Research Project: Enhancing Specialty Crop Tolerance to Saline Irrigation Waters

Location: Agricultural Water Efficiency and Salinity Research Unit

2020 Annual Report

Objectives
Objective 1: Generate new tools and techniques for studying and understanding plant responses to salt stress in high value specialty crop plants. Sub-objective 1A: Determine the importance of ion uptake and ion ratios during salinity stress, with emphasis on Na+ and Cl-. Sub-objective 1B: Evaluate the effect of endophytes on the salinity tolerance of horticultural crops. Sub-objective 1C: Determine the effect of priming using different biochemicals to increase salt tolerance in crop plants. Sub-objective 1D: Conduct expression analyses and characterize genes involved in salt tolerance in crop plants. Objective 2: Identify and develop plant material with improved salt tolerance, enabling use of low quality water/alternative waters for irrigation. Sub-objective 2A: Generate and screen alfalfa populations segregating for the component traits of the salt tolerance mechanism to select genotypes with high tolerance to salt. Sub-objective 2B: Identify markers (molecular or biochemical) for salt tolerance and use them in marker assisted selection (MAS) to improve alfalfa germplasm.

Approach
This project focuses on salinity responses and underlying mechanisms of high-value specialty crops that includes alfalfa, strawberry, almond, spinach, tomato, eggplant and pepper. In objective 1, we concentrate on understanding relative importance of sodium ions (Na+) and choride ions (Cl-) which will lead to improved prediction of plant response to salinity. Also, the relative importance of Na+ and Cl- may become instrumental in refining breeding or genetic improvement efforts of specific crops. Understanding the mechanism of how plants use Na+ to maintain growth and ion homeostasis may help in development of lines with higher Na+ tissue tolerance. We intend to explore new technologies such as effect of endophytes and priming on salinity tolerance of horticultural crops. The interactions of endophytes/priming with crops will increase knowledge of mechanisms used by plants against abiotic stresses. This knowledge has the potential to mitigate salinity effects on crops with rapid implementation. To understand the genetic changes happening in a genome in response to salinity, we plan to conduct expression and Ribonucleic acid sequencing (RNA-Seq) analyses followed by functional validation of selected genes using model plants. Studying expression of important genes characterized in model plants may help in identifying critical genes involved in salt tolerance of high-value crops. Global gene expression changes detected via RNA-Seq analysis may detect genes or mechanisms that are specific to a particular species. Furthermore, interactions among different pathways may provide a bigger picture of the whole process. Functional complementation of Arabidopsis mutants with candidate genes will confirm evolutionary conservation of the genes involved in the salt tolerance mechanism. This will facilitate development of molecular marker based assays for these genes to screen genotypes tolerant to salt. Additionally, these genes may be manipulated in alfalfa and strawberries for improved salt tolerance. In objective 2, we intend to generate and screen alfalfa populations segregating of the component traits of the salt tolerance mechanism to select genotypes with high tolerance to salt and develop markers for salt tolerance for marker assisted selection (MAS). Crossing genotypes differing for the component traits may lead to development of genotypes with combination of multiple component traits. Pyramiding genes for different component traits will provide enhanced salt tolerance in some of the alfalfa segregating lines. Screening these for salt tolerance will lead to identification of superior lines, which can then be molecularly tested for the presence of the component traits. In addition to selecting for salt tolerant lines we will be able to able to determine importance of different component traits of the salt tolerance mechanism. Once importance of the genes involved in the component traits of the salt tolerance mechanism has been established, molecular markers developed from these lines can be used in MAS. MAS will result in fast and efficient selection of genotypes for salt tolerance.

