ripening and biosynthesis of sugars, acids and phenylpropanoids. Selected genes can be used as functional markers for industry management and breeders. Once these genes are identified, the already commercially available treatments, such as calcium and UVB, will be applied to determine whether and how these treatments affect expression of the selected genes. The optimum treatments will be identified from two approaches and/or combination of two approaches if there is an additive or synergistic effect. Further, stable or transient transformation with silencing or over-expression gene constructs will be used to assess the function of specific genes in various aspects of fruit physiology and metabolism, including ripening, sensory parameters, responses to stresses, and accumulation and/or retention of health-beneficial secondary metabolites. For second objective, microgreens, young vegetable seedlings with rich nutrition, such as broccoli, red radish, amaranth and pea will be selected for this study. The seeds of microgreens will be subjected by physical treatments, such as cold plasma, UVC to control pathogen infection and promote seed germination. Seedlings will be treated with different lights, UVB, and calcium and carbon dioxide. Microgreen growth and quality at the production level will be evaluated to determine the best practice for microgreen yield and quality. In collaboration with NASA, microgreen growth and quality will be studied under microgravity and high carbon dioxide. Global gene expression analysis of microgreen responses to stress both in controlled environment systems on earth and in microgravity will be investigated to determine how stress relates to yield and quality at the gene and metabolic pathway level. Putative differentially expressed genes will be used to find which genes are the better markers for future use in industry.

Progress Report
Under Objective 1, the goal is to identify, characterize and manipulate key regulatory genes for antioxidant biosynthesis in pre- and post- harvest produce to optimize product quality and nutritive value. Objective 2 focuses on identification of pre-harvest parameters and develop commercially relevant treatments that enhance microgreen productivity, quality and nutritive value for urban and space farming. Objective 1 relies on the recently discovered growth regulators carboxamide derivative (CAD) and phenylacetamide derivative (PAD) (Patent pending; DN 85.20, DN 87.20), which have shown activity to accelerate or inhibit fruit development and ripening, as well as to enhance endodormancy or inhibit sprouting of potato tubers during storage. Based on transcriptome analyses and transient transformation with gain-of-function and knockout studies, three selected genes involving in abscisic acid/stress (ABA) action had clear impact on delaying or accelerating strawberry fruit ripening. Stable transformation was carried out, and the fruit development and quality traits of heterozygous mutants were analyzed and observed. Five to ten homozygous transformants have been obtained. Analyses of plant growth and fruit development patterns are on the way. In regard to the potato sprouting work, we had seen that application of gaseous CAD in small scale inhibited potato sprouting. The results obtained were as effective as isopropyl-N-(3-chlorophenyl) carbamate (CIPC), the most popular sprouting inhibitor in the market. This year, the treatment in a semi-commercial scale was done, and CAD showed consistent effectiveness. This research was supported by the ARS Potato Program. The potato industry in Washington State has shown interest for speeding utilization of CAD. An invention disclosure for CAD as a novel potato sprouting inhibitor is underway. Under Objective 2, optimization of microgreen growth conditions in controlled environment. Energy cost is the major concern for indoor farming. Low light intensity effect on microgreen growth was studied. It was found that some cultivars grew better under low light intensity, which reveals that screening and selection of cultivars that grow well in low light intensity could be an effective way to reduce the energy cost. Furthermore, human health beneficial compounds responding to low light were identified. To reduce the foodborne pathogens contamination, seed treatment with cold plasma, ozone and heat shock were investigated. Seed treatment with hot temperature showed most effective and less cost. These results will contribute to controlled environment agriculture. In addition, a multi-team project for dual use of peanut kernel supported by ARS headquarters was joined. The purpose of the project was to use just half kernel as seed, and another half for food which can increase split kernels by 174 million pounds into edible markets and reduce the land and energy use for peanut production. To keep the split kernel with similar growth and stress tolerance to the whole kernel, several growth regulators and minerals were added to the embryo only and split seeds. The effective dosages of the regulators were identified, and the recipes were provided for coating by other team.

Accomplishments
1. Effects of harvest day on broccoli and radish microgreens yield and quality. Microgreens are 10-20 days old young vegetables with rich nutrition. The harvest time for microgreens varies after first true leaf emerge. To determine the optimal harvest time, ARS researchers in Beltsville, Maryland, analyzed the microgreen yield and quality of two Brassicaceae vegetables, broccoli (Brassica oleracea L.) and radish (Raphanus sativus) after different harvest times. Plants were grown in hydroponic conditions in a controlled growth chamber at 25oC under a 16/8h photoperiod. The first true leaf of broccoli and radish began emerging on day 11 and day 8 respectively. Then the microgreens were harvested respectively at days 11, 12, 13 and 14 for broccoli or days 8, 9, 10, and 11 for radish. Broccoli harvested at 13 days and radish harvested at 10 days when about 75% of plants had first true leaf emergence showed significantly highest yield and chlorophyll content compared to plants harvested at other days. The overall visual quality of stored microgreens at 4 oC was evaluated for the extent of decay. There was less visible decay for broccoli harvested at day 13 and radish harvested at day 10. In addition, there was a significant decrease in anthocyanins for radish harvested at 11 days. The lowest glucosinolate content in broccoli was found on day 12. Overall, the results suggest that the best harvest time for radish and broccoli microgreens is when about 75% true leaf emerging. This research provides microgreen growers the basis for determining the optimal harvest time of microgreens. Significant activities: Acted as a co-chair of Microgreen Sub-Group of NASA and ARS and supervised a postdoc to organize bimonthly meeting. Participated USDA NE 2336 Fruit Crops Quality through Storage Technologies proposal renewal; Served as Chair of the S-294 Multi-institutional project; Supervised three postdocs and a visiting scientist from Republic of Korea to conduct research projects. 306 1 A 2019

