Functional Characterization Of Rice OsHAK17 In Potassium Distribution And Two Sodium(Potassium)/Proton Exchangers Of Jerusalem Artichoke In Enhancing Salt And Aluminum Stresses

Potassium(K+)is a most abundant essential cation for plant growth and development,which weights 2-8%of plant dry weight or about 100-150 mM in plant cytosol.Sufficient K+supply and acquisition contributes to enhance salt tolerance by limiting sodium(Na+)accumulation in plants.In addition,aluminum(Al)toxicity to plants is the major withdraw in acid soil,and one of the most quick and prominent effects is its inhibition of the expansion at transition zone of root tip.Since K+creates cell turgor pressure which is essential for cell expansion,improving K+availability may alleviate the Al inhibitory effect on root growth.Plant root uptake K+from soil solution and K+distribution inside the plant is mediated mainly by K+ channel and high affinity K+transporters,while monovalent cation carriers and sodium(Na+)-K+/proton exchangers(known as NHXs)are also playing the roles in transmembrane K+ transport and K+compartmentation.Plant HAK/KUP/KT is the largest family,it has at least 27 and 13 members in rice and Arabidopsis genome,respectively,which are divided into four clusters.During past two decades,a few members of HAK/KUP/KT belonging to cluster Ⅰ and cluster Ⅱ have been characterized in K+uptake and distribution.In rice,three genes that encode HAK/KUP/KT protein,OsHAK1,OsHAK5 and OsHAK21,have been shown to contribute root K+acquisition and salt tolerance.In this thesis,the physiological function of one of rice HAK/KUP/KT member,OsHAK17,was characterized by using CRISPR-Cas9 generated mutant lines,ubiquitin promoter driven over-expression lines,and heterologous expression in yeast.In addition,the mechanism of the function of two Jerusalem Artichoke NHXs(HtNHX1 and HtNHX2)in improving salt tolerance,as well as the tolerance to aluminum stress,were elucidated.The following are the summarization of my major results:1.The signals of both C-terminal and N-terminal GFP tagged OsHAK17 were located at plasma membrane of rice protoplasts,indicating that OsHAK17 is a plasma membrane protein.Real time qRT-PCR analysis demonstrated that the expression of OsHAK17 was up-regulated by limited K+supply or high salt(NaCl)stress in culture medium,which resembles the expression patterns of most of the characterized members of plant HAK/KUP/KT family.The expression pattern analysis of OsHAK17 showed that it was mainly expressed in the leaves throughout entire growth of rice.However,in the late stage of rice growth,in addition to leaves,it was also expressed in leaf sheaths and floral organs.GUS staining signals driven by OsHAK17 promoter were detected at phloem of vascular.and parenchyma of leaf sheath and mesophyll cells of leaf blade,indicating OsHAK17 mainly paticipates K+transport in the shoot.2.OsHAK17 was able to complemental the growth of yeast R5421,a strain lack of endogenous high affinity K+transporters,in either low K+ or high Na+medium,confirming that OsHAK17 contributes to cell K+acquisition.3.Neither knockout(KO)or over-expression of OsHAK1 7 did not affect rice root uptake K+,K+content in xylem sap,and K+concentration in both root and shoot when grown at the medium containing normal K+(1 mM).In comparison to wild type(WT),OsHAK1 7 OX lines showed significant higher K+concentration in phloem sap and K+accumulation in the shoot grown at low K+(0.1 mM)or high Na+solution.Based on these findings,it can be suggested that OsHAK17 is not directly involved in the rice root K+acquisition and root to shoot translocation,but it mainly mediates K+redistribution and K+transport in the shoot.4.The other members in our lab had previously found that expression of two NHX genes cloned from salt tolerance plant species,Jerusalem Artichoke(Helianthus tuberosus L.),HtNHX1 and HtNHX2,could enhance rice salt tolerant,while their functional mechanism was not clear.In this study,both HtNHXl and HtNHX2 were found being able to complement the Arabidopsis salt sensitive atnhx5nhx6 double mutants,but only HtNHX1 not HtNHX2 could rescue the mutant growth in hygromycin included medium.5.The subcellular localizations of two NHXs expressed in rice protoplast were different,HtNHX1 was at vacuolar membrane(tonoplast)while HtNHX2 was at the membrane of endosome.Take the advantage of HtNHX2 almost identity to HtNHX1(only missing 114 amino acids),a patch of peptide which is conserved in vacuolar located NHX was identified.The detailed analyses of expression localization of these transporters indicated that a conserved 8 amino acid among known plant NHXs was essential for targeting of HtNHX1 to vacuolar tonoplast.6.Since both expression of HtNHX1 and HtNHX2 in rice could enhance the plant K+uptake,the effect of these two transporters on lifting Al toxicity to rice was also examined in this study.In comparison to WT,the transgenic lines expressing either HtNHX1 or HtNHX1 showed much longer roots,blunted Al induced root elongation inhibition in rice.Only the lines expressing HtNHX1 not expressing HtNHX2 showed enhanced citric acid secretion of the roots at Al stressed condition.In addition,Al inhibited K+influx rate significantly at the root tips of HtNHX1 OX lines and WT,but not at the root tips of HtNHX1 OX lines.The results clearly indicated that HtNHX1 and HtNHX2 functioned in improving rice tolerance to Al stress in different mechanisms.