| Potassium (K) is one ofthe essential macronutrients for plant growth and development,widely involved in a series of plant physiological and biochemical processes, such as adjustment of plant cell turgor pressure and osmotic, stomatal opening and closing, ion balance, enzyme activation and resistance of biological and abiotic stress etc. K can easily be fixed and leaching in the soil. Low available K in tropical and subtropical highly wreathing soil severely hampers agricultural production. Plants have two systems for absorbing K from soil solution depending on K concentration. It is generally assumued that K Channels and transporters play important role in acquiring K under high and low K conditions, respectively. Plants have evolved many strategies to cope with low Kstress,including the cell membrane hyperpolarization, changing root morphology, enhancing K flow in various tissues, extracellular acidification and inducing and increasing high affinity transporters expression. KUP/HAK/KT (K Uptake Permease / High Affinity K transporter/ K Transporters) family is the largest K transporter family in rice. Ithas been predicted that there are 27 KUP/HAK/KTmembers in rice genome. However, up to the beginning of this study, none of rice KUP/HAK/KT members was reported physiological function. In this thesis, we focused on charactering the one of the known Arabidopsis HAK5 homolog.OsHAK5 in rice.In this study, we detected the expression pattern of OsHAK5 by RT-PCR and using OsHAK5-promoter-GUS transgenic rice. Subcellular localization of OsHAK5 was detected by transformingOsHAK5 fused GFP in rice protoplast. Yeast mutants were used to detect function of OsHAK5 in heterogeneous system. A series of molecular and physiological experiments with OsHAK5 over-expression (OX) and knockout (KO) of T-DNA insertion mutants together with wild type (WT) were used to identify the physiological function of OsHAK5. The main results were summarized as follows:1. The expression level of sHAK5was up-regulated by K starvation. Promoter of OsHAK5 fused GUS reporter gene showed that OsHAK5 was expressed in the root epidermis, lateral root primordium, lateral root, stele, root-shoot junction, leaf, anther,embryo, seeds and shell. The GUS staining was enhanced by K deficiency in the junction of root and shoot, root and leaf. Subcellular localization showed OsHAK5 located on the plasma membrane of rice protoplast. Promoter deletion analysis showed that its promoter region from -1479 to -1776 bp of OsHAK5 gene coding sequence exists K starvation cis-elements. The expression of OsHAK5 gene was up-regulated or down regulated by different plant hormones treatments. In hydroponic experiment with exogenous added 10-μM IAA, GUS expression was significantly enhanced in various tissues of the transgenic lines. An auxin responding cis-element ASF1MOTIFCAMV, was predicted in OsHAK5 promoter region from -1479 to-1776 bp.2. To validate the function of OsHAK5 in K uptake and salt tolerance, OsHAK5 was expressed in a heterogeneous system, the K uptake-defective yeast mutant R5421, and Na+sensitive yeast mutant AXT3, respectively. The results showed that there was no significant growth differences between pYES2-OsHAK5-yeast and pYES2-yeast grown on high K mediums (20 mM and 50mM K+) ,while the growth of pYES2-OsHAK5 was much better than pYES2 on low K mediums (the range from 0.05 mM K+ to ImM K+) .Grown on high salt culture medium,pYES2-OsHAK5-yeast displayed a higher K+/Na+ ratio than pYES2-yeast by accumulating more K+ and less Na+.3.We planted OsHAK5 OX, KO and WT lines in hydroponic solution with different K concentrations.The results showed that when the K concentration was ImM, there was no significant growth difference between OX and KO lines. Compared with WT lines, the biomass, K absorption amount and K net absorption rate of KO lines were significantly decreased. The results with a certain time of low K (0.3 mMK+) and removal K (0mM K+) showed that the biomass, K absorption amount and K net absorption rate of OX lines were higher than WT lines, while the K concentration of the transgenic root was lower than WT. In contrast to OX lines, compared with WT lines, the biomass, K absorption amount and K net absorption rate of KO lines were significantly reduced, while the K concentration of root was higher than WT lines.When the K concentration was 1 mM, there was no significant difference in the net K influx of OX, KO,WT lines.When the K concentration supplied with 0.1mMK+, the net K influx in roots of OX lines was significantly larger than that in the WT lines, while the net K influx in roots of KO lines was much lower than that in WT lines. In addition, to verify whether OsHAK5is involved in K transport from roots to shoots, the xylem sap of OX, WT and KO lines were collected.It showed that the K concentration and K efflux rate in roots of OX lines were higher than that of WT lines,which was opposite in roots of KO lines. Our results demonstrated that OsHAK5 not only could absorb K from low K environment, but also could transport K from roots to shoots when K was at deficiency condition.4. The OsHAK5 OX lines displayed stronger salt tolerance with higher biomass, more K content accumulation, higher Na+/K+ in shoot compared with WT lines in hydroponic experiment with 100 mM NaCl treatment. While KO lines obtained higher Na+concentration and Na+/K+ with lower biomass, less K accumulation in the shoot, displayed a weaker salt tolerance of OsHAK5 overexpression in comparison to WT.5. In rice, OsHAK5 OX and KO lines showed different architecture with WT lines. The total tiller number and effective tiller number of OX lines were much higher than that of WT lines, while the height of both OX and KO lines were lower than that of WT lines.Auxin content in the root tips of OX and KO lines were only half of that in WT. However,auxin content in the root-shoot-junction of OX lines was significant higher than WT lines.In contrast, The KO lines showed much lower concentration of auxinin the root-shoot-junction. Overexpreesion and knockout ofOsHAK5 gene affected the expression of OsPIN family members and AUX1.The alteration of OsPIN genes and OsAUX1 expression was opposite in OX lines and KO lines comparied with WT. The genes related to the rice tiller (OsFC1) and tiller angle (OsLazyl and OsTAC) were also significantly changed by overexpreesion and knock out of OsHAK5 gene in rice. Moreover,the [3H]-IAApolar transportation in OX and KO lines were also affected in the opposite way. The roots of OX lines developedmuch larger than that of WT in pH 4.5 culture medium, while the roots of KO lines were worse than that of WT grown on pH 6.5 and 5.5 mediums. We speculated that OsHAK5 affected rice architecture through influencing the pH homeostasis between the cells.Taken together, OsHAK5 gene was not only involved in absorbing K, but also could transport K from roots to shoots at low K supply levels.OsHAK5 enhanced the salt tolerance of rice plants through high selectively absorbing K+,while not sensitive to Na+,thus gained a higher K+/Na+ in the shoot. In addition, our collected evidences suggested that OsHAK5 may affect the content and distribution of auxin in rice plant to regulate rice architecture. |
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