| Symbiotic nitrogen fixation is a process of interaction between microorganisms and plants.During the symbiosis,plants provide carbon sources for microorganisms,and in return,microorganisms convert molecular N2 in the air into ammonia available to plants.It has been documented that symbiotic nitrogen fixation contributes more than 60%of the totol amount of nitrogen to the world’s biological nitrogen fixation,providing a large amount of nitrogen nutrition for the agricultural system.However,it is also a process that requires a considerable amount of potassium(K),the most abundant cation in living plant cells,which plays crucial roles in many physiological processes,including photosynthesis,osmoregulation,membrane transport,enzyme activation.The K content in most soils is about 0.1-1mM.As a result of leaching losses and chemical fixation in soil,the availability of K+drastically fluctuates in large areas of agricultural land,and is often a limiting factor to crop quality and yields in the world.Transport of K+through plant membranes is mainly mediated by a number of transporters with different affinities that located in the plasma or organelle membranes.Plant K+transporters could be classified into four major families,KT/HAK/KUP,HKT,KEA and CHX families,in which KT/KUP/HAK family comprises most of the plant K+transporters identified so far,For example,a total of 27 and 13 KUP/HAK/KT genes have been identified in the model plants,rice and Arabidopsis,respectively.In Arabidopsis,multiple KT/HAK/KUP paralogous genes have been functionally characterized,including KUP1,KUP2,KUP4,KUP7 and HAK5.There are 32 KUP/HAK/KT genes in soybean,but limited information regarding the function of these genes is available to date.Moreover,the mechanism of K uptake and translocation in soybean nodules is unknown.Therefore,research on the physiological function of soybean K transporter is of great significance for revealing the physiological and molecular mechanisms of K uptake and translocation in soybean nodules and the cultivation of new K high-efficiency soybean varieties.In this study,the soybean(Glycine max,cv.Williams-82)was used to study the effect of K nutrition on nodule development and nitrogen fixation activity.A gene encoding a putative high-affinity K transporter of the KT/HAK/KUP family that was highly expressed in nodules was identified.Detailed expression and functional analysis of this gene was performed,The main findings were summarized as follows.1.Supply of appropriate amount of potassium can promote the growth of soybean plants inoculated with rhizobium(USDA110)and significantly increase the fresh weight,size and nitrogenase activity of nodules.However,treatment with different concentrations of K had no effect on the number of nodules.2.GmHAK5 was mainly expressed in roots and nodules,and to a less extent in stems.The expression of GmHAK5 in inoculated roots was higher than that of non-inoculated root.The expression of GmHAK5 showed a rapid response to the inoculation of USDA110,and the highest level was observed at 1d after the inoculation.Its expression in nodules was up-regulated by high potassium supply.Subcellular localization analysis showed that GmHAK5 encoding a plasma membrane-localized protein.Histochemical staining showed that the promoter of GmHAK5 could direct the GUS reporter gene expression strongly in the root steel,vascular bundles and epidermal cells of the nodule.GmHAK5 can restore the potassium absorption capacity of the K-absorbed yeast mutant at relatively low potassium concentations(0.5mM and 1mM K+).3.The potassium contents in roots and nodules of the gmhak5 mutant lines were significantly decreased as compared with those in the WT plants.Consistent with this,the nodulation of the gmhak5 mutants was also inhibited.In the case of low nitrogen(50μM)supply,the shoot biomass of the gmhak5 mutant lines was significantly reduced,and the potassium content in the roots,shoots and nodules was also significantly decreased.Compared with the control lines,the K content in the shoots of the gmhak5 mutant lines was reduced by 21.9%when treated with high potassium(2mM K+),and no significant difference was observed between the WT and mutant plants grown under the low K(20μM K+)conditions.The K content in the roots of the gmhak5 mutant lines was reduced by 26.6%,58.7%when treated with high K and low K,respectively.The difference could also be observed even in the absence of USDA110 infection.The K content in nodules of the GmHAK5 mutant lines decreased by 28.8%and 35.5%at high K and low K,respectively.The K content of the gmhak5 mutant lines decreased slightly compared to the WT with no inoculation.The number,size,fresh weight and nitrogenase activity of the gmhak5 mutant lines were significantly inhibited.In the high K treatment,the number,size,fresh weight and nitrogenase activity of the gmhak5 mutant lines decreased by 28.2%,36.2%,50.8%,and 29.8%,respectively,compared with the control lines.Moreover,the number,size,fresh weight and nitrogenase activity of the gmhak5 mutant lines grown under high K did not differ significantly from those grown under low K.4.Overexpression of GmHAK5 in soybean significantly promoted the plant growth and the K content in roots and nodules.The K contents in the shoots of the inoculated and non-inoculated overexpressing lines increased by 52.1%under the low K(50μM)supply conditions,and increased by 40.1%under the high K(2mM K+)supply conditions,compared to the control lines.The K content in roots of the overexpressing lines increased by 50%and 45.7%,respectively.The fresh weight,number,and nitrogenase activity of GmHAK5 overexpressing lines grown under high K increased by 40.1%,22.7%and 73.2%,respectively,compared with the control plants.The GmHAK5 overexpressing lines showed significantly higher N content in the inoculated soybean,plants especially under the high K conditions.The N content in the nodules was increased by more than one fold than that in the control plants.In summary,in this study,we demonstrated that K nutrition is important for nodule development and nitrogen fixation.GmHAK5 a putative high-affinity potassium transporter that is highly expressed in both the roots and nodules,might be involved in the uptake of K+by the roots and translocation of K+from the roots to nodules,which thus performs an essential role in regulating nodule development and nitrogenase activity. |