Progress Report
In support of Sub-objective 1A, our research continued on understanding the genetic basis of salt-tolerance in almonds. In FY 20, 15 almond rootstocks were evaluated to determine their tolerance to a range of salt concentrations and characterized almond genotypes based on different component traits of the salt-tolerance mechanism. The top-performing rootstocks under salinity that were identified included Rootpac 40, Empyrean 1, Cornerstone, and BB 106. These rootstocks also had the least amount of tissue accumulation of sodium and chloride, suggesting that ion exclusion may be the main component trait of the salt tolerance mechanism in almond. Leaf proline content showed a significant correlation with the ability of plants to exclude sodium (Na) and Chlorine (Cl) and a negative correlation with the survival rate. These findings suggest that proline can be used as a useful biochemical marker for screening genotypes tolerant to salinity. Additional research in support of Sub-objective 1A included work on salinity tolerance in spinach. Two spinach cultivars (‘Raccoon’ and ‘Gazelle’) were evaluated for their responses to deficient and sufficient potassium (K) levels combined with salinities of 5, 30, 60, 90, and 120 millimoles of charge per liter (mmolc L-1) of sodium chloride. Plants substituted potassium for sodium proportionally with salinity within each K dose. Spinach favored potassium over sodium, accumulating significantly less sodium when plants were provided sufficient potassium compared to deficient potassium. Salinity did not affect shoot nitrogen, phosphorus, and potassium accumulation, indicating that spinach plants can maintain homeostasis of important minerals even when soil potassium is deficient. Although tissue calcium and magnesium decreased with salinity, but plants showed no deficiency symptoms. There was no sodium to potassium or chloride to nitrate competition and shoot biomass decrease was attributed to excessive sodium chloride accumulation in plant tissues. Overall, ‘Raccoon’ and ‘Gazelle’ biomasses were similar regardless of potassium dose, but ‘Raccoon’ outproduced ‘Gazelle’ with sufficient potassium at the two highest salinity levels, indicating that ‘Raccoon’ may outperform ‘Gazelle’ at higher sodium chloride concentrations. At low potassium, sodium may be required by ‘Raccoon’, but not ‘Gazelle’. This study suggested that spinach can be cultivated with recycled waters of moderate salinity, and less potassium than recommended, leading to savings on crop input and decreasing crop environmental footprint. In a study with Turkish cooperators, improvement in salt tolerance was demonstrated in spinach with the addition of selenium (Se). Additional work focusing on Sub-objective 1A, included our continuous research on vegetables. In FY 19, we evaluated eight varieties each of tomato, eggplant, and pepper and selected the most salt-tolerant and the most salt-sensitive for each. In FY 20, we evaluated these selected varieties under seven treatments of irrigation waters differing in salinity levels and ion compositions. This analysis will allow us to understand the regulation of salinity stress in three species of the same family (Solanaceae) and help us decipher the genetic basis for these differences. In support of Sub-objective 1B, a project on the effect of endophytes on the salinity tolerance of strawberry was initiated. Strawberries are one of the main California crops that are susceptible to salinity. Although we have found a couple of salinity-tolerant cultivars in our past research, they still showed decreased biomass and fruit production under low salinity (2.5 deciSiemens per meter) of irrigation water. In an effort to assess fungal and bacterial endophytes as mitigators of salinity damage on strawberry plants, diploid and polyploid plants were inoculated with endophytes and subjected to a moderate salinity of 7 deciSiemens per meter. In both experiments, bacterial endophytes seemed to provide an advantage to produce significantly higher shoot biomass both for diploid and polyploid species. However, when plants were exposed to salinity, neither fungal or bacterial endophytes provided any benefit for plants to tolerate salinity, and all had significantly lower biomass than control plants. However, these experiments need to be repeated before any conclusions can be drawn. With regard to Sub-objective 1C, experiments were initiated to evaluate the effect of priming agents on salt-tolerance in different almond rootstocks. The experiment evaluated the effect of two salinity treatments (control and high salinity) and five priming agent treatments (No agent, Melatonin, H2S, Salicylic Acid, and Zinc Sulfate) on change in trunk diameter. Data analyses and