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Research Project: Genetic Improvement of Crop Plants for Use with Low Quality Irrigation Waters: Physiological, Biochemical and Molecular Approaches


2018 Annual Report

Objective 1: Characterize plant responses to salt stress and isolate important genes associated with salt tolerance [NP301, C1, PS 1A]. Objective 2: Evaluate and develop germplasm with increased tolerance to salt stress [NP301, C1, PS 1A].

This project will focus primarily on two important California based crops, alfalfa and strawberry. Alfalfa is moderately tolerant to salinity, while strawberry is very sensitive to salt. Objective 1. We propose to dissect salt tolerance mechanism into its components and add genetic, physiological and biochemical tools to our selection approach, which earlier considered only biomass production and ion accumulation. For crops such as alfalfa that are polyploid and highly heterozygous, it is important to reduce variation by developing clonal material. We plan to use this alfalfa clonal material for the physiological, biochemical and genetic studies and study long term response to salt stress. We also propose to determine the levels of total phenolics, antioxidant capacity, quantification of photosynthetic activity, stomatal conductance, and use these biochemical/physiological indicators to characterize genotypes that are tolerant to high salt concentrations. We have identified 20 genes from the model plants such as Arabidopsis that are involved in different components of salt tolerance mechanism including i) ion efflux from root to soil, ii) ion accumulation in vacuoles, iii) retrieval of ions from xylem, iv) increased tissue tolerance to high concentrations of toxic ions and accumulation of compatible solutes. Strawberry genome has been sequenced, so homologs of Arabidopsis genes can be easily identified. For alfalfa that has not been sequenced, RNA-seq atlas can be used to design primers for PCR. We propose to study expression of these 20 genes in roots and leaves of the control and salt stressed plants using quantitative Reverse Transcription – PCR (qRT-PCR). Based on the correlation between salt tolerance index and expression profiles of genes involved in different components of salt tolerance mechanism, major genes that play important role in salinity stress will be identified. This analysis will not only help us in determining relative importance of different components of salt tolerance mechanism but will also facilitate identification of genes that can then be used to screen alfalfa and strawberry germplasms for salt tolerance. Objective 2. Expression analysis of genes will allow us to classify genotypes based on their ability to regulate different components of salt tolerance mechanism. For example, a genotype may be really good in excluding Na+ from root to soil but may not be as good in tissue tolerance to excessive sodium in the leaf tissue. We propose to make crosses between selected genotypes to combine different components of salt tolerance. Segregating populations will be subjected to physiological studies to select individual plants with enhanced salt tolerance. Gene expression analyses will be performed on these selected plants to confirm presence of multiple components of salt tolerance mechanism. This knowledge will be particularly important for breeders and geneticists in isolating genes or quantitative trait loci (QTL) important in salt tolerance. In addition, some of the salt tolerant genotypes developed and evaluated in this project may be integrated by alfalfa and strawberry breeders in their breeding programs.

