Location: Agricultural Water Efficiency and Salinity Research Unit
2025 Annual Report
Objectives
Objective 1: Evaluate select crop germplasm under high salinity conditions to identify accessions for genetic and molecular analyses and improvement of salinity-tolerant crops.
Sub-objective 1.A: Evaluate crop germplasm for salinity tolerance using morphological traits and tissue ion analyses.
Sub-objective 1.B: Evaluate crop germplasm for salinity tolerance using gene expression analysis.
Sub-objective 1.C: Evaluate crop germplasm for salinity tolerance using biochemical parameters.
Sub-objective 1.D: Screen different almond rootstocks for quantitative responses to drought and salinity stress parameters.
Objective 2: Determine the genetic, molecular, and physiological mechanisms responsible for salinity tolerance in selected crops using genetic and molecular approaches.
Sub-objective 2.A: Decipher roles of nanomaterials in alleviating salinity stress during seed germination.
Sub-objective 2.B: Validate candidate genes for their roles in salinity tolerance.
Sub-objective 2.C: Examine the role of the SOS pathway in Prunus using protein-protein interaction (PPI) studies and In vitro reconstitution assay of the SOS pathway.
Approach
This project focuses on salinity responses and underlying mechanisms of high-value specialty crops that include almond, spinach, and guar.
In objective 1, we intend to evaluate crop germplasms for salinity tolerance by analyzing various aspects such as morphological traits, tissue ion concentration, gene expression, and biochemical parameters. By understanding how genotypes respond to salinity and identifying key ions that play a role in salt toxicity, we aim to improve tools and approaches used in salinity studies, leading to better predictions of plant responses. We will also investigate how plants maintain the balance of essential macronutrients such as potassium under elevated salinity and mineral nutrient deprivation conditions to understand the importance of different traits in salt tolerance mechanisms. Additionally, by analyzing the correlation between salinity tolerance and changes in gene expression levels, we aim to identify genes that can be used as markers for efficient screening of crop germplasm for salinity tolerance. Furthermore, we will develop suitable biochemical markers for salinity tolerance through a targeted-metabolomic approach. Lastly, we will study quantitative responses to drought and salinity stress parameters. Identifying genetic mechanisms that are common or unique during drought and salt tolerance will be the key in developing genetic material tolerant to these stresses.
Objective 2 of this project focuses on uncovering the genetic, molecular, and physiological mechanisms of salinity tolerance in selected crops using genetic and molecular methods. In our preliminary study, we demonstrated improved wheat seed germination under salinity stress by treating seeds with cerium oxide nanoparticles. The proposed project aims to study the expression differences between nanoparticle-treated and non-treated seeds during seed germination under controlled and saline conditions. By conducting transcriptome analyses, we hope to identify differentially expressed genes between the two groups, which will provide insights into the genes and pathways that regulate the enhanced effects of cerium oxide nanoparticles during seedling germination and growth. Understanding these mechanisms will enable successful wheat cultivation in salt-affected soils. We will also validate candidate genes for salinity tolerance in Prunus, Medicago, and spinach. As these species lack genetic transformation tools and single gene mutants, functional validation of genes involved in salinity tolerance is not feasible. By complementing the salinity tolerance function in Arabidopsis mutants with a particular crop gene, we will be able to validate the gene's role in salinity tolerance. These validated genes will facilitate the development of molecular markers for marker-assisted selection and can be manipulated to improve salt tolerance. Additionally, we will investigate the role of the salt overly sensitive (SOS) pathway in Prunus. Understanding how different SOS proteins interact with each other in regulating ion concentrations in plant cells will be crucial in determining plant responses to salinity stress.
Progress Report
This report documents the FY 2025 progress of project 2036-13210-013-000D, titled, “Understanding and Improving Salinity Tolerance in Specialty Crops” which began in March 2023.