interpretations are in process. For Sub-objective 1D, gene expression analyses in almonds was expanded to 25 genes. Results indicate that the genes involved in sodium exclusion from roots play important roles in the rootstock tolerance to salinity stress. To study global changes in the gene expression profiles under normal- versus salt-stress conditions in almond rootstocks, RNA sequencing (RNA-seq) analysis was performed on the most salt-tolerant and the most salt-sensitive genotype. The RNA-seq analysis resulted in the identification of several genes involved in different pathways that are induced by salt treatment and also differentially expressed between the sensitive and resistant rootstocks. Some differentially expressed genes include genes encoding transporter proteins such as SOS2, NHX2, and SOS3. Also, genes coding for transmembrane receptors, ion transporters, organic solutes, and hormones were also differentially expressed. Additionally, functional validation of Prunus genes was conducted by transforming PpHKT1 and PpSOS2 into Arabidopsis mutants. The HKT1 gene is known to regulate the movement of sodium from root to shoot, and SOS2 plays a role in excluding sodium from the root into the soil. Both genes fully complemented the lost salt-tolerance function in Arabidopsis mutants. These candidate genes will be further explored to develop a link between the predicted function and their functional relevance to the physiological or the biochemical mechanisms involved in salt tolerance. Additional research in support of Sub-objective 1D, focused on determining if the Salt Overly Sensitive (SOS) pathway is conserved in spinach. Exposure of an organism to abiotic stresses such as high salinity may lead to the excessive accumulation of certain molecules, causing cellular imbalance or failure. Under a high-salinity environment, the SOS pathway is critical in extruding sodium ions from plant cells. In a model plant, Arabidopsis, the SOS pathway is comprised of SOS1, SOS2, and SOS3 proteins. The objectives of this study were to investigate if the SOS pathway functions similarly in spinach, a member of the Amaranthaceae family, and to understand the evolution of SOS pathway-related genes in Amaranthaceae species. Genome-wide identification and family analyses of SOS1-, SOS2-, and SOS3-like genes were conducted in four Amaranthaceae species. Most Amaranthaceae genes identified, exhibited orthologous relationships with Arabidopsis and/or rice. Single spinach proteins for SOS1, SOS2, and SOS3 were identified. Protein-protein interaction assays showed that SOS3 and SOS2 interact with each other; however, SOS2 and SOS1 do not, suggesting some differences in modes of action between Spinach and Arabidopsis. Spinach SOS3 gene was expressed at higher levels in roots emphasizing its more critical role in roots. Spinach SOS3 was upregulated under salinity in both leaves and roots. Research in support of Sub-objective 2A on alfalfa, focused on evaluating a population generated by crossing two salt-tolerant lines different in component traits of their salt tolerance mechanism. Several progenies in the population had significantly improved salt tolerance under high-salinity conditions (irrigation water salinity (ECiw) of 18 deciSiemens per meter (dS/m)). Next year, these selected lines will be evaluated at a higher salinity of 24 dS/m to confirm that we have successfully transferred salt-tolerance genes from both parental clones to the new progeny. In support of Sub-objective 2B, research on genome-wide association studies resulted in the identification of genetic markers associated with salinity tolerance in maize. Maize is moderately sensitive to salinity; therefore, soils or irrigation water with high salt concentrations pose a severe threat to global maize production, particularly in arid and semi-arid regions. Due to the complex nature of the trait, the most efficient approach for improving salt tolerance in modern cultivars is through the exploitation of the available naturally diverse germplasm. Here we used 399 maize lines to study genome-wide associations between salt tolerance and early vigor traits, including shoot length, shoot weight, root length, and root weight. This analysis resulted in the detection of 57 markers associated with early vigor under salinity stress and allowed us to identify candidate genes. Characterization of these genes will provide novel sources for salt tolerance in maize. In addition, this study sheds light on the understanding of the genetic mechanisms regulating salt tolerance in maize and other related crops. These findings will be useful to breeders and geneticists in developing new salt-tolerant commercial cultivars that are suitable for marginal lands high in salinity.

