Characteristics Of Na～+,K～+ Uptake And The Responses Of The Vacuolar Proton Pumps Of Wheat Seedlings To NaCl Stress

The growth of salt-tolerant wheat cultivar DK961 is markedly better than that ofsalt-sensitive wheat cultivar LM15, and the study of salt-tolerant mechanisms of wheat isbenificial for the breeding salt-tolerant wheat cultivars. Firstly, we examined whether DK961possessed more effective K⁺ uptake mechanisms compared with LM15, whether NaCltreatments of low concentraions caused more powerful induction on the high-affinity K⁺uptake system, whether nonselective cation channels (NSSCs) were the main pathways forNa⁺ entry into root of wheat. Secondly, if K⁺ nutrition is the key factor that improves the salttolerance of wheat based on the study of root proton extrusion was investigated. Finally, theresponses of PM H⁺-ATPase, V-ATPase and V-PPase to NaCl stress and their contributions tolimiting of Na⁺ influx, promoting of Na⁺ extrusion and compartmentation of Na+ into thevacuole were studied. The main results were shown as follows.1. Effects of NaCl treatments on the physiological index such as freshweight of two wheat cultivars200 mmol/L NaCl treatments led to a marked decrease in fresh weight of roots andleaves of two wheat cultivars, but the degree of the growth inhibition for DK961 was lowerthan that for LM15. Na⁺ content in roots and leaves of two wheat cultivars markedly increased,but K⁺ content and K⁺/Na⁺ ratio markedly decreased under NaCl stress. Na⁺ content in roots ofDK961 was higher than that in its leaves, however, Na⁺ content in leaves of LM15 was higherthan that in its roots. The degree of decrease in K⁺ content in roots and leaves of DK961 waslower than that in LM15. The K⁺/Na⁺ ratios in roots of two wheat cultivars under NaCl stresswere similar, but the K⁺/Na⁺ ratios in leaves of DK961 were 1.65, 1.82 and 1.83 times of thosein leaves of LM15 treated by 200 mmol/L NaCl for 1 d, 2d and 3d. These results suggestedthat DK961 root possessed more powerful ability of K⁺/Na⁺ selective uptake compared withLM15. NaCl treatment caused a marked decrease in Ca²⁺ content in roots and leaves, and thedegree of decrease increased with time at 200 mmol/L NaCl. The degree of decrease of LM15was markedly more than that of DK961, and Ca²⁺ content in leaves dereased more than that inroots. NaCl treatments induced a marked decrease in the osmotic potential in roots and leavesof two wheat cultivars, but the osmotic adjustment abilities in roots and leaves of DK961were stronger than those of LM15. Constitutive activities of SOD in roots and leaves ofDK961 were markedly higher than those of LM15. SOD activities in roots and leaves of twowheat cultivars increased under the conditions of NaCl treatments for 1 d and 2 d anddecreased on the third day, but SOD activities on the third day were higher than control.Under the conditions of NaCl treatments for 1 d, 2 d and 3 d, SOD activities in roots ofDK961 were 1.25, 1.29 and 1.26 times of those of LM15, and SOD activities in leaves ofDK961 were 1.23, 1.2, 1.24 times of those of LM15. The MDA content and plasmamembrane relative permeability in roots and leaves markedly increased under NaCltreatments, but the MDA content and plasma membrane relative permeability in roots andleaves of LM15 were markedly higher than those of DK961 under the same NaClconcentrations and time, which indicated that DK961 possessed stronger system ofscavenging ROS compared with LM15.2. Characteristics of K+ and Na+ uptake2.1 K+ Uptake curve The K+ uptake of wheat was biphasic, mediated by a high affinity (<1 mmol/L) and a lowaffinity (>1 mmol/L) transport system. Michaelis-Menten parameter values for K961 wereKm=22.26 μmol/L, Vmax=26.31 μmol/gDW/h and k=2.32 μmol g-1 DW h-1 mmol L-1 for thehigh-and low-affinity systems respectively, and Michaelis-Menten parameter values forLM15 were Km=27.15 μmol/L, Vmax=19.51 μmol/gDW/h and k=2.03 μmol g-1 DW h-1 mmolL-1. Compared with LM15, DK961 possessed more effective K+ uptake mechanisms undernormal conditions.2.2 