Research Project: Enhancing Specialty Crop Tolerance to Saline Irrigation Waters

Location: Agricultural Water Efficiency and Salinity Research Unit

2021 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 involved the evaluation of four diverse guar genotypes in a salinity experiment using a greenhouse lysimeter system. Based on the salt tolerance index (STI) for shoot biomass, root biomass, shoot length, and root length, Matador and PI 268229 were classified as salt-tolerant and PI 340261 and PI 537281 as salt-sensitive genotypes. Leaf sodium (Na) concentrations were 4- to 5.5-fold higher, and leaf chlorine (Cl) concentrations were 1.6- to 1.9-fold higher in salt-sensitive lines than salt-tolerant lines under salinity. The strong associations between the leaf potassium (K) concentrations under salinity compared to the control (K-salinity/K-control) ratio and STI for stem and root length advocate higher importance of K-salinity/K-control than total leaf K concentrations. Additional work focusing on Sub-objective 1A included evaluation of yellow passion fruit under irrigation waters of various salinities. Our results revealed that salinity did not affect the antioxidant capacity of passion fruit leaves. Leaf mineral analysis showed that an increase in irrigation water salinity resulted in significant increases in Na and Cl leaf concentrations, but levels of macro and micronutrients were maintained in the leaves. At the highest salinity levels (Electrical conductivity (ECiw) = 12 deciSiemens per meter (dS/m), plants showed a significant reduction in biomass and visual symptoms of leaf edge scorching. In additional research supporting Sub-objective 1A, 15 diverse spinach genotypes were screened for salinity tolerance in a greenhouse study. Genotypes were ranked based on their STI for the shoot and root biomasses. In addition, the relative change in proline concentration was determined in different genotypes under the control and salinity treatments. In support of Sub-objective 1C, our experiment on the effect of different priming agents on the performance of almond rootstocks under salinity is complete. The effect of four priming agents (melatonin, sodium hydrogen sulfide, salicylic acid, and zinc sulfate) compared to water (control) was tested on grafted almond plants ('Nonpareil' onto 'Cornerstone', 'Monterey' onto 'Cornerstone', 'Nonpareil' onto 'Nemaguard', and 'Monterey' onto 'Nemaguard') submitted to the irrigation water salinity (ECiw) of 3.57 dS/m compared to the control salinity of ECiw = 0.65 dS/m. Our results indicated that irrespective of the rootstock/scion combination used, there was no effect of any of the primers tested in any of the grafted plants based on trunk diameter. In general, relative trunk diameter was reduced for every rootstock/scion combination when salinity increased from 0.65 to 3.57 dS/m. Research in support of Sub-objective 1D focused on comparing RNA expression profiles in roots and leaves of two spinach varieties differing in their salt tolerance, exposed to either high- or low-salinity irrigation-water treatments. The RNA of 24 spinach samples was sequenced. RNA sequencing (RNA-seq) analyses of two spinach genotypes, 'Monstrans Viroflag' and 'Palek', subjected to high-salinity irrigation, revealed that a higher degree of differential gene expression (DEG) was caused by salinity rather than by genotype. Genotypic comparisons suggest that the low salt-tolerance index for root and shoot biomass of 'Palek', compared to 'Monstrans Viroflag', may be due to the differential expression of genes involved in water/nutrient uptake rather than to tissue salt accumulation. The 'Montrans Viroflag' genotype had better Cl exclusion than Palek and was more efficient in restricting Na from entering its roots, thus protecting leaves from ion toxicity. Further analyses resulted in the identification of several genes involved in different physiological pathways, including ion uptake, ion movement, calcium signaling, and hormonal signaling. Additionally, research to support Sub-objective 1D included the expression of candidate genes known to be involved in different components of the salt tolerance mechanism in 15 spinach genotypes. Leaf ion compositions are being determined. We are in the process of studying associations among biomass, ion compositions, proline concentrations, and gene expression. The results will help us determine the importance of different component traits in salt tolerance in spinach and related species. Additional work focusing on Sub-objective 1D involved the expression analyses of genes regulating sodium ions (Na+) and choride ions (Cl-) transport in guar. This work revealed the importance of different component traits of salinity tolerance mechanisms, such as the exclusion of Na+/Cl- from the root, sequestration of Cl- in root vacuoles, retrieval of Na+/Cl- from xylem during salinity stress, root-to-shoot Na+/Cl- translocation, and K+-Na+ homeostasis (where K+ is potassium ions). Additional research in support of Sub-objective 1D included expression analyses of 12 transporter genes in passion fruit. Six genes involved in Na+ transport and six involved in Cl- transport had higher expressions in roots compared to leaves, indicating a more critical role of roots (compared to shoots) in ion transport. Na+ exclusion from roots to soil was vital for controlling Na concentration in plant tissues, and xylem loading of Cl- and compartmentalization of Cl- into vacuoles were two important component traits regulating tissue Cl concentration under salinity stress in passion fruit. Additional research toward Sub-objective 1D focused on gene expressions for 12 genes involved in Na+ and Cl- transports using a salt-tolerant and a salt-sensitive cultivar for each of the three Solanaceae species: eggplant, tomato, and pepper. Expression analysis revealed that salinity treatment-specific induction of genes was more important than genotype-specific expression in Solanaceae. Na+ exclusion was a vital component trait for eggplant and pepper, while sequestration of Na+ into vacuoles was critical for tomato plants. The differences in relative fruit yield were not explainable by one single mechanism; rather, several component traits were critical during salinity stress. The primary component traits included the ability to exclude Na, restrict Na transport to leaves, sequester Na+ and Cl- in vacuoles, and accumulate K. The high variability for salt tolerance found in heirloom cultivars helped characterize genotypes based on component traits of salt tolerance and emphasized a potential of diverse genetic pool in generating salt-tolerant varieties in Solanaceae. In support of Sub-objective 2A, our research on the evaluation of an alfalfa F2 population between two elite parents under control and high salinity ECiw = 18 dS/m has been completed. We have identified a few highly salt-tolerant genotypes currently being tested at ECiw = 24 dS/m. Additional work focusing on Sub-objective 2A, involving screening other crop genotypes for tolerance to salt is currently under way. In FY20, 45 almond rootstocks from breeding populations were screened for salinity tolerance. These rootstocks were previously evaluated by our collaborating breeders at USDA-ARS at Davis, California and at the University of California at Davis for various traits, including performance, vigor, biotic stresses, and abiotic stresses. Our research showed that the relative change in trunk diameter varied from 0.79 to 1.11 among rootstocks under salinity. Thirteen genotypes showed a relative change of 1.0 or above, indicating their salinity tolerance. To evaluate the survivability of the genotypes, each plant was given a score of 1-6. (1-Completely dead plant; 2-Dying/mostly dead; 3-Few green leaves/new sprouts present in the plant; 4-Few dead leaves; 5-Good plants; 6-Very good plants). Out of the 45 genotypes, 16 of them had a score of 3.0 or above. For 18 rootstocks, the average scores were 2.0 or below, and in 11 of them, all the plants were dead or in the process of dying. Of the 16 rootstocks with a survival score of 3.0 or above, seven had a relative change in trunk diameter of more than 1.0, suggesting that these rootstocks outperformed others both for survivability and growth rate. We have selected 16 rootstocks with high survivability under salinity and will continue monitoring their salinity tolerance for FY22. In support of Sub-objective 2B, our research established that leaf-K concentrations under salinity compared to leaf-K under control conditions (K-salinity/K-control) was highly correlated with the salt tolerance index in both almond rootstocks and guar. These results indicate that leaf-K-salinity/leaf-K-control can be a more useful marker to select for salt-tolerant genotypes than the widely used leaf-Na/leaf-K comparison between control and salinity. In addition, the ratio of leaf proline under salinity compared to control (leaf-proline-salinity/leaf-proline-control) has an inverse correlation with salt tolerance in almond rootstocks and can be used as a biochemical marker for screening diverse genotypes.