In support of Sub-objective 1.A, ARS researchers in Riverside, California, evaluated the performance of the widely cultivated and commercially significant 'Nonpareil' almond variety when grafted onto two promising rootstocks developed by University of California, Davis. These rootstocks had previously demonstrated favorable traits in earlier trials. The current experiment revealed significant rootstock-dependent differences in performance, providing valuable insights that may guide breeders in selecting suitable rootstocks for cultivation in salt-affected regions.
Also as part of Sub-objective 1.A, research was conducted on alfalfa. In FY 24, nine F3 alfalfa plants demonstrating the ability to survive seawater-level salinity were identified. These plants were vegetatively propagated to produce multiple clones of each. The resulting clones were then intercrossed to generate seed populations for future field evaluation of important agronomic traits.
As well for Sub-objective 1.A, research focused on the genetic and molecular basis of salt stress tolerance in wheat. Researchers evaluated a diverse panel of 228 spring wheat accessions under control (ECiw = 1.46 deciSiemens per meter (dS/m)) and saline (ECiw = 14 dS/m) irrigation treatments using a greenhouse lysimeter system. Phenotypic screening revealed distinct differences in salinity response. Salt-tolerant lines were predominantly landraces from coastal or saline environments, while sensitive lines were mainly modern cultivars or landraces from freshwater regions. Genome-wide association studies (GWAS) identified 25 high-confidence quantitative trait loci (QTLs) linked to salinity tolerance. Candidate gene analysis revealed several salt-responsive genes, including a sodium symporter and a peptide transporter with favorable haplotypes primarily found in tolerant landraces. These genetic resources offer valuable tools for breeding salt-resilient wheat varieties for saline soils.
In support of Sub-objective 1.B, ARS researchers, provided the first evidence of epidermal bladder cells (EBCs) in spinach—a feature previously thought to be limited to halophytic species. Using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), they demonstrated for the first time that spinach EBCs accumulate sodium (Na), chloride (Cl), and potassium (K), with Na and Cl signals significantly elevated under salinity stress. Transcriptomic analysis further revealed differential expression of several ion transporter genes in EBCs compared to adjacent leaf tissue. A physiological comparison with quinoa under the same salinity conditions established that spinach tolerated and compartmentalized higher salt loads in leaves than quinoa, indicating that spinach salt tolerance and potential for food production in saline areas was previously underestimated.
Also, in support of Sub-objective 1.B, ARS researchers investigated salinity tolerance mechanisms in two tomato cultivars (‘Sanibel’ and ‘Tasti-Lee’) with contrasting responses to salinity. Plants were grown under four irrigation-water salinities (ECiw = 1.5, 4, 8, and 12 dS/m) to evaluate morphological, ionic, and gene expression responses. Salinity stress led to significant declines in biomass, plant height, root length, and leaf number, with the most severe reductions observed at the highest salinity level. Ion profiling revealed increased Na and Cl accumulation in both roots and shoots. K levels declined in aboveground tissues but increased in roots under severe stress. Gene expression analysis showed that SOS1, SOS2, and NHX1 were upregulated in roots of both cultivars, supporting their roles in Na exclusion and vacuolar sequestration. Several genes involved in Cl transport and signaling (SAL1, CLCg, NPF2.4, and NPF2.5) were downregulated in leaves. These findings underscore both common and cultivar-specific strategies for managing ion homeostasis under salt stress and offer promising targets for developing salt-tolerant tomato cultivars.
In support of Sub-objective 1.C, ARS researchers investigated the salinity responses of two contrasting maize inbred lines, C68 (salt-sensitive) and NC326 (salt-tolerant), using a controlled greenhouse lysimeter experiment. Plants were irrigated with half-strength Hoagland’s solution, and salinity was gradually increased to an electrical conductivity (EC) of 16 dS/m to minimize osmotic shock. Untargeted metabolomic analysis identified 56 key metabolites exhibiting either constitutive or salt-inducible accumulation patterns. The tolerant line, NC326, displayed higher basal levels of flavonoids and osmoprotectants, including proline and raffinose, indicating a pre-adapted antioxidant and osmotic defense system. In contrast, salt-induced accumulation of specific lipids and sterols, particularly lanosterol, was observed in both genotypes, suggesting a conserved mechanism for membrane remodeling under salt stress. These findings reveal critical biochemical and physiological adaptations that underpin salinity tolerance in maize.
