Comparative analysis of root ionic relation between Oryza sativa and Oryza coarctata species in the context of differential salinity stress tolerance

Soil salinity seriously limits global agricultural production resulting in over US$27 billion per year economic losses. Rice (Oryza sativa) is a basic staple food that feeds about 50 % of the global population but is considered to be highly salinity sensitive among all cereal crops. While wild relative of rice possesses salinity tolerance, this trait was lost during domestication of Oryza sativa species. Understanding the mechanisms conferring salinity tolerance in wild relatives is essential to introduce efficient traits into elite rice cultivars, thus contributing to future global food security. This project aimed to reveal some of these traits in Oryza coarctata, a wild relative of Oryza sativa, and arguably the most tolerant of all rice species. The focus of my work was on the rootrelated traits, focusing on kinetics of xylem ion loading; regulation of Na⁺ and K⁺ homeostasis; and ROS detoxification and signalling. The comparison was made between cultivated rice (O. sativa cv. Koshihikari) and halophytic relative of wild rice (O. coarctata). We have also compared some of these traits in O. sativa with those present in barley, one of the most salt-tolerant cereal crop species.

Although control of xylem Na⁺ loading is recognised as an important mechanism of salt stress tolerance, it remained to be answered to what extent the difference in this trait between species can determine differential salinity tolerance. In the Chapter 3, changes in root, shoot and xylem sap Na⁺ and K⁺ content were compared between rice and barley species at different time points after salinity stress onset. Salt-exposed rice plants prevented xylem Na⁺ loading at the beginning of salt stress, but later failed to control this process in the longer term, ultimately resulting in a massive Na⁺ shoot loading. Barley plants quickly increased xylem Na⁺ concentration and its delivery to the shoot (most likely for the purpose of osmotic adjustment) but were able to reduce this process later on, keeping most of accumulated Na⁺ in the root, thus maintaining non-toxic shoot Na⁺ level. Rice plants increased shoot K⁺ concentration, while barley plants maintained higher root K⁺ concentration. Control of xylem Na⁺ loading is remarkably different between rice and barley; this difference may differentiate the extent of the salinity tolerance between species. This trait should be investigated in more details to be used in the breeding programs aimed to improve salinity tolerance in crops.

Na⁺ toxicity is one of the major causes of physiological constraint imposed by salinity, however Na uptake may be beneficial under some circumstances as an easily accessible inorganic ion that can be used for increasing osmotic potential and maintaining cell turgor. As shown in Chapter 4, cultivated (salt-sensitive) and wild (salt-tolerant) rice species demonstrated different strategies in controlling Na⁺ uptake. Glasshouse experiments and gene expression analysis suggested that salt-treated wild rice quickly increased xylem Na⁺ loading for osmotic adjustment but maintained non-toxic level of stable shoot Na⁺ concentration by increased activity of HKT1;5 (essential for xylem Na⁺ unloading) and NHX (for sequestering Na⁺ into root vacuoles). Cultivated rice prevented Na⁺ uptake and transport to the shoot at the beginning of salt treatment, but failed to do it in a long-term. While electrophysiological assays revealed greater net Na⁺ uptake upon salt application in cultivated rice, O. sativa plants showed much stronger activation of the root plasma membrane Na⁺/H⁺ exchanger (SOS1). Thus, it appears that wild rice limits passive Na⁺ entry into root cells while cultivated rice relies heavily on SOS1-mediating Na⁺ exclusion, with major penalties imposed by the existence of the “futile cycle” at the plasma membrane.

