increasing salinization of agricultural soils is one of the most challenging issues faced by modern agriculture . in excess of 30% of cultivated soils
are affected by salinity ( epstein et al . , 1980 ; zhu et al . , 1997
much of this salinization is attributable to the infiltration and accumulation of nacl ( zhu , 2001 ; munns , 2005 ) , often resulting in soil na concentrations above 40 mm , and growth suppression in most crops ( munns , 2005 ) .
one of the key physiological processes disrupted by na supply in this toxic range is the maintenance of cellular and whole - plant potassium homeostasis ( rains and epstein , 1967 ; flowers and luchli , 1983 ; watad et al . , 1991 ; gaxiola et al . , 1992 ;
, 1998 ; santa - mara and epstein , 2001 ; peng et al . , 2004 ; cakmak , 2005 ; kader and lindberg , 2005 ; kronzucker et al . , 2006
the ratio of k to na is considered an excellent indicator of plant tolerance to salinity ; the higher the ratio , the higher the plant 's tolerance ( flowers and hajibagheri , 2001 ; cakmak , 2005 ; chen et al .
as a result of this observation , selection or breeding cultivars that maintain high k : na ratios has emerged as an important strategy to counteract the detrimental effects of soil salinity ( deal et al . , 1999 ;
a more precise proposal has been that a high k : na ratio specifically in the cytosolic compartment is critical to plant survival under sodium challenge , while a decrease in this ratio will predict the onset of salinity stress and growth decline ( hajibagheri et al . , 1987 , 1989 ; maathuis and amtmann , 1999 ; flowers and hajibagheri , 2001 ; carden et al . , 2003 ; peng et al .
, 2004 ; kader et al . , 2006 ; james et al . , 2006
; chen et al . , 2007a ; davenport et al . , 2007 ; obata et al . , 2007 ; takahashi et al . , 2007
this proposal has gained wide acceptance , even though cytosolic k : na ratios are , in fact , rarely measured . in a recent study in barley ( hordeum vulgare l. ) , it was shown that , at low to intermediate levels of external k supply ( [ k]ext=0.11.5 mm ) , and at varying salinity levels , the ratio did not in fact correlate with seedling growth in this major cereal ( kronzucker et al . , 2006 ) .
on the contrary , no difference in growth was observed in the presence of a > 5-fold variation in the cytosolic k : na ratio .
the study further demonstrated that na suppressed k influx across the plasma membrane to a similar extent at 0.1 and 1.5 mm [ k]ext , concentrations at which high - affinity transport systems for k predominate ( epstein et al .
, 1963 ; kochian and lucas , 1982 ; szczerba et al . , 2006 ) , while na influxes and cytosolic pools were unaffected by k. however , a significant suppression of the cytosolic k pool by na was seen only at the higher [ k]ext , suggesting that different cellular responses may come into effect as high - affinity k transport gives way to low - affinity transport ( see szczerba et al . , 2006 ) .
in the present study , to examine further the proposed pivotal role of k homeostasis in salinity stress and tolerance , the effects of k supply across the low - affinity transport range of k ( up to 40 mm [ k]ext ) upon the primary fluxes and cytosolic pools of na , and , conversely , the effects of na upon k fluxes and pools in this range were investigated .
such an examination was particularly necessary in the light of recent disagreements in the literature pertaining to ( i ) the size of cytosolic na pools ( flowers and hajibagheri , 2001 ; carden et al . , 2003 ; james et al .
( ii ) the proposed , but as yet unresolved , roles of molecular candidates for toxic na influx into the plant ( tester and davenport , 2003 ; flowers , 2006 ; wang et al . , 2007 ) .
the primary candidates proposed are k - specific channels ( wang et al . , 2007 ) , non - selective cation channels ( nsccs ; demidchik et al . , 2002 ) , hkt - type transporters ( rodriguez - navarro and rubio , 2006 ) , and the low - affinity cation transporter lct ( amtmann et al . , 2001 ) .
indeed , were these candidates to catalyse toxicologically significant fluxes of sodium , they should be competitively influenced by the presence of potassium . for these reasons , a detailed study of the interactions between the two ions was carried out along a wide gradient of external k supply .