Also in support of Sub-objective 1.C, ARS researchers focused on evaluating the physiological and biochemical responses of Artemisia annua to saline irrigation and potassium deficiency. This greenhouse study examined how high-salinity water, applied alone or in combination with potassium deficiency, affected plant growth, leaf biomass, and the accumulation of artemisinin and its biochemical precursors, the key compounds used in antimalarial treatment. Results indicated that A. annua is moderately tolerant to both salinity and potassium deficiency. Notably, neither stress condition caused a significant reduction in artemisinin content, suggesting the crop's resilience under suboptimal growing conditions. Given that A. annua is not only the primary commercial source of artemisinin for antimalarial drugs but also widely consumed as a medicinal tea in over 25 countries, these findings have important practical implications. Farmers may be able to irrigate Artemisia using lower-quality saline water, thereby conserving high-quality freshwater resources for staple food crops.
In support of Sub-objective 1.D, ARS researchers investigated the combined effects of drought and salinity stress on almond trees, two major environmental challenges that frequently co-occur in agricultural systems. This study aimed to better understand the physiological and developmental responses of almond trees under these combined stresses, providing insights that could enhance breeding strategies and management practices for improving resilience in water-limited and salt-affected regions.
To support Sub-objective 2.B, ARS researchers expanded their investigation into the functional role of the Medicago sativa (alfalfa) MsSOS2 gene in salinity tolerance. Detailed physiological and molecular characterization of transgenic Arabidopsis lines expressing the MsSOS2 alfalfa gene was conducted. Ion profiling of these lines under salinity stress revealed significantly lower Na accumulation and higher K retention in both roots and shoots compared to the atsos2 mutant, indicating improved ionic homeostasis. The results showed that the expression of MsSOS2 in the atsos2 background led to enhanced transcription of AtSOS1, a plasma membrane Na/H antiporter, suggesting activation of Na efflux. These findings support a model in which MsSOS2 not only restores the function of AtSOS2 but also enhances downstream ionic transport activities under salinity stress.
To support Sub-objective 2.C, ARS researchers, continued their investigation of the Salt Overly Sensitive (SOS) signaling pathway, extending their work from Prunus persica to Medicago sativa (alfalfa). The SOS pathway plays a key role in regulating intracellular sodium ion homeostasis under salt stress and involves three core components: SOS1 (a Na/H exchanger), SOS2 (a CBL-interacting protein kinase), and SOS3 (a calcineurin B-like calcium sensor). This year, researchers focused on characterizing the protein–protein interactions among the SOS1, SOS2, and SOS3 components in alfalfa. Using yeast two-hybrid assays, they demonstrated that MsSOS3 directly interacts with MsSOS2, and that MsSOS2 interacts with the C-terminal region of MsSOS1. These interactions confirm the presence of a conserved SOS signaling module in Medicago sativa, suggesting that alfalfa possesses the molecular machinery necessary for SOS pathway-mediated salinity tolerance.
Also, to support Sub-objective 2.C, ARS researchers applied a refined Plant Phase Extraction (PPE) method to generate the first comprehensive map of RNA-binding proteins (RBPs) in soybean. Using this method, over 1,000 high-confidence RBPs were identified. These included not only conserved RBPs known to participate in key aspects of RNA metabolism but also novel RBPs with unconventional RNA-binding domains whose functions in RNA biology remain to be elucidated. The study revealed both constitutively expressed RBPs and tissue-specific RBPs that are likely essential for normal growth and development. Notably, when soybean plants were exposed to high salinity, researchers uncovered an expanded set of RBPs that appear to be specifically involved in the plant's salinity stress response. These findings offer a new perspective on how RBPs regulate gene expression under stress at the post-transcriptional level. This work establishes a foundation for understanding RBP–RNA interactions in soybean and highlights the critical role of RBPs in mediating stress adaptation. It adds a novel molecular layer to the salinity tolerance framework and provides valuable targets for future functional studies and stress-resilience breeding strategies in legume crops.