Accomplishments
1. Genetic regulation of salinity tolerance in almond rootstocks. ARS researchers in Riverside, California, identified various salt-tolerant almond rootstocks by screening several commercial rootstocks. This work also established an understanding of genetic and biochemical mechanisms regulating the salt tolerance process in almonds. The results showed that sodium and, to a lesser extent, chloride concentration in irrigation water are the most critical toxic ions for almond rootstocks. Irrigation waters dominant in chloride or sodium induced the majority of salt-associated genes during salt stress, suggesting the importance of chloride and sodium toxicities in almonds. The RNA sequencing (RNA-seq) analysis of one tolerant and one sensitive rootstock resulted in the identification of several candidate genes involved in salinity tolerance. In a function-complementation experiment with an Arabidopsis mutant, two Prunus genes, PpHKT1 and PpSOS2, fully complemented the salt tolerance function in the Arabidopsis mutants. The information on salt tolerance of different rootstocks was shared with almond breeders, extension specialists, and commercial nurseries, and will help in developing and recommending suitable rootstocks for salinity-affected areas. The knowledge generated in this project is vital to ensure sustainable almond production in areas with scarce or expensive freshwater where the alternative water sources available (well water and municipal recycled wastewaters) are higher in salts.

2. Determinants of salinity tolerance in Solanaceae. To understand salinity tolerance mechanisms in the members of the Solanaceae family, ARS researchers in Riverside, California, evaluated 24 cultivars of tomato, eggplant, and pepper under control and saline conditions and examined them for ion composition, physiological parameters, and gene expression that may relate to salt tolerance. The cultivars were ranked based on their salt tolerance for fruit yield. One salt-tolerant and one salt-sensitive genotype were selected for each of the crops for gene expression analyses of 12 genes associated with plant salt tolerance. Expression data showed that for eggplant and pepper, Na exclusion was a vital component trait, and for tomato, sequestration of Na into the vacuole was critical. The differences in relative yield under salinity were regulated to several component traits, which was consistent with the gene expression of relevant genes. This information is of direct use to growers selecting suitable cultivars as well as for plant geneticists seeking to develop new, more salt-tolerant cultivars of these species.

