Research Project: Enhancing Specialty Crop Tolerance to Saline Irrigation Waters

Location: Agricultural Water Efficiency and Salinity Research Unit

2022 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 investigated the effect of saline water and potassium availability on the phenolic profile and soluble sugars of spinach cultivars ‘Raccoon’ and ‘Gazelle’ by comparing colorimetric and chromatographic assays for the quantification of phenolic compounds. Results revealed that salinity reduced the antioxidant capacity of spinach leaves for ‘Raccoon’ but slightly increased total phenolic concentrations in leaves of ‘Gazelle’. Although salinity reduced the concentration of phenolics, it did not alter the phenolic signature in spinach leaves and 13 polyphenols were quantified by chromatography. Also, glucose was the most abundant sugar (50%) in leaves of both cultivars. Additional work focusing on Sub-objective 1A involved screening selected genotypes of three Solanaceae species, eggplant, tomato, and pepper for salinity tolerance. From our previous experiment, where we screened eight cultivars of each Solanaceae species for salinity tolerance, we identified cultivars with the greatest and least salt tolerance. We conducted a new field experiment with a non-saline control, three salinity levels, and two water types with three replicates. Three treatments varied in salinity (3, 4.5, and 6 decisiemens per meter (dS m-1)) but contained primarily sodium (Na) and chlorine (Cl) salts, and three others also varied in salinity but were predominantly sodium sulfate with lesser amounts of Cl. We are currently analyzing the data. These experiments will provide specific information about factors limiting salt tolerance of these crops and genes controlling these factors, such as chloride ions (Cl-) exclusion. Also, in support of Sub-objective 1A, our research continues to investigate genetic mechanisms regulating salinity tolerance in Prunus. To understand the interaction between a rootstock and scion in mediating salinity stress, we evaluated grafted plants. Based on biochemical and genetic analyses in our previous study, we selected the seven best salt-tolerant rootstocks. Plants of seven selected rootstocks, grafted with ‘Nonpareil’ and ‘Monterey’ as scions, were field evaluated under irrigation waters with electrical conductivities (ECiw) = 1.4 (control) and ECiw = 3.5 (salinity treatment). Survival rates and stem girth readings were recorded. The ‘Hansen 536’ rootstock performed best under saline conditions while ‘Nemaguard’ was the worst performer. Of the scions, ‘Monterey’ performed better than ‘Nonpareil’ under salinity. Understanding the roles played by sodium ions (Na+) or Cl- in salt toxicity and the genetic interactions between rootstock and scion will help refine tools and techniques for studying salt tolerance, which in turn will enhance subsequent breeding efforts for almonds rootstocks adapted to irrigation with saline waters. Research in support of Sub-objective 1D, focused on functional complementation of the peach Salt Overly Sensitive 2 (PpSOS2) gene from the almond rootstock ‘Nemaguard’ in the Arabidopsis thaliana atsos2 mutant. Transgenic lines of PpSOS2 showed significantly higher germination and survival rates, and greater dry weights than the atsos2 mutant under salinity treatment. The atsos2 mutant displayed inhibition of primary and lateral roots under salinity. Root growth inhibition was restored by PpSOS2 complementation. The transgenic lines showed a significant decrease in electrolyte leakage compared to atsos2 under salinity. These observations suggest that PpSOS2 modulates and restores salt tolerance in the atsos2 mutant. Also, the salt overly sensitive (SOS) pathway is conserved in Prunus, suggesting that exclusion of Na+ is an important trait for salt tolerance. Additional research toward Sub-objective 1D, focused on comparing RNA expression profiles in roots and leaves of two guar genotypes differing in their salt tolerance and exposed to either high- or low-salinity irrigation-water treatments. RNA was sequenced for 24 guar samples. RNA-Seq analyses of two genotypes, 'Matador' and 'PI340261', subjected to high-salinity irrigation, revealed that a higher degree of differential gene expression (DEG) was caused by the salinity treatment than by genotype. Differentially expressed genes associated with stress-signaling pathways, transporters, chromatin remodeling, microRNA biogenesis, and translational machinery play critical roles in guar salinity tolerance. This study revealed the importance of various biological pathways during salinity stress and identified several candidate genes to use for developing salt-tolerant guar genotypes adapted to cultivation in marginal soils with moderate to high salinity or for irrigation with degraded waters of moderate to high salinity. In support of Sub-objective 2A, our research continued to screen an F2 population of two salt-tolerant alfalfa parents for salinity tolerance. In our initial screen with saline water of ECiw = 18 dS m-1 we selected 24 salt-tolerant segregants. These 24 genotypes were further evaluated at ECiw = 27 dS m-1. Few genotypes survived these high levels of salinity without showing symptoms of ion toxicity, indicating these survivors can be used by breeding programs to develop alfalfa genotypes for salt-affected regions in the United States.

Accomplishments
1. Spinach has efficient mechanisms for salt tolerance. ARS researchers in Riverside, California, evaluated the relationship between sodium and potassium in spinach under salinity. This work established that spinach plant substituted potassium (K) for sodium (Na) in a similar proportion but favored K over Na when K was abundant in the growing medium. When K was provided at 20 and 40 times less than the recommended dose, spinach plants supported K levels for growth and biomass accumulation, regardless of the salinity level applied. Although Na is not considered an essential nutrient for glycophytic plants, this investigation showed that Na promoted plant growth, mainly when potassium was low in the growth medium. These results suggest growing spinach with recycled waters of low to moderate salinity does not pose a risk to spinach production. While salinity reduced the antioxidant capacity of spinach, it produced enough flavonoids and sugars to remain a nutritious crop under saline conditions. Additionally, genetic analyses showed that the Salt Overly Sensitive (SOS) pathway, critical in extruding sodium from plant cells, is conserved in spinach. Protein-protein interaction assays showed that SOS3 and SOS2 interact with each other; however, SOS2 and SOS1 do not, suggesting differences in modes of action between Spinach and the model plant Arabidopsis. The knowledge generated in this project is important to spinach growers experiencing high soil or water salinities and plant geneticists seeking to develop new, more salt-tolerant spinach cultivars.

