Involvement Of Potassium Transporter Genes Belonging To Hak Family In The Regulation Of Rice Growth And Development And Nitrate Nutrition

Potassium(K)is the most abundant cation in plants,which participate in many physiological and biochemical processes;while nitrate(NO3-)together with ammonium are the major form of nitrogen(N)acquired by plant roots.K+ is considered as charge balance for plant NO3-uptake and translocation.In plants,the K channels and K transporters are the major pathways for K+ uptake and redistribution.KUP/KT/HAK family of K transporters is the largest members of K+ transporters which mediate mainly the high-affinity K+ uptake in plants.Our previous studies demonstrated that both OsHAK1 and OsHAK5 K transporters play important roles in K acquisition and redistribution at low K supply condition.We observed that OsHAK1 knockout(KO)in rice resulted in root growth inhibition,lower effective tiller number and plant height and seeds setting rate.Since plant morphology is established and defined largely by polar auxin transport,therefore,we compared auxin distributions and polar transport in OsHAKl overexpresson(OX)and KO lines vs.their respective WT lines.The expression levels of auxin efflux carriers,PINs,which are considered as the limit factor for auxin polar transport were also detected in these transgenic lines and WTs.In addition,we explored the mechanism of OsHAK1 effects on rice seed setting ratio by comparing floral morphology and pollen germination rate between the oshakl KO lines and WT.Moreover,the roles of OsHAKl and OsHAK5 in rice NO3-uptake and distribution were examined by tracing 15N labeled NO3-uptake and distribution in the OX,KO line and respective WTs.The main findings are listed as follows:1.Knock out of OsHAK1 decreased the root hair density,the lateral root density and lateral root length at low K(0.1 mM)supply condition,while K concentrations in the lateral root of KO lines and WT were similar.This result implicates that the effect of OsHAK1 on the root morphology is not only due to the reduction in K concentration.2.It is wildly accepted that auxin polar transport patterns plant architecture.We compared auxin polar transport in OsHAK1 KO and OX lines to that of WT by using Non-invasive Micro-test Technique(NMT)to detect IAA flux rate.We found that OsHAK1KO reduced IAA flux at the root tip while OsHAK1 OX enhanced this process.We also found that the transcripts abundance of the auxin efflux transporter genes OsPIN1 and O.sPIN2 were altered in OsHAK1 KO lines in comparison to WT.By tracing[3H]-IAA polar transport in the root,we provided the direct evidences that OsHAK1 KO significantly reduced auxin basipeta1 transport but had little effect on its acropetal transport.3.Root gravitropism is regulated by auxin polar transport.We detected that OsHAKl KO impaired the root gravitropism and this phenomenal was modulated by pH of the culture medium.In view of the fact that IAA influx is conducted by IAA transporters,while protonated IAA may enter the cell by free diffusing and HAKs are H+/K+ cotransporters,we propose that alteration of auxin polar transport by OsHAKl in rice is due to the effect of this transporter on rice cell pH homeostasis.4.In comparison to WT,OsHAK1 KO postponed the rice flower development,reduced the length of stigma,and generated the malformed anther.The pollens from KO lines contained much less starch grains and their viability dropped by about 50%.In addition,the elongation rate of pollen tube from KO rice was much slower than that of WT.5.We quantified the effects of OsHAK1 KO on rice root NO3-acquisition.After growing the plants in media containing 0.3 mM K or 1 mM K,a short-term acquisition experiment of 15N-NO3-was conducted by exposing the root to 1.25 mM of labeled NO3-for 5min.Comparing to WT,OsHAKl knockout mutant showed significant lower NO3-uptake irrelevant with the K supply levels.6.We have previously shown that OsHAK5 is the rice high affinity K transporter sharing the highest homolog to OsHAK1 at both DNA and protein levels.In this study,we confirmed that OsHAK5 also affected the rice root NO3-uptake but not in the same level as that of OsHAKl,Unlike OsHAK1 KO,OsHAK5 KO reduced the short term NO3-uptake and translocation from leaf sheath to leaf blades only when the plants were grown at low K supply condition(0.3 mM).Taken together,we conclude that OsHAKl affects rice growth and development which can be explained by the following mechanisms:(I)OsHAK1 alters rice architecture likely due to its effect on cellular pH homeostasis which controls the auxin transport,(2)OsHAKl affects the seed setting rate possibly by its integrated effects on flower development and K controlled turgor maintenance.In addition,the different of OsHAK1 and OsHAK5 in regulating rice NO3-absorptions might be due to their difference in the K acquisition and translocation for providing K+ and NO3-charge balance and K+-NU3--malate shuttle.