Ion homeostasis and regulation of membrane-transport activity in hypoxia-stressed Arabidopsis roots
thesis
posted on 2023-05-27, 02:27authored byWang, F
Waterlogging is a widespread abiotic stress to land plants, influencing nearly 10% of the global land area and reducing up to 80% of crop yields. There is a series of changes in physical, chemical, and biological properties in soil that ultimately inhibit plant growth under waterlogging stress. Waterlogging leads to a hypoxic stress which causes a rapid elevation of cytosolic free calcium ([Ca2+]cyt) in the cells of various species, and this elevation is fundamental for gene activation and acclimation responses at the cellular, tissue, as well as organismal levels. Reducing [Ca2+]cyt to the resting level requires activation of Ca2+ transporters such as high-affinity Ca2+-ATPases [autoinhibited Ca2+-ATPases (ACAs) and ER-type Ca2+-ATPases)] and the low-affinity Ca2+/H+ antiporter (CAXs) which are localized in both the plasma membrane and endomembranes. However, the specific roles and regulatory modes of CAX and ACA transporters in plant sensing and adaptation to hypoxia remains largely unexplored. Early studies showed that a root's ability to retain K+ under hypoxic conditions is strongly correlated with waterlogging tolerance in barley, and this ability also exhibits root tissue specificity. However, little is known about the molecular identity of ion transporters mediating the above phenomenon. It was shown before that depolarization-activated outward-rectifying K+ (GORK) channels mediate stress-induced K+ efflux from root cells in response to salinity and oxidative stress. The role of GORK channel in plant adaptive responses to hypoxia remains elusive. Hypoxic stress also interferes with the mitochondrial respiration pathway where it leads to the saturation of redox chains, accumulation of NAD(P)H (nicotinamide adenine dinucleotide phosphate), decreased synthesis of ATP, and generation of ROS (reactive oxygen species). Also, the knowledge of hypoxia-induced transport processes is largely restricted to ion exchange at the root surface, which is easily accessible. However, little is known about the patterns of hypoxic signal transduction in specific tissues and cell types of the roots. In the light of above, the aims of this project were: (1) to reveal essentiality of the CAX and ACA calcium transport systems in hypoxia response in different tissues (epidermis and stele) and zones (elongation and mature); (2) to investigate the potential roles of GORK and RBOHD (respiratory burst oxidase homologue D) in plant adaptive responses to hypoxia in Arabidopsis roots; (3) to understand tissue-specificity of hypoxic effects on cell ionic relations and the mode of their regulation; (4) to investigate interaction between salinity and waterlogging stress (two abiotic stresses that often occur together in the nature); and (5) to elucidate the potential ion channel as oxygen sensor in plants. The crucial role of Ca2+ as a second messenger in response to abiotic and biotic stimuli has been widely recognized in plants. However, the physiological and molecular mechanisms of Ca2+ distribution within specific cell types in different root zones under hypoxia are poorly understood. In the first experimental chapter, the whole-plant physiological and tissue-specific Ca2+ changes were studied using several ACA and CAX knock-out Arabidopsis mutants subjected to waterlogging treatment. In the wild-type (WT) plants, several days of hypoxia decreased the expression of ACA8, CAX4, and CAX11 by 33% and 50% respectively ecompared with the control. The hypoxic treatment also resulted in an up to 11-fold tissue-dependent increase in Ca2+ accumulation in root tissues as revealed by confocal microscopy. The increase was much higher in stelar cells in the mature zone of Arabidopsis mutants with loss of function for ACA8, ACA11, CAX4, and CAX11. In addition, a significantly increased Ca2+ concentration was found in the cytosol of stelar cells in the mature zone after hypoxic treatment. Three weeks of waterlogging resulted in a dramatic loss of shoot biomass in cax11 plants (67% loss in shoot dry weight), while in the WT and other transport mutants this decline was only 14-22%. These results were also consistent with a decline in leaf chlorophyll fluorescence (Fv/Fm). Regulation of root cell K+ is essential for acclimation to low oxygen levels. ROS are important signaling molecules mediating a broad range of developmental and adaptive responses. The roles and interaction between GORK channels and NADPH oxidase in hypoxic responses of roots requires elucidation since both can impact cellular signaling and K+ homeostasis and therefore possibly acclimation to hypoxia. In the second experimental chapter, the potential roles of GORK channels and RBOHD in plant adaptive responses to hypoxia were investigated in the context of tissue specificity (epidermis versus stele; elongation versus mature zone) in roots of Arabidopsis. The expression of GORK and RBOHD was