Review Publications
Kim, B.F., Lupolt, S.N., Santo, R.E., Bachman, G., Zhu, X., Yang, T., Fukagawa, N.K., Richardson, M.L., Green, C.E., Phillips, K.M., Nachman, K.E. 2024. Nutrients and non-essential metals in darkibor kale grown at urban and rural farms. PLOS ONE. 19(4). https://doi.org/10.1371/journal.pone.0296840.
Gu, G., Ding, Q., Redding, M., Yang, Y., O'Brien, R., Gu, T., Zhang, B., Zhou, B., Micallef, S.A., Luo, Y., Fonseca, J.M., Nou, X. 2024. Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2024.110665.
Peng, H., Luo, Y., Teng, Z., Zhou, B., Pearlstein, D.J., Wang, D., Turner, E.R., Nou, X., Wang, T.T., Tao, Y., Fonseca, J.M., Simko, I. 2024. Genome-wide association mapping reveals loci for enzymatic discoloration on cut lettuce. Postharvest Biology and Technology. 207. Article 112577. https://doi.org/10.1016/j.postharvbio.2023.112577.
Teng, Z., Luo, Y., Sun, J., Pearlstein, D.J., Oehler, M., Fitzwater, J.D., Zhou, B., Hussan, M.A., Chang, C.Y., Chen, P., Wang, Q., Fonseca, J.M. 2024. Effect of far-red light on biomass accumulation, plant morphology, and phytonutrient composition of ruby streaks mustard at microgreen, baby leaf, and flowering stages. Journal of Agricultural and Food Chemistry. 72(17):9587–9598. https://doi.org/10.1021/acs.jafc.3c06834.
Luciano-Rosario, D., Peng, H., Gaskins, V.L., Fonseca, J.M., Keller, N.P., Jurick II, W.M. 2023. Mining the penicillium expansum genome for virulence genes: A functional-based approach to discover novel loci mediating blue mold decay of apple fruit. The Journal of Fungi. 9(11). Article e1066. https://doi.org/10.3390/jof9111066.
Ortiz, I.N., Zhu, X., Shakoomahally, S., Wu, W., Kunle-Rabiu, O., Turner, E.R., Yang, T. 2024. Effects of harvest day after first true leaf emergence of broccoli and radish microgreens. Technology in Horticulture. 4. Article e003. https://doi.org/10.48130/tihort-0023-0031 .
Xu, W., Li, M., Li, W., Liu, H., Xu, X., Yang, T., Ma, M. 2024. Effect of H2 treatment under UV-B irradiation on the enrichment of germinated soybean isoflavones and mechanisms based on growth state, antioxidant system, and metabolomics. LWT - Food Science and Technology. 195. Article e115821. https://doi.org/10.1016/j.lwt.2024.115821.
Teplitski, M., Fonseca, J.M. 2024. Biotechnologies and bioinspired approaches for reducing loss and waste of foods of plant origin. Current Opinion in Biotechnology. 85. Article e103028. https://doi.org/10.1016/j.copbio.2023.103028.
Park, E., Luo, Y., Zhou, B., Fonseca, J.M., Stommel, J.R. 2024. Varied attributes of jalapeño pepper cultivars influence fresh-cut product quality. Journal of the American Society for Horticultural Science. 149(3):152-161. https://doi.org/10.21273/JASHS05346-23.
Bartholomew, H.P., Luciano-Rosario, D., Bradshaw, M., Gaskins, V.L., Peng, H., Jurick II, W.M., Fonseca, J.M. 2023. Avirulent isolates of Penicillium chrysogenum to control blue mold of apple caused by P. expansum. Microorganisms. 11(11). Article e2792. https://doi.org/10.3390/microorganisms11112792.
Bartholomew, H.P., Lichtner, F., Bradshaw, M., Gaskins, V.L., Fonseca, J.M., Bennett, J., Jurick II, W.M. 2022. Comparative Penicillium spp. transcriptomics: conserved pathways and processes revealed in ungerminated conidia and during postharvest apple fruit decay. Microorganisms. 10(12). Article e2414. https://doi.org/10.3390/microorganisms10122414.