The function of potassium and sodium transporters in the maintenance of cellular ion homeostasis in rice (Oryza sativa. L) under hostile environmental conditions

The world population is growing alarmingly fast and imposes more pressure than ever towards increasing food production. However, this task is restrained by various abiotic stresses. One of these stresses is salinity. Salinity affects 230 million ha of irrigated land and results in over $27B in annual losses to agricultural sector. Salinity also poses a major threat to the food security, significantly reducing plant growth and yield. This reduction is especially pronounced in salt sensitive glycophytic crops such as rice. Rice is a staple food of more than half of the world population since as much as 20% of the world's dietary energy is supplied by it. Salt stress tolerance in plants is a multigenic and physiologically complex trait which is dependent upon a numerous cross-interacting mechanisms involving an orchestrated series of molecular, cellular, metabolic and physiological responses. In order to perceive, and respond to abiotic stress, plants have evolved a complex CBL-CIPK signaling network, which is `Ca^(2+)`-dependent and generate the secondary messengers, such as `Ca^(2+)` and reactive oxygen species (ROS). This signaling network initially alters protein activity within a cell, such as the activation of ion transporters and transcription factors. It is these interactions which then may up- or down-regulate the expression of responsive genes to induce adaptive responses. One such adaptive response is the plant's ability to retain or take up `K^+`, one of the most abundant and essential inorganic nutrients. Some members of the high affinity `K^+` transporter (HAK) family are suggested to be involved in `K^+` homeostasis under `K^+` deficiency condition, and salt stress has also been found to alter their transcript levels. The Shaker-type AKT1 channel is another important component of the K+ uptake system. It has been targeted by plant breeders in order to improve `K^+` nutrition. Several studies have already reported that `K^+` channels may mediate ion uptake of `Na^+` and `NH4^+`, particularly under a `K^+`-deficient conditions. Given that salinity stress results in acute `K^+` deficiency, understanding of regulation of `K^+` transporters under stress conditions may open prospects for genetic improvement of rice and minimising NaCl induced yield losses. Sodium exclusion from uptake and control over long-distance transport of `Na^+` within plants are two components of a vital mechanism that plants have evolved to mitigate the adverse effects of salinity. The function of the high affinity potassium transporter HKT1;5 in the retrieval of `Na^+` from shoots has already been identified in many species including rice. The overall aim of this work was to elucidate the physiological role and functional expression of some of above ion transporters and signal transduction components (OsCIPK9, OsHAK1, OsHAK5, OsAKT1, and OsHKT1;5) in rice plants under stress conditions. This was achieved in a series of electrophysiological and whole-plant experiments conducted on the loss- or gain-of function rice mutants. The first experimental chapter identified the critical regulatory role played by the calcineurin B-like protein-interacting protein kinase 9 (CIPK9) in rice plants, particularly under the K+-deficient condition (e.g. mimicking the situation when K+ availability is reduced in salinized soils). It was found that loss of function of Oscipk9 increased plant sensitivity under the K+-deficient condition. Moreover, under salt stress, the Oscipk9 mutant plants had a lower `K^+:Na^+` ratio than the wild type. In response to oxidative treatment (one of components of salt stress), the root of Oscipk9 mutant plants grown under a low `Ca^(2+)` and `K^+` condition experienced higher `K^+` efflux compared to the wild type. This negative influence on the `K^+` efflux was mitigated by an increase in the external `Ca^(2+)` concentration in the growth medium. However, when the rice plants were grown in a medium containing a high `K^+` concentration, no significant difference in `K^+` flux was observed between the lines. Therefore, these results suggest that OsCIPK9 plays a critical role in regulation of `K^+` homeostasis under the K+-deficient conditions. The second experimental chapter investigated the role of HAK transporters in maintaining `K^+` homeostasis during salt stress by studying the responses of rice (Oryza sativa) Oshak1 and Oshak5 mutants to salt and oxidative stresses. It was found that the loss of function of the Oshak1 and Oshak5 mutants caused (1) an increase in the amount of `K^+` loss from roots both under the `K^+` deficiency (200 ˜í¬¿M KCl) and saline (40 mM NaCl) condition, and (2) a decrease in the expression level of the respiratory burst oxidase homolog OsRboH genes in plants exposed to low `K^+`. This decrease attenuated stress-induced `K^+` loss from Oshak mutants as compared with the WT. It is concluded that the loss of function of Oshak1 and Oshak5 increased the sensitivity of the mutant lines to salt stress, thereby revealing the crucial role of OsHAK1 and OsHAK5 in `K^+` acquisition in response to hostile environmental conditions, such as low potassium and high soil salinity. The third experimental chapter was focused used gain- and loss-of-function of OsAKT1 gene to reveal the role of AKT1 channel on the ability of rice plants to uptake `Na^+`, `NH^4+` and `K^+` ions. The results showed that in the presence of `NH^4+` the loss of function in akt1 did not change the growth rate of the mutant plants. Furthermore, mutation in the akt1 altered the `Na^+` and `K^+` content of the mutant plants, but had no significant effect on their growth. It was also found that AKT1 mediated `Na^+` uptake, both under the `K^+`-deficient and low external `NH^4+` concentration condition. Additionally, the study also identified the role of the AKT1 channel in `NH^4+` uptake as it was found that increasing the external `K^+` concentration until it was in the millimolar range stimulated stronger `NH^4+` influx in the elongation zone of the overexpressor compared to the wild type. Under `K^+`-deficient conditions, the presence of 2mM `NH^4+` inhibited AKT1 permeability to `Na^+` ions in the overexpressing line. Overall, this study has proven that AKT1 plays an important role in ion homeostasis in response to hostile environmental conditions, particularly under `K^+`-deficiency. The fourth experimental chapter has assessed the physiological role of OsHKT1;5 in the xylem `Na^+` unloading in response to salt and drought stresses by comparing knocked-down OsHKT1;5 (KD) line with its wild type. The phenotyping experiments showed that knocking-down of OsHKT1;5 expression resulted in excessive level of `Na^+` accumulated in the mutant plants KD under severe salt stress (80 mM NaCl), but much smaller amounts of `K^+` accumulated in its shoots. As a result, its growth was significantly impaired. However, the electrophysiological data showed that the KD plants had a greater capacity for exclusion of `Na^+` from the elongation cells accompanied by a lower `K^+` uptake. This difference has resulted from higher activity of the `H^+` -ATPase in the KD line. Such activation may also have enhanced the activity both of SOS1 (the \\(Na^+/H^+\\) antiporter), and the HAKs \\(K^+/H^+\\) symporter genes in the epidermal cells of the elongation zone of rice roots. Knocking-down of OsHKT1;5 expression did not alter the growth of the mutant compared to the wild type during drought stress, suggesting OsHKT1;5 has no role to play during drought stress. Overall, the finding of these studies has elucidated the crucial roles of the OsCIPK9 signaling network, the high-affinity `K^+` transporters (HAKs), the AKT1 channels and the `Na^+` transporter (HKT1;5) in ions homeostasis in rice plants in response to abiotic stresses. The information obtained from these studies could be used for further breeding program or to produce new lines in order to study the interactive effects of ion transporters in rice plants.