down-regulated by 2- to 3-folds within 1 h and 24 h of hypoxia treatment in Arabidopsis wild-type roots. Interestingly, the loss of the functional GORK channel resulted in a waterlogging-tolerant phenotype, while rbohD knockout was sensitive to waterlogging. To understand their functions under hypoxia stress, we studied K+, Ca2+, and ROS distribution in various root cell types. gork1-1 plants had better K+ retention ability in both the elongation and mature zones compared to the WT and rbohD under hypoxia. Hypoxia induced a Ca2+ increase in each cell type after 72 h, and the increase was much less pronounced in rbohD than in the WT. In most tissues except the elongation zone in rbohD, the H2O2 concentration had decreased after 1 h of hypoxia, but then increased significantly after 24 h of hypoxia in each zone and tissue, further suggesting that RBOHD may shape hypoxia-specific Ca2+ signatures via the modulation of apoplastic H2O2 production. When combining salinity with hypoxia stress, more Na+ and Cl- are transported from roots to shoots, whereas K+ transport decreases even further compared with salinity under normoxic conditions. Very few studies have elaborated Na+ and K+ distribution in specific cell types during combined salinity and hypoxia stress. Based on our findings in Chapter 4 indicating that RBOHD mediates hypoxia-induced Ca2+ signaling in Arabidopsis root, the third experimental chapter investigated the role of RBOHD adaptive responses to combined hypoxia and salinity stress. Three-week old Arabidopsis were exposed to two weeks of waterlogging, salinity and combined waterlogging and salinity stress. We found among these three kinds of treatments that waterlogging stress caused the most severe effect on plants growth and biomass accumulation. rbohD mutant was more sensitive to all the treatments compared with WT. After 48 h of salinity stress, both WT and rbohD showed significant K+ efflux from elongation zone and mature zone with rbohD losing more K+ than WT in both zones. WT and rbohD pretreated with 48 h of salinity stress then exposed to hypoxia stress. This transient hypoxia stress induced rbohD absorbing more Na+ and Cl- than WT in both elongation and mature zone. Briefly, these results indicate that rbohD mutant has less K+ retention ability under combined stress and may be more sensitive to salinity stress than WT. The RBOHD expression level was not significantly changed in WT between control and salinity stress, while the expression of RBOHD was significantly decreased after combined stress. A similar trend was found in the WT for the expression of GORK in the mature zone. However, in rbohD mutant, GORK expression was higher in salt-grown plants compared with control. Interestingly, the SOS1 expression in rbohD is much lower than in WT under control, salinity stress and combined stress. These results suggested that knocking out RBOHD makes Arabidopsis more sensitive to salinity stress than WT, and RBOHD may play functions in Na+ distribution in Arabidopsis root cells under both salinity and combined stress. Until now, the molecular identity of putative oxygen sensors in plants remains elusive. Here, we hypothesizes that one of the K+ or Ca2+ channels may potentially fulfill this role. Accordingly, in the fourth experimental chapter we reviewed the current knowledge about the oxygen sensing and signaling pathway in mammalian and plant systems and used bioinformatics tools to identify candidate genes for the potential role of ion channels as putative oxygen sensors in plant roots. Based on literature analysis, several known candidates for oxygen sensing in the mammalian systems were identified. This includes transient receptor potential (TRP) channels; K+-permeable channels (Kv; BK; TASK); Ca2+ channels (RyR; TPC); and various chemo- and ROS-dependent oxygen sensors. Identified key oxygen sensing domains (PAS; GCS; GAF; PHD) in mammalian systems were used to predict the potential plant counterparts in Arabidopsis. Then the sequences of known mammalian ion channels with reported roles in oxygen sensing were employed to BLAST the Arabidopsis genome for the candidate genes. Several plasma membrane and tonoplast ion channels (such as TPC; AKT; KCO) and oxygen domain-containing proteins with predicted oxygen sensing ability were identified and discussed. A testable model for potential roles of ion channels in plant hypoxia sensing is proposed. In conclusion, this study suggested that (1) CAX11 plays essential role in maintaining cytosolic Ca2+ homeostasis and/or signaling in root cells under hypoxic conditions; (2) plants lacking functional GORK channels are more capable of retaining K+ for their better performance under hypoxia, and that RBOHD is crucial in hypoxia-induced Ca2+ signaling for stress sensing and acclimation mechanism; (3) RBOHD may also play important roles in regulation of Na+ distribution in Arabidopsis root cells under both salinity and combined stresses; (4) TPC, AKT, and KCO ion channels may operate as potential oxygen sensors in plants.