Review Publications
Kaundal, R., Duhan, N., Acharya, B.R., Pudussery, M.V., Ferreira, J.F., Suarez, D.L., Sandhu, D. 2021. Transcriptional profiling of two contrasting genotypes uncovers molecular mechanisms underlying salt tolerance in alfalfa. Scientific Reports. 11. Article 5210. https://doi.org/10.1038/s41598-021-84461-w.
Sandhu, D., Kaundal, A., Acharya, B.R., Forest, T., Pudussery, M.V., Liu, X., Ferreira, J.F.S., Suarez, D.L. 2020. Linking diverse salinity responses of 14 almond rootstocks with physiological, biochemical, and genetic determinants. Scientific Reports. 10. Article 21087. https://doi.org/10.1038/s41598-020-78036-4.
Uçgun, K., Ferreira, J.F.S., Liu, X., Filho, J.B., Suarez, D.L., Lacerda, C.F., Sandhu, D. 2020. Germination and growth of spinach under potassium deficiency and irrigation with high-salinity water. Plants. 9(12). Article 1739. https://doi.org/10.3390/plants9121739.
Zhao, C., Sandhu, D., Ferreira, J.F. 2021. Transcript analysis of two spinach cultivars reveals the complexity of salt tolerance mechanisms. ACS Agricultural Science and Technology. 1(2):64-75. https://doi.org/10.1021/acsagscitech.0c00063.
Tareq, F.S., Kotha, R.R., Ferreira, J.F., Sandhu, D., Luthria, D.L. 2021. Influence of moderate-to-high salinity on the phytochemical profile of two salinity-tolerant spinach genotypes. ACS Food Science and Technology. 1 205–214. https://doi.org/10.1021/acsfoodscitech.0c00034.
Lima, R.E.M., Farias, L.F.L., Ferreira, J.F.S., Suarez, D.L., Bezerra, M.A. 2020. Translocation of photoassimilates in melon vines and fruits under salinity using 13C isotope. Scientia Horticulturae. 274. Article 109659. https://doi.org/10.1016/j.scienta.2020.109659.
Dias, N.D.S., Fernandes, C.D.S., Neto, O.N.S., Silva, C.R.S., Ferreira, J.F.S., Sá, F.V.S., Cosme, C.R., Souza, A.C.M., Oliveira, A.M., Batista, C.N.0. 2021. Potential agricultural use of reject brine from desalination plants in family farming areas. In: Taleisnik E., Lavado R.S., editors. Saline and Alkaline Soils in Latin America. Cham, Switzerland: Springer. p. 101-118. https://doi.org/10.1007/978-3-030-52592-7_5.
Dias, N.D.S., Silva, J.F., Moreno-Pizani, M.A., Lima, M.C.F., Ferreira, J.F.S., Linhares, E.L.R., Neto, O.N.S., Portela, J.C., Silva, M.R.F., Neto, M.F., Fernandes, C.D.S. 2021. Environmental, agricultural, and socioeconomic impacts of salinization to family-based irrigated agriculture in the Brazilian semiarid regions. In: Taleisnik E., Lavado R.S., editors. Saline and Alkaline Soils in Latin America. Cham, Switzerland: Springer. p. 37-48. https://doi.org/10.1007/978-3-030-52592-7_2.
Cavalcante, L.F., Bezerra, F.T.C., Souto, A.G., Bezerra, M.A.F., Lima, G.S., Gheyi, H.R., Ferreira, J.F.S., Beckmann-Cavalcante, M.Z. 2019. Biofertilizers in horticultural crops. Comunicata Science - Horticultural Journal. 10(4):415-428. https://doi.org/10.14295/cs.v10i4.3058.
Sandhu, D., Pallete, A., Pudussery, M.V., Grover, K.K. 2021. Contrasting responses of guar genotypes shed light on multiple component traits of salinity tolerance mechanisms. Agronomy. 11(6). Article 1068. https://doi.org/10.3390/agronomy11061068.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L. 2021. Influence of seasonal changes and salinity on spinach phyllosphere bacterial functional assemblage. PLoS ONE. 16(6). Article e0252242. https://doi.org/10.1371/journal.pone.0252242.
Acharya, B., Sandhu, D., Ferreira, J.F.S. 2021. Physiological, morphological, biochemical, and genetic responses of alfalfa to salinity. In: Yu, L.X., Kole, C., editors. The Alfalfa Genome. Switzerland: Springer Nature Switzerland AG. p. 145-157. https://doi.org/10.1007/978-3-030-74466-3_9.