Review Publications
Lima, B.L.C., Silva, E.F.F., Zonta, J.H., Neto, C.P.C.T., Lacerda, C.F., Ferreira, J.F.S., Cruz, F.J.R. 2021. Irrigation with wastewater and K fertilization ensure the yield and quality of coloured cotton in a semiarid climate. Agronomy. 11(12). Article 2370. https://doi.org/10.3390/agronomy11122370.
Liu, X., Suarez, D.L. 2021. Lima bean growth, leaf stomatal and nonstomatal limitations to photosynthesis, and 13C discrimination in response to saline irrigation. Journal of the American Society for Horticultural Science. 146(2):132-144. https://doi.org/10.21273/JASHS04996-20.
Kaundal, A., Sandhu, D., Singh, V., Duenas, M., Acharya, B.R., Nelson, B., Ferreira, J.F.S., Litt, A. 2022. Transgenic expression of Prunus persica Salt Overly Sensitive 2 (PpSOS2) in atsos2 mutant imparts salt tolerance in Arabidopsis. ACS Agricultural Science and Technology. 2(1):153-164. https://doi.org/10.1021/acsagscitech.1c00276.
Acharya, B.R., Sandhu, D., Dueñas, C., Dueñas, M., Pudussery, M.V., Kaundal, A., Ferreira, J.F., Suarez, D.L., Skaggs, T.H. 2022. Morphological, physiological, biochemical, and transcriptome studies reveal the importance of transporters and stress signaling pathways during salinity stress in Prunus. Scientific Reports. 12. Article 1274. https://doi.org/10.1038/s41598-022-05202-1.
Huang, L., Liu, Y., Ferreira, J.F.S., Wang, M., Na, J., Huang, J., Liang, Z. 2021. Long-term combined effects of tillage and rice cultivation with phosphogypsum or farmyard manure on the concentration of salts, minerals, and heavy metals of saline-sodic paddy fields in Northeast China. Soil & Tillage Research. 215. Article 105222. https://doi.org/10.1016/j.still.2021.105222.
Acharya, B.R., Sandhu, D., Dueñas, C., Ferreira, J.F., Grover, K.K. 2022. Deciphering molecular mechanisms involved in salinity tolerance in guar (Cyamopsis tetragonoloba (L.) Taub.) using transcriptome analyses. Plants. 11(3). Article 291. https://doi.org/10.3390/plants11030291.
Cavalcante, E.S., Lacerda, C.F., Mesquita, R.O., Melo, A.S.D., Ferreira, J.F.S., Teixeira, A.D.S., Lima, S.C.R.V., Sales, J.R.D.S., Silva, J.D.S., Gheyi, H.R. 2022. Supplemental irrigation with brackish water improves carbon assimilation and water use efficiency in maize under tropical dryland conditions. Agriculture. 12(4). Article 544. https://doi.org/10.3390/agriculture12040544.
Suarez, D.L., Celis, N., Ferreira, J.F.S., Reynolds, T., Sandhu, D. 2021. Linking genetic determinants with salinity tolerance and ion relationships in eggplant, tomato and pepper. Scientific Reports. 11. Article 16298. https://doi.org/10.1038/s41598-021-95506-5.
Chen, Y., Liu, S., Ferreira, J.F., Xiao, L., Gu, M., Luo, Y., Zhang, T., Zhang, X., Liu, Z., Huang, J., Tian, N. 2021. Development and application of a fast gas chromatographic method offer new insights into L-theanine production regulation in Camellia sinensis L. Journal of Agricultural and Food Chemistry. 69(37):11142-11150. https://doi.org/10.1021/acs.jafc.1c04093.
Jin, K., Tian, N., Ferreira, J.F.S., Sandhu, D., Xiao, L., Gu, M., Luo, Y., Zhang, X., Liu, G., Liu, Z., Huang, J., Liu, S. 2022. Comparative transcriptome analysis of Agrobacterium tumefaciens reveals the molecular basis for the recalcitrant genetic transformation of Camellia sinensis L. Biomolecules. 12(5). Article 688. https://doi.org/10.3390/biom12050688.
Pacheco, P.A., Louvandini, H., Giglioti, R., Wedy, B.C.R., Ribeiro, J.C., Verissimo, C.J., Ferreira, J.F.S., Amarante, A.F.T.D., Katiki, L.M. 2022. Phytochemical modulation of P-Glycoprotein and its gene expression in an ivermectin-resistant Haemonchus contortus isolate in vitro. Veterinary Parasitology. 305. Article 109713. https://doi.org/10.1016/j.vetpar.2022.109713.
Kucukyumuk, Z., Suarez, D.L. 2021. The effect of selenium on salinity stress and selenate - sulfate comparison in kale. Journal of Plant Nutrition. 44(20):2996-3004. https://doi.org/10.1080/01904167.2021.1936034.