Inductive and suppressive effects of NaCl treatments on K+ uptake system Km values for the high-affinity K+ uptake system of two wheat cultivars decreased andVmax increased under NaCl treatments of low concentrations (15 mmol/L and 25 mmol/L),which demonstrated that NaCl treatments of low concentrations had induction on thehigh-affinity K+ uptake system of two wheat cultivars, especially for DK961. However, NaCltreatments of high concentrations (35 mmol/L) inhibited the high-affnity K+ uptake system oftwo wheat cultivars, and NaCl treatments of low and high concentrations inhibited thelow-affinity K+ uptake system of roots of two wheat cultivars, but the inhibitive degree forDK961 was lower than for LM15.2.3 NSSCs were the main pathway for Na+ entry into wheat roots Experiments using inhibitors for ionic channels showed that the high-affinity K+ uptakesystem didn't mediate Na+ uptake into roots of wheat under low concentrations of K+and Na+.However, NSSCs were the main pathways for Na+ entry into wheat roots, and K+ channelswere the important pathways for Na+ entry into wheat roots under high concentrations of K+and Na+. Additionally, KCl treatments of low concentrations stimulated Na+ uptake.2.4 The sheath of DK961 possessed more powerful ability of selective transport of K+over Na+Compared with that of LM15, the sheath of DK961 possessed more powerful ability ofselective transport of K+ over Na+, i.e. limiting the transport of Na+ and promoting thetransport of K+ from sheaths to leaves, which contributed to maintain lower Na+/K+ ratio ofleaves under conditions of NaCl treatments. Therefore, sheath played a vital role in saltresistance of wheat.3. K+ nutrition markedly alleviated the inhibition of NaCl stress on rootproton extrusion NaCl treatments markedly reduced the ability of root proton extrusion of two wheatcultivars, especially under the conditions of K+ deficiency. Under the same concentrations ofNaCl, 1 mmol/L KCl in led to more powerful ability of proton extrusion. Intrestingly, it hadmore effective on DK961 than LM15. The PM H+-ATPase activity was to some extentdepedent on the external K+ based on the study of PM H+-ATPase hydrolysis activity underthe conditions of different concentrations of NaCl and KCl and of proton extrusion at low K+concentrations. These results showed that K+ nutrition played a vital role in the salt toleranceof wheat, especially for DK961. We obtained only one polypeptide exhibiting molecularmasses of 100 kDa by Western blot immunostaining in roots of the two wheat cultivars. Thechanges of PM H+-ATPase activity were consistent with the changes of protein amount of PMH+-ATPase.4. Responses of PM H+-ATPase to NaCl stress PM H+-ATPase activities in roots and leaves of two wheat cultivars increased on the firstday and decreased later under the conditions of 200 mmol/L NaCl treatments. Moreover, PMH+-ATPase activities of DK961 were higher than those of LM15 under the same treatmentsand time. We obtained only one polypeptide exhibiting molecular masses of 100 kDa by Westernblot immunostaining in roots and leaves of the two wheat cultivars. NaCl stress increased thePM H+-ATPase protein amount, especially under 200 mmol/L NaCl treatment for 1 d,although it did not induce new subunit. Western-blotting ananlysis showed that the increase inPM H+-ATPase activities in roots and leaves under 200 mmol/L NaCl treatments wasconsistent with the increase in the protein amount of PM H+-ATPase, additionally, PMH+-ATPase activities were regulated by other elements.5. The responses of V-ATPase and V-PPase to NaCl stress 200 mmol/L NaCl treatments led to enhancement of the V-ATPase and V-PPase activityof two wheat cultivars on the first and second day and inhibition later, and enhancement ofV-ATPase and V-PPase activity was partly due to the increase of their protein amount. AndV-ATPase and V-PPase activities in roots and leaves of DK961 were higher than those ofLM15 under the same treatments and time. These results suggested that DK961 possessed...