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 Agricultural Water Efficiency and Salinity Research Unit: Riverside, CA
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ARS Home Pacific West Area Riverside, California Agricultural Water Efficiency and Salinity Research Unit Research





- Sandhu, Devinder
- Ferreira, Jorge
- 2023
- Publications
- Water Availability and Watershed Management (211)
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Understanding and Improving Salinity Tolerance in Specialty Crops **Location:
                            Agricultural Water Efficiency and Salinity Research Unit** 2023 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 is the first annual report for project 2036-13210-013-000D, “Understanding and Improving Salinity Tolerance in Specialty Crops”, which began March 15, 2023.

In support of Sub-objective 1A, researchers in Riverside, California, evaluated a selected set of 33 elite hybrids derived from almond breeding programs conducted by collaborating breeders. These hybrids were assessed for their salinity tolerance, building upon previous evaluations of almond rootstock breeding lines for various traits such as performance, vigor, and stress resistance. The hybrids were classified based on the relative change in trunk diameter between the salinity treatment and the control. Trunk diameter changes ranged from 0.83 to 1.10, indicating varying responses to salinity stress. Significant correlations were found between the proline ratio (under salinity compared to the control) and shoot Sodium concentration, as well as between the proline ratio and shoot Chloride concentration, indicating that the proline ratio could serve as a valuable biochemical marker for screening almond rootstocks in terms of their salinity tolerance. The salinity screening process allowed for the identification of the most suitable parental combinations that can be utilized to develop hybrids with enhanced salinity tolerance.

In support of Sub-objective 1B, tissue samples were collected from Prunus rootstocks, and RNA isolation was performed. Primers were designed for key genes involved in salinity tolerance in plants. Currently, we are conducting Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis to investigate the gene expression patterns under both control and salinity conditions in different Prunus breeding lines. This analysis aims to deepen our understanding of the relationship between gene expression and salinity tolerance across various genotypes. Furthermore, it will provide insights into the significance of different component traits within the salinity tolerance mechanism across different genotypes. These findings will contribute to the development of a comprehensive understanding of salinity tolerance in Prunus and facilitate the identification of crucial genes and traits for breeding improved salt-tolerant varieties.

For Sub-objective 2A, ARS researchers have embarked on a study investigating the potential benefits of cerium oxide (CeO2) nanoparticles on wheat seed germination under salinity conditions. The initial phase of the research aimed to identify the optimal properties and concentration of nanoceria for enhancing seed germination in the presence of salinity stress. The initial results determined that treating wheat seeds with 500 mg L-1 of cerium oxide nanoparticles for a duration of 24 hours yielded the most favorable results in terms of germination. Researchers are now conducting a time-course experiment, which will compare seed germination with and without nanoparticle treatment under both control conditions and salinity stress. By monitoring the germination progress over time, researchers aim to evaluate whether the nanoparticles are capable of mitigating the detrimental effects caused by salinity stress. Such findings could have implications for developing novel strategies to improve crop establishment and productivity in salt-affected environments.

To support Sub-objective 2B, ARS researchers have taken a crucial step in validating the MsSOS2 gene derived from alfalfa. This gene is of great interest due to its potential involvement in salinity tolerance. The first stage of the validation process involved cloning the MsSOS2 gene into a vector, which serves as a carrier for introducing the gene into another organism. With the successful construction of the gene vector, the researchers are currently focused on the next phase: transforming the vector carrying the MsSOS2 gene into Arabidopsis, specifically the atsos2 mutant. The atsos2 mutant, lacking the native SOS2 gene, provides an ideal background for testing the function of the MsSOS2 gene. Once successfully transformed, the plants will undergo careful analysis and observation to assess whether the MsSOS2 gene effectively complements the salt tolerance function in the atsos2 mutant. This validation step is crucial in establishing the role of the MsSOS2 gene in enhancing salinity tolerance and further understanding its mechanism of action.

In support of Sub-objective 2C, ARS researchers in Riverside, California, have made significant advancements by developing a groundbreaking plant phase extraction (PPE) method for the isolation of RNA binding proteins (RBPs) from plant tissues. This innovative approach has demonstrated numerous technical advantages compared to existing methods, setting it apart in the field. While several techniques exist for identifying RBPs in human cell lines, mouse brains, and bacteria, none have successfully uncovered RBPs in plants until now. By leveraging the power of PPE, the researchers achieved comprehensive identification of RBPs in plants, providing a deeper understanding of the intricate dynamics between RBPs and RNA in various developmental processes and responses to environmental stimuli. This breakthrough has opened new avenues to explore and characterize novel RBPs, shedding light on their functions under different physiological and stress conditions. These significant findings hold tremendous value for plant biologists and geneticists as they unravel the complex mechanisms underlying RBP-mediated regulation of gene expression in plants. Such knowledge will prove crucial for plant breeders in their pursuit of developing elite genetic material capable of withstanding diverse stresses, including the challenges posed by salinity stress. **Accomplishments** **Review Publications**
                            *Zhang, Y., Xu, Y., Skaggs, T.H., Ferreira, J.F., Chen, X., Sandhu, D. 2023. Plant phase extraction: A method for enhanced discovery of the RNA-binding proteome and its dynamics in plants. The Plant Cell. 35(8):2750-2772. https://doi.org/10.1093/plcell/koad124.*


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