Progress Report
This is the final report for this project which began in June of 2016 under National Program 301, Plant Microbial and Insect Genetic Resources, Genomics, and Genetic Improvement. The goals were substantially realized, and good results were obtained over the course of the project. This project will be replaced by 2036-13210-012-00D, “Enhancing Specialty Crop Tolerance to Saline Irrigation Waters”, which begins August 2018. Alfalfa: Over the course of the project, significant new information has been developed on the response of alfalfa under salinity stress, including establishing the threshold of the tested varieties to salinity. The roles of several genes have been established in the salinity tolerance of alfalfa and the expression patterns of these genes were used to select the best salt-tolerant genotypes to be crossed and to generate new genotypes with increased tolerance to salinity. More specifically, 1) It was established that salinity caused reduced biomass accumulation in most genotypes and increases in biomass yield showed a strong positive correlation with the salt tolerance index, height and shoot number; 2) The analysis of net photosynthetic parameters may not directly explain the poor performance of a genotype under salinity stress; 3) The accumulation of tissue sodium (Na+), per se, is not a reliable parameter to select alfalfa genotypes for salt tolerance; 4) The forage quality and mineral composition of alfalfa was not altered by salinity; and 5) Five alfalfa genotypes were selected based on different genes that represented the salt tolerance mechanisms (Na+ exclusion, Na+ sequestration in vacuoles, retrieval of Na+ from xylem, antioxidants and organic solutes synthesis and signal transduction). These genotypes were crossed in all possible combinations to produce F1 and F2 seeds that are expected to have some progenies more salt tolerant than the respective parents. One of the segregating populations is in the process of getting screened for salt tolerance. Medicago truncatula: Medicago truncatula is an ideal model to study genetic networks involved in salt tolerance in alfalfa. One important mechanism plants develop to cope with salinity is to keep the cytosolic Na+ concentration low by sequestering Na+ in vacuoles, a process facilitated by sodium–hydrogen (Na+/H+) exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) characterized in Arabidopsis. Bioinformatics analysis of the known Arabidopsis genes was performed and corresponding M. truncatula genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6 and MtNHX7) were identified. Domains that are known to be important for NHX function were conserved in five out of six MtNHX proteins. The results revealed that under salt stress, Na+ exclusion may be responsible for the relatively smaller increase in Na+ concentration as compared to chloride ions (Cl-). Expression analysis results suggest that in M. truncatula sequestering Na+ into vacuoles may not be the principle component trait of the salt tolerance mechanism and other component traits may be pivotal. The information generated in this project will be available and useful for geneticists to explore specific genes critical for salt tolerance in alfalfa in their breeding programs. Strawberry: New information was uncovered on the tolerance of strawberries to salinity when 13 strawberry genotypes were field-grown and irrigated for an extended time with saline waters of electrical conductivity (ECiw) of 7.5 deciSiemens per meter (dS/m). The main findings of this experiment are as follows: 1) Sodium did not increase in leaves, only in petioles and roots; 2) Chloride, not sodium, was the ion toxic to strawberry plants. The genotypes that were efficient in controlling chloride tissue accumulation were able to maintain biomass production (Objective 1); and 3) Levels of leaf total antioxidant capacity were also maintained under saline irrigation. The remarkable variation in response to salinity indicates that some sub-species can be potential donors of genes involved in salinity tolerance. As commercial strawberries are polyploids, it is difficult to dissect the genetic mechanisms regulating salt tolerance due to multiple copies of each gene. Hence, to link physiological and biochemical responses to genetic mechanisms, the recent salt tolerance trial was conducted on eight asexually propagated diploid strawberry (Fragaria vesca) genotypes. The results suggest that the salinity responses were genotype dependent. Strawberry genotypes maintained their highest amount of Na+ in the roots, followed by petioles and leaves indicating that strawberries have a mechanism in place to reduce the amount of Na+ from reaching leaf blades, by storing high amounts of Na+ in roots and petioles. However, chlorine is not very well regulated in strawberry. Expression analysis of several genes involved in Na+ and Cl- regulation suggested that genes involved in exclusion of ions from roots (SOS2), retrieving ions from xylem (AKT1) and chloride transporters (CLC_G and SLAH3) are critical in providing salt tolerance in strawberry. Grape: A four-year field experiment on the interaction of salinity and drought stress was completed on three purported salt tolerant grape rootstock grafted to cabernet sauvignon scion. Physiological factors including yield, sugar content, carbon (C) fixation, stomatal conductance and vine growth parameters were examined. The variety with the highest yield in control had a large yield loss with salinity, with all three having comparable yields under moderate-high salt stress. Fruit yield loss was observed at the first water stress level (80 percent of water needed to meet evapotranspiration (ET) needs of an unstressed vine). Reducing water application below vine ET resulted in a proportional decrease in yield. Under moderate salt stress, reduced water application did not further reduce yield. This research contributes to development of predictive models for plant response to abiotic stress and assists wine grape growers managing salinity and water scarcity. Spinach: To further understand mechanisms utilized by spinach plants to cope with high sodium during their growth and development, plants were irrigated with water containing increasing concentrations of sodium, chloride, and potassium. Even at 80 milliequivalents per liter (meq L-1) of Na+ and Cl- (ECiw = 10 dS/m), spinach plants had no visual toxicity symptoms and no significant effect on growth. Expression analyses of 10 genes known to play important roles in salt tolerance indicated no significant upregulation in spinach plants in any salinity treatment. The results reveal that spinach can cope with, and may benefit from, moderate level of Na+ present in irrigation water. Mineral nutrient levels, antioxidant capacity, and oxalic acid in leaves were maintained through the salinity treatments. These results indicate that salinity does not reduce spinach nutritional or anti-nutritional (oxalic acid) characteristics. Passion fruit: The yellow passion fruit is a specialty crop in Brazil and has the potential to become a high-value crop in southern California. A salinity tolerance trial displayed significant differences in growth between control and salinity treatments at 15 dS/m. However, under 6.8 dS/m salinity, fruit production was not impacted. But, if high temperatures were combined with low air humidity, there was almost no fruit yield. The results so far indicate that the yellow passion fruit could be a crop for coastal areas of southern California and could be cultivated with recycled waters of moderate salinity with no detriment to fruit production. This cultivar produces fruits 2-3 times larger than the purple sweet passion fruit exported from New Zealand. The lower Brix and pH compared to the purple passion fruit indicate that this cultivar is more suitable for industrial uses (juices, ice-cream) than the purple passion fruit. An ARS scientist in Riverside, California, collaborated with the University of California Agricultural and Natural Resources (UCANR) to hold a field day in grape, avocado, and passion fruit production under salinity. There were 25 in attendance, and most were very interested in the adaptation of the crop in southern California. Jerusalem artichoke: Jerusalem artichoke is a rich source of inulin that is considered as a soluble fiber and a prebiotic that can be used in the food industry or can be used to produce biofuels. Plants grown under salinities of irrigation water ranging from 1.2 to 12 dS/m were evaluated for their inulin content and degree of polymerization. After the extraction of tuber juice, the bagasse was analyzed and the results are as follows: 1) the bagasse is an added source of inulin that holds an additional 15 percent inulin that can be hydrolyzed by microorganisms to release sucrose and fructose for fermentation; 2) degree of polymerization of inulin was not altered by salinity; 3) tuber production was maintained up to 6.6 dS/m; and 4) due to the high hemicellulose and pectin concentration in the bagasse, addition of the enzymes xylanase and pectinase to cellulase substantially improved sugar yield from enzymatic hydrolysis compared to adding cellulase alone. These findings can lead to low-cost production of ethanol for transportation fuel with a crop that can be irrigated with recycled waters of moderate salinity and that will not compete with staple crops, such as corn, which is currently the source of raw materials for bioethanol.


