Function Analysis Of Rice Oshak Genes In Maintaining Gamete Fertility,Potassium And Sodium Homeostasis

Potassium(K)is the most abundant essential cation in plants.K concentration in plant cytoplasm is usually maintained between 100-150 mM,which is the main osmotic regulator and plays an important role in maintaining cellular turgor presure,membrane potential,stability of DNA and proteins(enzymes)hydration structure.In addition,K plays an important role in the distribution of photosynthetic products from source to sink for organs generation,thus crop development and yield.Due to soil K fixation,intensive cultivation and mismatch of K fertilizer with nitrogen(N)and phosphorus(P),the available K in the soil is commonly low in many parts of arable lands.In addition,climate change,seawater intrusion and increasing of irrigation all lead to aggravation of soil salinity.Therefore,K deficiency and salt stress including osmotic stress and sodium(Na)toxicity are limiting factors for agricultural production.K concentration in soil soluiton is in the range of 0.01-1 mM.To cope with varibal K supply,plants have evolved two mechanisms for K absorption:low affinity absorption at high K level and high affinity K absorption at low K level.HAK/KUP/KT is the largest K transporter family in plants.There are 13 and 27 members predicted in Arabidopsis and rice,respectively,which can be divided into four clusters.So far,OsHAK1,OsHAK5,OsHAK16,and OsHAK21 of the cluster I in rice have been reported to be functional in root K acquisition,K distribution and plant tolerance to salt stress,while the physiological functions of other subfamily members are still poorly understood.Our research group has previously demonstrated OsHAK1 and OsHAK5 of Cluster I which function in rice K uptake and transport affect also on rice yield.In this study,we characterized the different effects of plasma membrane localized OsHAK1,OsHAK5 and Golgi apparatus localized OsHAK26 of Cluster VI on gamete fertility and seed setting.In addition,we also found that OsHAK27,a member of Cluster I,plays an important role in maintaining Na and K homeostasis in plants.The main results are summarized as follows:1.The analysis of gene tissue localization and quantity expression in floral organs of rice plants indicate that expression of OsHAK26 was mainly in mature pollen grains,leaf sheath,and node at late developing stage.OsHAK26 expression was not detectable in entire rice at seedling stage.The cellular location assay showed that OsHAK26 was specifically expressed in Golgi apparatus of rice protoplast.Knockout of OsHAK26 either by T-DNA insertion in the background of Japonica cultivar Dongjin or by CRISPR/Cas9 gene editing in the background of Japonica cultivar Nipponbare did not affect K content in the vegetative tissues under normal K supply.2.Knockout of OsHAK1,OsHAK5 or OsHAK26 resulted in low seed-setting rate and grain yield.The yields of oshak1,oshak5 and oshak26 were about 20%,50%and 30%of wild type,respectively.K content in peduncle was reduced only by inactivation of OsHAK1,not by OsHAK5 or OsHAK26,meanwhile the total K content in the spikelet of oshak1 was also lower than that of the wild type,indicating that OsHAK1 was involved in K distribution from vegetative organs to reproductive tissues.Anthers of oshakl and oshak5 are smaller and twist,while mutation of OsHAK26 did not affect development of anthers.However,knockout of any one of these OsHAK members decreased pollen numbers,pollen activity and germination rate.Elongation of pollen tubes was not affected by OsHAK1 mutation,but was severe retarded by knockout of either OsHAK5 or OsHAK26.Large portion of the pollen tubes of oshak5 plants was ruptured after extending to about 1-2 folds length of pollen diameter in vitro germination.Although mutation of these HAK genes reduced the number of pollen grains on stigma,the germination of these grains as observed in vivo was not severe affected.3.OsHAK1,OsHAK5 or OsHAK26 mutation on the activity of male or female gamete was conducted by analyzing the morphology,developing process of pollen and pistil and pollination success of cross hybridization using the gamete either from wild type or one of the mutants.Knockout of OsHAK1 or OsHAK5 affected the fertility of both male and female gametes to a similar extent,while OsHAK26 mutation showed the largest effect on the activity of male gametes.The cell wall of pollen can protect pollen grains against dessicate stress and invasion of harmful microbes.In addition,the oil layer contained in the pollen wall can further prevent the pollen grains from losing water,ensuring timely germination of pollens.Electrical microscope observation of pollen grains revealed that oshak26 plants displayed much shorter baculum and thinner oil layer than wild type.Relative to oshakl and oshak5,oshak26 showed the worst pollen fertility and most sensitive of male gamete activity to drought stress at booting stage,indicating the critical role of OsHAK26 in maintaining pollen activity particularly at dessicate environment.In conclusion,OsHAK1,OsHAK5 and OsHAK26 all affected pollen activity and pollen germination,and the mutations of OsHAK5 and OsHAK26 also inhibited pollen tube elongation,while the mutations of OsHAK26 resulted in the shorter baculum of pollen wall.4.The transcriptional expression of OsHAK27 was up-regulated by multiple abiotic stresses including K deficiency,high salts,osmotic stress,drought,and external ABA.The drought stress could enhance OsHAK27 expression in leaves by 120 times.The GUS staining of the transgenic line expressing OsHAK27 promoter fusion with GUS indicated that OsHAK27 was expressed in the parenchyma cells of root xylem,mesophyll cells and vascular bundle cells of the leaf blades,vascular bundles of the leaf sheath and enlarge vascular bundles(EVB)of the nodes.Subcellular localization analysis showed that OsHAK27 was localized in the plasma membrane and trans-Golgi network(TGN)as well.5.Functional analysis of OsHAK27 expressing in heterologous yeast cells indicated that OsHAK27 can selectively transport Na.For functional characterization of OsHAK27 in rice,knockout mutants of OsHAK27 by CRISPR/Cas9 gene editing and overexpressing lines of OsHAK27 driven by a constitutive promoter(35S)were generated in the background of Japonica cultivar Nipponbare.Relative to wild type,both the mutants and over-expressing lines accumulated more Na in the shoot grown at both high-salt(100 mM Na)and low-salt(20 mM Na)solutions,resulting in decrease of K/Na ratio in the shoot.However,Na content enriched in OsHAK27 over-expression lines could decreased to normal levels as that content in wild type,while Na content in crispr27 plants was still maintained high after transferring the plants to Na-free solution for 3-d.The mutation OsHAK27 increased the Na concentration in xylem sap,while no significant difference of K concentration in xylem sap and concentration of K and Na in phloem sap.OsHAK27 may reduce the accumulation of Na in the shoots by promoting the retrieval of Na in xylem vessels and reducing the distribution of Na to leaves in EVB of the nodes.The results suggest that OsHAK27 might be involved in perception of osmotic regulation in the shoot for K and Na balancing in addition to serve as Na transporter.Taking all the above results together,we conclude that OsHAK1,OsHAK5 and OsHAK26 are involved in maintaining the activity of pollen gamete fertility and OsHAK27 function in enhancing salt tolerance by balancing K to Na ratio.This study revealed that the OsHAK family genes,encoding the largest numbers of K transporters in rice,have different subcellular and tissue localization and functional diversification.The identification for the first time that OsHAK26 gene located in golgi body plays an important role in maintaining fertility and seed setting rate of rice,expanding the connotation of the molecular mechanism of K regulation in plant development.