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


2017 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
Alfalfa. We used 12 alfalfa genotypes previously selected for different levels of salt tolerance, difference in tissue sodium (Na+) and chlorine (Cl) accumulation, and potassium (K) +/Na+ ratio. These cloned genotypes were evaluated for various physiological, biochemical, and genetic traits under salt stress (EC 16.6 dS m-1). Salinity caused reduced biomass accumulation in most genotypes. Biomass showed a strong positive correlation with salt tolerance index, height and shoot number. Individual genotypes were selected based on tissue Na+ and Cl- accumulation, high tissue tolerance of Na+ and Cl-, high Na+/K+ ratio, and biomass yield. Our analysis of net photosynthesis, transpiration rates and stomatal conductance demonstrated that gas exchange parameters may not directly explain the poor performance under salinity stress. Most genotypes had increased oxygen radical absorbance capacity and total phenolic values under salinity treatment. These results fulfill objective 1 of this project by increasing the knowledge on how biochemical markers and agronomic performance of alfalfa under salinity can aid in the selection of genotypes with higher tolerance to salinity. To establish a relationship between genes involved in salt stress and phenotypic differences among genotypes, we evaluated expression of candidate genes known to be involved in different components of the salt tolerance mechanism. We were able to determine which components of the salt tolerance mechanism (Na+ exclusion, Na+ sequestration in vacuoles, retrieval of Na+ from xylem, antioxidants and organic solutes synthesis and signal transduction) were important in a particular genotype during salinity stress. Combining attributes from different genotypes by crossing and selecting for lines with multiple components of salt tolerance mechanism may help in developing alfalfa lines with enhanced salt tolerance. We have selected five genotypes representing different components of the salt tolerance mechanism for genetic crossing. We have generated crosses between two parents to produce F1 and F2 seeds. This fulfills objective 2 of this project and the information generated will be very valuable to alfalfa breeders to develop salt tolerant varieties. Medicago truncatula. Medicago truncatula is an ideal model legume and can be used to study genetic networks involved in salt tolerance in alfalfa. One important mechanism plants develop to cope with salinity is keeping 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) identified and characterized in Arabidopsis. We performed bioinformatic analysis of the known Arabidopsis genes and identified corresponding Medicago truncatula genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6 and MtNHX7). Domains that are known to be important for NHX function were conserved in five out of six MtNHX proteins. Our results revealed that under salt stress, Na+ exclusion may be responsible for the relatively smaller increase in Na+ concentration as compared to 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. This study is in line with objective 1 of this project. The information generated will be useful to geneticists to characterize genes critical for salt tolerance in alfalfa. Strawberry. Twelve wild type accessions and one traditional fruit bearing cultivar, Lassen, were evaluated at moderately-high salinity of irrigation with waters of electrical conductivity (ECiw) of 0.7 and 7.5 deci Siemens per meter (dS/m). At 7.5 dS/m, number of runners and shoot weight in Lassen reduced by 59 and 63%, respectively, while root dry weight was reduced by only 5%. The other 12 accessions had very different responses to salinity and the reduction in shoot dry weight and runner number varied greatly. However, this remarkable variation in response to salinity indicates that some sub-species can be potential donors of genes involved in salinity tolerance. For instance, within the species Fragaria chiloensis, we found striking differences in salinity response with F. chiloensis sub-species c.f.c. losing only 20% of their biomass while F. chiloensis sp. Lucida CA losing 65% by the end of the experiment. These findings suggest that the above mentioned species can be mined for differences in salinity response at their sub-species level. Although the antioxidant activity of shoots and roots of most accessions were very high, the differences in tissue antioxidant capacity at the end of the experiment could not explain their susceptibility or tolerance to salinity. Plant tissues will be analyzed for their concentration of mineral nutrients to try to explain their tolerance/susceptibility to salinity. We recently acquired 20 different genotypes of diploid strawberry (Fragaria vesca) and propagated these in the greenhouse. The genotypes that produced runners were asexually propagated. For the remaining genotypes, seeds were harvested for future salinity studies. These genotypes will be compared for their salt tolerance and agronomic, physiological, biochemical and genetic parameters in 2018. Grape. We are in the fourth year of a field experiment on the interaction of salinity and drought stress on three purported salt tolerant grape rootstock grafted to cabernet sauvignon scion. We examined physiological factors including yield, sugar content, carbon (C) fixation, stomatal conductance and vine growth parameters. 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% 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. Almond. In recent years many producers of perennial crops in California and elsewhere have faced fresh water scarcity requiring irrigation with lower quality ground water with limited pumping capacity, hence both water and salt stress. We completed a two year outdoor experiment on the physiological response of two grafted almond scions to three levels of salinity stress, 2 levels of water stress and treatments where both stresses were imposed in all combinations. As trees were not bearing fruit, response was related to changes in trunk diameter, photosynthesis, transpiration, leaf water potential, leaf area index and leaf water potential. This research is part of an effort to evaluate previously developed predictive models for plant response to abiotic stress and to provide almond growers with practical advice on management options and likely outcomes. 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. NaCl treatments of 2, 20, 40, and 80 milli equivalant liter (meq L) meq L-1 were tested in combination with 3, 5, and 7 meq L-1 of K. Biomass data and visual observations indicated that, even at 80 meq L-1 of Na+ and Cl-, spinach plants had no visual toxicity symptoms and their growth was not significantly affected after three weeks under salinity. Expression analyses of 10 genes known to play important role in salt tolerance in Arabidopsis indicated no significant upregulation in spinach plants in any salinity treatment. These observations suggest that 80 meq L-1 of Na+ and Cl- may not be critical to affect plant growth or gene expression during salt stress. Our results clearly show that spinach can cope with, and may actually benefit from, moderate level of Na+ present in irrigation water. Mineral nutrient levels and oxalic acid in leaves were maintained through the salinity treatments showing that salinity does not reduce spinach nutritional or anti-nutritional (oxalic acid) characteristics. Hence, we have met objective 1 of this project. Figuring out the mechanism by which Na+ becomes beneficial for spinach growth may help geneticists to take advantage of this knowledge to develop salt tolerant lines in other plant species. Passion fruit. Our first year of experimentation with a Brazilian yellow passion fruit tested for growth, development and fruit yield under salinity of irrigation water up to 6.8 dS/m resulted in no significant difference in plant growth or fruit yield. This indicates that the Brazilian yellow passion fruit may be a new fruit crop for Southern California that can be grown with recycled waters with no detriment to fruit production. This cultivar has fruits 2-3 times larger than the purple sweet passion fruit. The juice characteristics measured also were not altered by salinity. The lower Brix and potential Hydrogen (pH) compared to the purple passion fruit indicate that this cultivar is more suitable for industrial (juices, ice-cream) than the purple passion fruit imported from New Zealand. The cultivar shows a good adaptation to the climate of Southern California. Currently, the plants are being tested at a much higher salinity to verify the effects on plant growth, development and fruit production.