Accomplishments
1. Spinach leaves found to contain specialized bladder cells that accumulate sodium, chloride and potassium. Spinach is a widely consumed leafy vegetable considered to be salt-sensitive. Salinity is a major constraint in agricultural production, especially in arid and semiarid regions with limited access to high-quality water. ARS researchers in Riverside, California, discovered for the first time that spinach produces epidermal bladder cells (EBCs) — salt-accumulating structures previously thought to exist only in salt-tolerant plants. These structures were identified in all 13 spinach cultivars evaluated. Using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), researchers found that spinach EBCs accumulate high concentrations of sodium (Na), chloride (Cl), and potassium (K), particularly under salinity stress. Importantly, spinach accumulated over four times more Na than quinoa, a well-known salt-tolerant crop, while maintaining leaf integrity and function. This suggests that spinach has high tissue-level salt tolerance, not previously recognized. The study also revealed that several salt-related ion transporter genes are differentially expressed in EBCs compared to leaf tissue lacking EBCs, offering new insights into spinach’s molecular response to salinity. Furthermore, a new experiment comparing spinach and quinoa under multiple water-salinity levels showed comparable growth and physiological performance, challenging the traditional classification of spinach as salt-sensitive. This research highlights a previously unknown trait in spinach that may be leveraged to develop cultivars suitable for saline agriculture, enabling farmers to produce food with marginal water resources and reducing competition for freshwater in the production of specialty crops.
Review Publications
Vieira, C.B., Silva, G.H.M.C., de Almeida, B.G., Pessoa, L.G.M., Freire, F.J., de Souza Junior, V.S., de Melo, H.F., de Lima, L.G.G., do Nascimento Paiva, R.F., Ferreira, J.F.S., dos Santos Freire, M.B.G. 2025. Saturated hydraulic conductivity of nine soils according to water quality, soil texture, and clay mineralogy. Agronomy. 15(4). Article 864. https://doi.org/10.3390/agronomy15040864.
Acharya, B., Gill, S.P., Kaundal, A., Sandhu, D. 2024. Strategies for combating plant salinity stress: The potential of plant growth-promoting microorganisms. Frontiers in Plant Science. 15. Article 1406913. https://doi.org/10.3389/fpls.2024.1406913.
Scudiero, E., Schmidt, M.P., Skaggs, T.H., Ferreira, J.F., Zaccaria, D., Pourreza, A., Corwin, D.L. 2025. Apparent soil electrical conductivity and gamma-ray spectrometry to map particle size fraction in micro-irrigated citrus orchards in California. Frontiers in Plant Science. 16. Article 1512598. https://doi.org/10.3389/fpls.2025.1512598.
Pessoa, U.C., de Franca e Silva, E.F., de Oliveira, T.F., Ferreira, J.F., Rodrigues de Souza, E., Rolim, M.M., Oliveira da Silva, A., Santos Junior, J.A. 2025. Cilantro photosynthetic parameters in response to different flows of nutrient solutions prepared with brackish waters dominant in Na+, Cl-, or Ca2+. Water. 17(11). Article 1640. https://doi.org/10.3390/w17111640.
Gudi, S., Gill, H., Collins, S., Singh, J., Sandhu, D., Sehgal, S., Upinder, G., Gupta, R. 2025. Association analysis identified superior haplotypes for improved salt stress tolerance in wheat (Triticum aestivum L.). Plant Stress. 16. Article 100900. https://doi.org/10.1016/j.stress.2025.100900.
Gudi, S., Gill, H.S., Collins, S., Singh, J., Sandhu, D., Sehgal, S., Upinder, G., Gupta, R. 2025. Phenotypic data related to seedling traits of hexaploid spring wheat panel evaluated under salinity. Data in Brief. 61.Article 111801. https://doi.org/10.1016/j.dib.2025.111801.