Accomplishments

Review Publications
Semiz, G.D., Suarez, D.L. 2019. Impact of grafting, salinity and irrigation water composition on eggplant fruit yield and ion relations. Scientific Reports. 9. https://doi.org/10.1038/s41598-019-55841-0.
Filho, J.B., Fontes, P.C., Cecon, P.R., Ferreira, J.F., McGiffen, M.E., Montgomery, J.F. 2020. Yield of potato minitubers under aeroponics, optimized for nozzle type and spray direction. HortScience. 55(1):14-22. https://doi.org/10.21273/HORTSCI13971-19.
Suddarth, S.R., Ferreira, J.F., Cavalcante, L.F., Fraga, V.S., Anderson, R.G., Halvorson, J.J., Bezerra, F.T., Medeiros, S.A., Costa, C.R., Dias, N.S. 2019. Can humic substances improve soil fertility under salt stress and drought conditions? Journal of Environmental Quality. 48(6):1605-1613. https://doi.org/10.2134/jeq2019.02.0071.
Filho, F.G., Dias, N.D., Suddarth, S.R., Ferreira, J.F., Anderson, R.G., Fernandes, C.D., Lira, R.B., Neto, M.F., Cosme, C.R. 2020. Reclaiming tropical saline-sodic soils with gypsum and cow manure. Water. 12(1). https://doi.org/10.3390/w12010057.
Wetzstein, H.Y., Janick, J., Ferreira, J.F. 2019. Germplasm release of four high-artemisinin clones of Artemisia annua L. HortScience. 54(11):2081-2082. https://doi.org/10.21273/HORTSCI14385-19.
Ferreira, J.F., Filho, J.B., Liu, X., Sandhu, D. 2020. Spinach plants favor the absorption of K+ over Na+ regardless of salinity, and may benefit from Na+ when K+ is deficient in the soil. Plants. 9(4). https://doi.org/10.3390/plants9040507.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L., Ma, J., Ghasemimianaei, A., Yang, C. 2020. Functional relationships between aboveground and belowground spinach (Spinacia oleracea L., cv. Racoon) microbiomes impacted by salinity and drought. Science of the Total Environment. 717. https://doi.org/10.1016/j.scitotenv.2020.137207.
Zhao, C., William, D., Sandhu, D. 2021. Isolation and characterization of Salt Overly Sensitive family genes in spinach. Physiologia Plantarum. 171(4):520-532. https://doi.org/10.1111/ppl.13125.
Sandhu, D., Pudussery, M.V., Kumar, R., Pallete, A., Markley, P., Bridges, W.C., Sekhon, R.S. 2020. Characterization of natural genetic variation identifies multiple genes involved in salt tolerance in maize. Functional and Integrative Genomics. 20:261-275. https://doi.org/10.1007/s10142-019-00707-x.
Thu, S.W., Rai, K.M., Sandhu, D., Rajangam, A., Balasubramanian, V.K., Palmer, R.G., Mendu, V. 2019. Mutation in a PHD-finger protein MS4 causes male sterility in soybean. Biomed Central (BMC) Plant Biology. 19. https://doi.org/10.1186/s12870-019-1979-4.
Ribeiro, A.D., Lacerda, C.F., Neves, A.L., Sousa, C.H., Braz, R.D., Oliveira, A.C., Pereira, J.M., Ferreira, J.F. 2020. Uses and losses of nitrogen by maize and cotton plants under salt stress. Archives of Agronomy and Soil Science. https://doi.org/10.1080/03650340.2020.1779228.
Braz, R.S., Lacerda, C.F., Assis, R.N., Ferreira, J.F., Oliveira, A.C., Ribeiro, A.A. 2019. Growth and physiology of maize under water salinity stress and nitrogen fertilization in two soils. Revista Brasileira de Engenharia Agricola e Ambiental. 23(12):907-913.https://doi.org/10.1590/1807-1929/agriambi.v23n12p907-913.