1. A comprehensive approach to develop salt tolerant alfalfa. Alfalfa is a major forage crop that is vital to the dairy industry. California is the leader in alfalfa production in the U.S., but due to high water consumption and competition for water with the urban sector as well as with high value specialty crops, the future of alfalfa in semiarid areas, including California, is questionable. However, developing salt tolerant alfalfa will enable farmers to use recycled waters and brackish ground waters for irrigation. ARS researchers in Riverside, California, worked on understanding genetic/biochemical determinant of salt tolerance in alfalfa and used this information to develop markers for selecting salt tolerant genotypes. Using expression analyses of candidate genes during salinity stress, researchers were able to establish importance of different components of the salt tolerance mechanism in different genotypes. Combining attributes from different genotypes by crossing and selecting for lines with multiple components of salt tolerance mechanism may help in developing alfalfa lines for growers that are commercially viable on low quality waters for irrigation.

Review Publications
Sandhu, D., Cornacchione, M.V., Ferreira, J.F., Suarez, D.L. 2017. Variable salinity responses of 12 alfalfa genotypes and comparative expression analyses of salt-response genes. Scientific Reports. 7:42958. doi: 10.1038/srep42958.
Coleman, Z., Boelter, J., Espinosa, K., Goggi, S., Palmer, R.G., Sandhu, D. 2017. Isolation and characterization of the aconitate hydratase 4 (Aco4) gene from soybean. Canadian Journal of Plant Science. 97(4):648-691. doi: 10.1139/CJPS-2016-0363.
Ors, S., Suarez, D.L. 2017. Spinach biomass yield and physiological response to interactive salinity and water stress. Agricultural Water Management. 190:31-41. doi: 10.1016/j.agwat.2017.05.003.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L. 2016. Seasonal induced changes in spinach rhizosphere microbial community structure with varying salinity and drought. Science of the Total Environment. 579:1485-1495. doi: 10.1016/j.scitotenv.2016.11.151.
Cornacchione, M.V., Suarez, D.L. 2016. Evaluation of alfalfa (Medicago sativa L.) populations' response to salinity stress. Crop Science. 57:137-150. doi: 10.2135/cropsci2016.05.0371.
Dias, N.S., Blanco, F.F., Souza, E.R., Ferreira, J.F.S., Neto, O.N.S., Queirzo, Í.S.R. 2016. Efeitos dos sais na planta e tolerância das culturas à salinidade (Salinity effects on plants and tolerance of crops to salinity). In: Gheyi, H. R., Dias, N.D.S., Lacerda, C.F.D., Gomes Filho, E., editors. Manejo da Salinidade na Agricultura: Estudos Bàsicos e Aplicados. Fortaleza, CE, Brazil: Instituto Nacional em Ciência e Tecnologia em Salinidade (National Institute of Science and Technology Applied to Salinity). p. 151-162.
Katiki, L.M., Barbieri, A.M., Araujo, R.C., Verissimo, C.J., Louvandini, H., Ferreira, J.F. 2017. Synergistic interaction of ten essential oils against Haemonchus contortus in vitro. Veterinary Parasitology. 243(2017):47-51. doi: 10.1016/j.vetpar.2017.06.008.
Liu, S., Ferreira, J.F., Liu, L., Yuwei, T., Tian, D., Liu, Z., Tian, N. 2017. Isolation of dihydroatemisinic acid from the artemisia annua l. by-product by combining Ultrasound-assisted extraction with response surface methodology. Chemical and Pharmaceutical Bulletin. 65(8):746-753. doi: 10.1248/cpb.c17-00192.
Lima, B.L., Lacerda, C.F., Neto, M.F., Ferreira, J.F., Bezerra, A.M., Marques, E.C. 2017. Physiological and ionic changes in dwarf coconut seedlings irrigated with saline water. Revista Brasileira de Engenharia Agricola e Ambiental. 21(2):122-127. doi: 10.1590/1807-1929/agriambi.v21n2p122-127.