Low-Affinity Na~+ Uptake And Accumulation In The Halophyte Suaeda Maritima

Salinity is an increasing problem for agricultural production worldwide as more and more field becomes salinized.If excessive amounts of Na⁺ enter the plant in the transpiration stream there will be injury to cells in the transpiring leaves and this may cause further reductions in growth.Plants,especially wild plants,developed varying mechanisms to adapt to salt stress,of which,reducing Na⁺ influx must be the key and efficient approach to control Na⁺ accumulation in plants,and hence to improve plant salt tolerance to salty environments.Low affinity Na⁺ uptake is toxic to the cytoplasm of most crop plants.Nevertheless,the pathways for plant low affinity Na⁺ uptake are not certain by now.The precise molecular identities of NSCCs,LCT1 and HAK in plant low affinity Na⁺ uptake are not known;HKT has varying function in plant Na⁺ transport and uptake processes in different species and most of work about its role in Na⁺ uptake just rest on heterologous expressions;the function of AKT1-type channels in plant Na⁺ uptake is uncertain.Furthermore,there is no perfect system currently that can be used to investigate Na⁺ uptake and accumulation in higher plants although high-affinity Na⁺ uptake in plant roots can be tested by the depletion method.To date,low affinity Na⁺ uptake pathways by plant roots have been explored using glycophytes(e.g.Arabidopsis, rice,or wheat)and a few halophytes(e.g.Thellungiella halophila,Puccinellia tenuiflora,and Mesembryanthemum crystallinum).Most of the glycophytes and some halophytes,such as T.halophila and P.tenuiflora(Wang et al.,2002;Wang et al.,2004; Wang et al.,2008)are relatively salt-excluding plants and have a strong selectivity for K⁺ over Na⁺,which restricts considerable Na⁺ uptake.The genus of Chenopodiaceae, the family with the highest proportion of halophytes,offers potential physiological models.The species of Suaeda in this genus,belonging to salt-accumulating halophyte plants,can accumulate substantial amounts of Na⁺ in their succulent shoots(about 400 mM NaCl without injury).The aim of this Ph.D.project,therefore,to characterize the low affinity Na⁺ uptake pathways and Na⁺ accumulation mechanism in Suaeda maritima in response to controlled NaCl treatments,in order to supply some basic theory for identification of the genes involved and manipulation of their expression.The main founding and conclusions are as follows:1.Our experiments showed that Suaeda maritima is a valuable plant material for characterizing Na⁺ uptake pathways in roots and transport pathways from roots to shoot in higher plants.And this plant makes it convenient and direct to investigate Na⁺ uptake and Na⁺ transport pathways in higher plants.We identified two low-affinity Na⁺ uptake pathways by ²²Na⁺ influx procedure using Ca²⁺(blocker of nonselective cation channels and low-affinity cation transporter),Li⁺(a competitive inhibitor of Na⁺ uptake),and TEA⁺, Cs⁺ and Ba²⁺(inhibitors of K⁺ channels).Our results further confirmed that neither NSCCS nor LCT1 were supported to be the major pathways for Na⁺ entry into root cells in plants;HKT-type transporter is a prime candidate for mediating the uptake of low-affinity Na⁺ under low salinity conditions(around 25 mM NaCl);AKT1-type channel might be involved in the absorption of Na⁺ at high external salt concentration (around 150 mM NaCl).2.We further found that the turning-point of external NaCl concentrations for the two low-affinity Na⁺ uptake pathways is between 90 and 95 mM of external NaCl.And the affecting pattern of K⁺ on Na⁺ uptake was consistent with this turning-point.3.HKT type transporters contribute less to Na⁺ uptake in wheat and rice than in S. maritima and also contribute to slight Na⁺ exclusion in wheat and rice under low salinity(25 mM NaCl);under high salinity(125 or 150 mM NaCl),HKT mainly function more as Na⁺ exclusion in wheat and rice than in S.maritima.4.Reducing root cell transmembrane H⁺ electropotential gradient established by H⁺-ATPase by NH₄⁺ together with blocking HKT-type transporter by Ba²⁺could result in significantly decreased root Na⁺ exclusion;We propose that at least two kinds of HKT-type transporters exist in roots of S.maritima:one kind functions as root Na⁺ uptake under low salinity condition;the other one plays roles in root Na⁺ exclusion under both low and high salinity condition.ABC transporter also might be another candidate of Na⁺ extrusion pump conferring salt tolerance in plants under high salinity condition.5.CCC transporter is also a candidate for mediating Na⁺ uptake in S.maritima, especially under high salinity condition,and might function in regulating hydraulic conductance in planta under high external Na⁺ or K⁺ concentrations by using bumetanide(CCC inhibitor).6.Low concentration of K⁺(5 and 10 mM NaCl)stimulate Na⁺ uptake and might activate AKT1 and under high salinity(100-200 mM NaCl). 7.Root Na⁺ concentrations(mM)reached their peak values of 140,205 and 310 mM,which is 5.6,1.4 and 1.5 times of the corresponding external salt concentrations,at 192,240 and 360 h after 25,150 and 200 mM NaCl treatments,respectively,and then decreased with the time of salt stress treatment;however,shoot Na⁺ concentrations reached their peak values of 376,616 and 715 mM,which is 15,4 and 4 times of the corresponding external salt concentrations,at the end(20d)of the three kinds of salt stress treatments.Shoot Na⁺ concentration(mM)increased suddenly between 6-12 h of the three salinity treatments,and then increased slowly.The changing pattern of ²²Na⁺ influx was in accordance with that of Na⁺ concentration(mM)in roots and shoots of S. maritima.All these indicate that Na⁺ exclusion can also occur even in salt-accumulating halophyte when exposed to salt solution for a certain term and abundant Na⁺ is accumulated in the plant.8.As a whole,K⁺ concentrations in shoots decreased significantly by 37.04%, 6.4.04%and 54.49%respectively;Na⁺/K⁺ ratio in roots increased significantly by 41,38 and 47 times,at the end of the three salinity treatments(25,150 and 200 mM NaCl); Na⁺/K⁺ ratio in shoots increased much more steadily and significantly by 47,156 and 147 times at the end of the three salinity treatments.