Identification and characterization of genes from wild halophytic rice (Porteresia coarctata), for using in development of salt tolerant rice

Abstract

Soil salinity is a major abiotic constraint affecting rice cultivation in coastal and estuarine regions, where rising sea levels and irrigation-induced salinization increasingly threaten global food security. The halophytic wild rice species Oryza coarctata—the only naturally salt-tolerant species in the genus Oryza, which can set rice-like grains—offers a promising genetic reservoir for improving salt stress tolerance in cultivated rice (Oryza sativa). This study presents a comprehensive functional and molecular characterization of O. coarctata, along with its application in wide hybridization and genetic engineering strategies aimed at enhancing salt tolerance in rice. Oryza coarctata exhibits exceptional salt tolerance supported by unique physiological and anatomical features. Its leaf structure includes deep adaxial invaginations, multiple vascular bundles per ridge, and salt-secreting hairs, while its roots possess a thickened exodermis, large xylem vessel, and well-developed aerenchyma—traits that aid in ionic regulation and survival in saline, waterlogged conditions. Notably, O. coarctata reduced electrical conductivity (EC) of saline hydroponic media by 2.77–8.51 dS/m across 100–300 mM NaCl, demonstrating a desalination ability absent in salt-sensitive rice varieties. Co-cultivation of O. coarctata with BRRI Dhan28 improved the latter’s growth and yield under 100 mM salt stress, increasing yields from 34% to 67%. Gas exchange measurements in O. coarctata showed only modest declines in photosynthesis at 100–300 mM NaCl (14–26% reduction in CO₂ assimilation), with strong light response (r = 0.932) and moderate Jₘₐₓ reduction (19–32%, p < 0.05). Unlike O. sativa, which fails at 80 mM salt, O. coarctata maintains high photosynthetic efficiency and survival under extreme salinity. These traits underscore its potential for use in salt-affected rice ecosystems through ecological facilitation and genetic improvement strategies. To explore gene transfer from the tetraploid O. coarctata (4n=2x=48) into rice, a wide hybridization approach was adopted using a tetraploid O. sativa (var. Latisail 4n) as the maternal parent. This approach is referred to as the bulbosum technique, where chromosomal loss occurs resulting in half of the original chromosomes (2x). Despite high genomic divergence, two partial 18 hybrids were recovered out of 1,191 pollinated spikelets, indicating successful albeit low-frequency gene introgression. These partial hybrids exhibited intermediate phenotypes, including fibrous and tap root systems, variable seed morphology, and in some cases, the absence of leaf midribs—a signature trait of O. coarctata. Molecular analysis confirmed that the hybrids carried specific chromosomal segments from O. coarctata, particularly on chromosomes 3 and 12. Partial hybrid lines A2-06-01, A2-10-01, and B2-03-01 exhibited significantly higher chlorophyll content than diploid and tetraploid O. sativa following 100 mM salt stress. Lines B-02-01 and B-03-01 showed significantly greater plant height compared to O. sativa (2n), while A2-06-01 and A2-10-01 also demonstrated a significantly higher tiller number than O. sativa (2n). A significant advancement of this work was the development of O. coarctata-specific SSR markers to facilitate molecular screening of hybrids lines. Initially, known SSR markers from the Gramene database showed limited polymorphism between O. sativa and O. coarctata. Subsequently, 90 markers from the O. officinalis genome (CC genome), developed under the OMAP project, were tested. Of these, 19 markers were optimized to uniquely detect O. coarctata alleles across most chromosomes (except 5 and 8). In addition, seven new SSR markers were developed from O. coarctata genomic sequences. These 26 markers together form a robust toolkit for identifying and validating introgressions in future breeding programs. To directly assess the functional contribution of O. coarctata genes, three candidate genes—OcAsr1 (abscisic acid stress ripening protein), OcPVA1 (vacuolar H⁺-ATPase subunit c), and OcMT3 (metallothionein type 3)—were cloned and overexpressed in O. sativa using in planta Agrobacterium-mediated transformation method. This non-tissue culture-based transformation system enabled the successful generation of transgenic lines in the high-yielding indica background BRRI Dhan75 (for OcAsr1and OcPVA1) and BRRI Dhan67 (for OcMT3). Gene expression profiling revealed distinct stress-inducible expression patterns of the genes in O. coarctata: OcAsr1 peaked at 24 hours under 200 mM NaCl, OcPVA1 showed strong and consistent expression across both 100 and 200 mM NaCl, and OcMT3 exhibited a late response, peaking at 48 hours. Transgenic lines overexpressing OcAsr1 (notably P_73_2, P_70_1, and P_76_2) displayed enhanced root and shoot biomass, chlorophyll retention, and a 30–50% reduction in root and shoot Na⁺/K⁺ ratios. These lines maintained 40–50% higher grain yield under 100 mM NaCl 19 and exhibited minimal yield penalty under normal conditions. OcPVA1-expressing lines (MUH_113, MUH_117, MUH_99) demonstrated strong early responses, enhanced membrane stability, improved ionic balance, and significant grain yield retention under salinity. These results support its role in ion transport and vacuolar compartmentalization of sodium ions. Transgenic lines expressing OcMT3 (P-14-1, P-13-2) showed reduced oxidative damage (as measured by electrolyte leakage and histochemical staining), improved Na⁺/K⁺ balance, and stable yield under stress, highlighting OcMT3's role in ROS detoxification and metal ion sequestration. Importantly, all three gene constructs did not compromise yield under non-saline conditions, underscoring their suitability for future breeding or biotechnological applications. This study highlights the remarkable salt tolerance of Oryza coarctata and its potential to enhance salinity resilience in cultivated rice through wide hybridization and genetic engineering. Functional traits, molecular markers, and transgenic lines expressing O. coarctata genes demonstrated improved growth, yield, and stress tolerance under saline conditions, offering promising avenues for climate-resilient rice breeding.