K⁺ is an essential plant macronutrient that has important roles in many metabolic processes, and maintenance of the intracellular K⁺ homeostasis is considered as one of the key factors in plant salinity stress tolerance. Over the exposure to salt stress, wild rice possessed a superior root K⁺ retention while cultivated rice showed an overall decline in the root K+ concentration. This K⁺ decrease resulted in a considerable loss of root cell viability in cultivated rice while wild rice maintained normal root growth. Salinity induced K⁺ efflux from the elongation root zone was higher in wild rice, prompting a suggestion for its role as a “metabolic switch”. The magnitude of K⁺ efflux from mature root zone was lower in wild compared with cultivated rice and correlated to salinity tolerance between two species. Cultivated rice showed significantly greater H⁺-ATPase activation, but failed to maintain negative membrane potential, thus triggering massive K⁺ loss via depolarization-activated K⁺ efflux channels. Wild rice maintained more negative membrane potential with less activation of H⁺-ATPase and, hence, was much more energy efficient in its adaptation to salinity. These findings are described in detail in experimental Chapter 5.

Reactive oxygen species (ROS) production is induced by salinity and comes with a danger of causing oxidative damage. At the same time, ROS also play important signalling roles in plant adaptive responses. The differences in redox homeostasis between O. sativa and O. coarctata species were compared in Chapter 6. Root treatment with 10 mM H₂O₂ decreased cell viability in cultivated but not wild rice, and histochemical staining showed greater H₂O₂ accumulation in wild rice root under nonsaline condition. These observations point out at the likely signalling roles of Reactive oxygen species (ROS) production is induced by salinity and comes with a danger of causing oxidative damage. At the same time, ROS also play important signalling roles in plant adaptive responses. The differences in redox homeostasis between O. sativa and O. coarctata species were compared in Chapter 6. Root treatment with 10 mM H₂O₂ decreased cell viability in cultivated but not wild rice, and histochemical staining showed greater Hin wild rice. Wild rice also possessed superior ability to scavenge excessive ROS by having higher superoxide dismutase (SOD) activity; also lower was O₂^- accumulation. Cultivated rice showed greater K⁺ loss from root mature zone through ROS-activated NSCC accompanied by Ca²⁺ uptake in response to Reactive oxygen species (ROS) production is induced by salinity and comes with a danger of causing oxidative damage. At the same time, ROS also play important signalling roles in plant adaptive responses. The differences in redox homeostasis between O. sativa and O. coarctata species were compared in Chapter 6. Root treatment with 10 mM H₂O₂ application, while wild rice possessed better Ca²⁺ homeostasis with less Reactive oxygen species (ROS) production is induced by salinity and comes with a danger of causing oxidative damage. At the same time, ROS also play important signalling roles in plant adaptive responses. The differences in redox homeostasis between O. sativa and O. coarctata species were compared in Chapter 6. Root treatment with 10 mM H₂O₂ -induced K⁺ leakage. ^●OH application induced much greater K⁺ leakage in cultivated rice that clearly reflects its sensitivity to ROS and a lack of ability for its detoxification. Wild rice effectively controlled cytosolic Ca²⁺ homeostasis by Ca²⁺ efflux systems such as Ca²⁺-ATPase and CAX, while cultivated rice downregulated RBOH expression that may affect operation of “ROSCa²⁺ hub” and signalling cascades under salinity.

Over the last few decades, rice breeding for salinity tolerance has targeted predominantly Na⁺ excluding trait but achieved a very modest progress. This project has carried out a less biased approach and a comprehensive analysis of root ionic relations conferring salinity tolerance of wild rice, to be used as a blueprint in future breeding programs. The following traits were deemed as most critical: 1) efficient control of xylem Na⁺ loading and sequestration of Na⁺ into root vacuoles, 2) limiting passive Na⁺ uptake and avoiding a futile cycle of SOS1 operation; 3) maintenance of negative membrane potential by energy-efficient means; and 4) desensitisation of ROS-activated NSCC to confer K⁺ retention in mature root zone. It is highly suggested that above existing traits in wild relative of rice should be reintroduced into domesticated rice cultivars. To achieve this goal, the next step can be a further investigation to identify their candidate genes and QTLs. Once genes/ QTLs are identified and mapped, molecular breeding tools can be used to introduce these traits into O. sativa, to produce ultimately salinity tolerant rice cultivar.