Review Publications
Sandhu, D., Bhattacharyya, M.K. 2017. Transposon-based functional characterization of soybean genes. In: Nguyen, H.T., Bhattacharyya, M.K., editors. The Soybean Genome. Cham, Switzerland: Springer International Publishing. p. 183-192.
Sandhu, D., Pudussery, M.V., Kaundal, R., Suarez, D.L., Kaundal, A., Sekhon, R.S. 2018. Molecular characterization and expression analysis of the Na+/H+ exchanger gene family in Medicago truncatula. Functional and Integrative Genomics. 18(2):141-153.
Pacheco, P.A., Rodrigues, L.N., Ferreira, J.F., Gomes, A.C., Verissimo, C.J., Louvandini, H., Coasta, R.L., Katiki, L.M. 2018. Inclusion complex and nanoclusters of cyclodextrin to increase the solubility and efficacy of albendazole. Parasitology Research. 117(3):705-712.
Dias, N.S., Ferreira, J.F., Liu, X., Suarez, D.L. 2016. Jerusalem artichoke (Helianthus tuberosus, L.) maintains high inulin, tuber yield, and antioxidant capacity under moderately-saline irrigation waters. Industrial Crops and Products. 94:1009-1024.
Anderson, R.G., Ferreira, J.F., Jenkins, D.L., da Silva Dias, N., Suarez, D.L. 2017. Incorporating field wind data to improve crop evapotranspiration parameterization in heterogeneous regions. Irrigation Science. 35(6):533-547.
Huang, L., Liu, X., Wang, Z., Liang, Z., Wang, M., Liu, M., Suarez, D.L. 2017. Interactive effects of pH, EC and nitrogen on yields and nutrient absorption of rice (Oryza sativa L.). Agricultural Water Management. 194:48-57.
Bhagia, S., Ferreira, J.F., Kothari, N., Nunez, A., Liu, X., Dias, N.D., Suarez, D.L., Kumar, R., Wyman, C. 2018. Sugar yield and composition of tubers from Jerusalem Artichoke (Helianthus tuberosus) irrigated with saline waters. Biotechnology and Bioengineering. 115:1475-1484.
Zrig, A., Ferreira, J.F., Serrano, M., Valero, D., Tounekti, T., Khemira, H. 2018. Polyamines and other secondary metabolites of green-leaf and red-leaf almond rootstocks triggered in response to salinity. Pakistan Journal of Botany. 50(4):1273-1279.
Sandhu, D., Kaundal, A. 2018. Dynamics of salt tolerance: molecular perspectives. In: Gosal, S.S., Wani, S.H., editors. Biotechnologies of Crop Improvement. Volume 3. Cham, Switzerland: Springer International Publishing AG. p. 25-40.
Lacerda, C.F., Ferreira, J.F., Suarez, D.L., Freitas, E.D., Liu, X., Ribeiro, A.A. 2018. Evidence of nitrogen and potassium losses in soil columns cultivated with maize under salt stress. Revista Brasileira de Engenharia Agricola e Ambiental. 22(8):553-557.