Functional Characterization Of Potassium Transpoter OsHAK1 And Generating Potassium Efficient Rice By Using Promoters Of Potassium-deficient Responsive OsHAK Genes

Potassium(K)is the most abundant cation in plants,it plays a key role in a number of biochemical and physiological processes for plant growth and development.Due to strongadsorption and fixation by soil particles and low mobility in soil,K availability for plant roots is limited in large areas of agricultural land in the world(e.g.3/4 of paddy soils in China,2/3 of the wheatbelt in Southern Australia).The high demand for K needs to be met through efficient uptake from the soil solution by roots and further translocation into theaerial parts,distribution within cells into different compartments,and recycle from source to sink organs through various K transport systems.In plants,there are two transportsystems,channels and transporters,for K acquisition and distribution.The putative functions of the KT/HAK/KUP transporters have been predicted to maintain K and Na homeostasis in plants under low K and high salt conditions.Here,we report the expression patterns and physiological roles of OsHAKl,a member belonging to KT/KUP/HAK gene family in rice(Oryza sativa L).Potassium(K)deficiency in plants confines root growth and decreases root to shoot ratio,thus,limits root K acquisition in culture medium.A WUSCHEL-related Homeobox(WOX)gene,WOX11,has been reported as an integrator of auxin and cytokinin signaling that regulates root cell proliferation.The aim of the present study was to test if conditionally enlarging the root system and development of adventitious roots could improve K uptake efficiency at low K supply level.We used the promoter of OsHAK16,a K-deficiency up-regulated gene encoding a K-transporter,to drive the expression of OsWOX11 gene in rice roots.The transport of assimilates via the phloem is restricted in K deficient plants.Limited photoassimilate production coupled with transport inhibition can lead to smaller assimilate supply to heterotrophic organs,thus reducing their growth.CSA has been reported as a key transcriptional regulator for sugar partitioning in rice during male reproductive development.In this study,two promoters of K-deficiency up-regulated genes(OsHAK7 and OsHAK16)and the promoter of a constitutive expression gene(ubiquitin),with contrasting expression pattern,were used for the low potassium-inducible expression of the CSA gene in transgenic rice plants with the aim of optimizing transgene expression for carbon partitioning,thus increasing low K stress tolerance and grain yield.The main results are summarized as follows:1.Expression of OsHAKl is up-regulated by K deficiency or salt stress in various tissues,particularly in the root and shoot apical meristem,the epidermises and steles of root,and vascular bundles of shoot.Both oshakl knockout mutants in comparison to their respective Dongj in or Manan wild types showed dramatically reduction in K concentration and stunted root and shoot growth.Knockout of OsHAKl reduced the K absorption rate of unit root surface area by～50-55%and～30%,and total K uptake by～80%and～65%at 0.05-0.1 mM and 1 mM K supply level,respectively.The differences in growth,K uptake and K transport from roots to aerial parts between WTs and the mutants were further exacerbated by Na stress.The root net high affinity K uptake of oshakl mutants was sensitive to salt stress,but not to ammonium supply.Increasing K supply could not fully eliminate the phonotype of the growth defects and impairment of K uptake in the mutants.Over-expression of OsHAK1 increased K uptake and K/Na ratio in rice.The positive relationship between K concentration and shoot biomass in the mutants suggests that OsHAK1 plays an essential role in K-mediated rice growth and salt tolerance over low and high K concentration ranges.2.Ectopic expression of PWOX11 gene driven by the promoter of OsHAK16 encoding a low K-enhanced K transporter led to an extensive root system and adventitious roots,and more effective tiller numbers in rice.The WOX11 regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K deficiency enhanced expression of several RR genes encoding type-A cytokinin responsive regulators,PIN genes encoding auxin transporters and Aux/IAA genes.In comparison with WT,the transgenic lines showed increases of root biomass,root activity and K concentrations in the whole plants,and higher soluble sugar concentrations in roots particularly under low K supply condition.The improvement of sugar partitioning to the roots by expression of OsHAK16p:WOX11was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots.Expression of OsHAK16p:WOX11 in the rice grown in moderate K deficient soil increased total K uptake by 72%and grain yield by 24%-32%.3.Constitutive over-expression of CSA gene very significantly improved plant growth,elevated sugar metabolism and increased K accumulation at the vegetative stage of plant development irrespective of K supply condition,but led to tiller-spreading phenotypes and 4-week delays in flowering compared with control plants.OsHAK16p:CSA transgenic rice showed evidently higher expression of CSA mainly in roots and a similar phenotype to the nontransgenic controls.Ectopic expression of CSA,under the control of a low potassium-inducible promoter OsHAK7,led to more tillers,a larger tiller angle and delayed flowering in rice.OsHAK7p:CSA transgenic plants had suppressed the expression of OsTB1 in the leaf sheath,but increased expression of PROG1 and OsTAC1,which were reported as tiller number and tiller angle controllers in rice respectively.Enhancing the expression of CSA had activated the suppression pathway and suppressed the activation pathway in the LD condition,resulting in 2 weeks delayed flowering.In comparison withWT,the transgenic lines showed increases of sucrose-phosphate synthase(SPS)activity in source blades and acid invertase activity in sink organs such as roots and spikelets.Soluble sugar concentration in transportation and sink organs and K concentration in the whole plants were higher than WT particularly under low K supply condition.The elevated sugar partitioning and net K uptake rate by expression of OsHAK7p:CSA were further indicated by increasing the expression of several sugar transport related genes(OsSUTs and OsMSTs)and K acquisition related genes(OsAKT1 and OsHAK1),respectively.Due to the enhancement of sugar synthesis,partitioning and dry matter translocation,OsHAK7p:CSA transgenic rice grown in moderate K deficient soil increased total K uptake by 137%and grain yield by 147%.Taken together,all these results indicated that OsHAKl could be considered as a candidate gene for improving rice tolerance to potassium deficiency.Enlarging root growth and development by expression of WOX11 in roots or improving sugar metabolism by expression of CSA in shoots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.