klondike ) were surface - sterilized by immersing seeds in 1.0% sodium hypochlorite for 10 min .
seeds were then washed under running tap water for 3 h , placed on discs of plastic mesh , and covered by 2 cm of moist sand .
germination proceeded for the following 3 d in a walk - in growth chamber equipped with fluorescent lights ( philips econ - o - watt , f96t12 , with an irradiation of 200 mol photons m s at plant height , for 16 h d ) , and having a day / night temperature cycle of 20 c/15 c , and relative humidity of 70% .
following germination , seedlings were transferred to opaque plastic 4 l hydroponic vessels , filled with modified quarter - strength johnson 's solution , consisting of : 5 mm ca(no3)2 , 0.5 mm nah2po4 , 0.25 mm mgso4 , 0.2 mm caso4 , 0.125 m na2moo4 , 20 m feedta , 25 m h3bo3 , 2 m znso4 , 0.5 m mnso4 , and 0.5 m cuso4 .
control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) .
potassium concentrations were adjusted to treatments of 1.5 , 5 , 10 , 20 , and 40 mm by addition of k2so4 .
solutions were replaced after 2 d. plants remained in hydroponic solutions for 4 d prior to experimentation . in select treatments ( 1.5 mm and 40 mm k , at low and high nacl ) , plants were also grown for 2 weeks ( 11 d in solution ; see fig .
compartmental analysis by tracer efflux was conducted as described in detail elsewhere ( kronzucker et al .
roots of intact seedlings were immersed for 1 h in a nutrient solution identical to the growth solution , except that it contained na or k in addition to non - radioactive na or k. roots were desorbed of radioactivity in tracer - free solutions for the monitoring of na or k efflux , by periodic washing with a timed series of non - radioactive aliquots of nutrient solution ( kronzucker et al . , 1999 , 2006 ;
the time course of aliquots was as follows : 15 s ( 4 ) , 20 s ( 3 ) , 30 s ( 2 ) , 40 s ( 1 ) , 50 s ( 1 ) , 1 min ( 5 ) , 1.25 min ( 1 ) , 1.5 min ( 1 ) , 1.75 min ( 1 ) , and 2 min ( 8 ) .
radioactivity was measured in aliquots and plant tissues by gamma counting , using a canberra - packard counter , quantum cobra series ii , model 5003 .
unidirectional na or k fluxes were determined using standard analyses ( for further details , see kronzucker et al .
efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) .
1).(ii )  net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux.(iii )  cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where  is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume .
activity coefficients (  ) for cytosolic concentrations were estimated using the debye  hckel  onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) .
( i )  efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) .
( ii )  net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux .
( iii )  cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where  is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume .
activity coefficients (  ) for cytosolic concentrations were estimated using the debye  hckel  onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) .
representative plots of k and na ( inset ) efflux from roots of intact barley seedlings , under varying ionic conditions .
roots had been preloaded for 60 min in radioactive solution , then eluted of radioactivity in a timed series of non - radioactive growth - solution aliquots .
dashed lines represent the slowest exchanging compartment ( cytosolic ) , with a minimum of 12 time points used for linear regression .
roots of barley seedlings were desorbed for 5 min in 10 mm caso4 to remove extracellular k and na .
roots and shoots were then separated , weighed , and oven dried for a minimum of 72 h at 8085 c , then pulverized and digested with 30% hno3 for a minimum of 72 h. k and na concentration was determined using a single - channel flame photometer ( digital flame analyzer model 2655 - 00 ; cole - parmer , anjou , qubec ) .
statistical analyses were conducted using one - way analysis of variance ( anova ) with the statistical package spss ( ver .
klondike ) were surface - sterilized by immersing seeds in 1.0% sodium hypochlorite for 10 min .
seeds were then washed under running tap water for 3 h , placed on discs of plastic mesh , and covered by 2 cm of moist sand .
germination proceeded for the following 3 d in a walk - in growth chamber equipped with fluorescent lights ( philips econ - o - watt , f96t12 , with an irradiation of 200 mol photons m s at plant height , for 16 h d ) , and having a day / night temperature cycle of 20 c/15 c , and relative humidity of 70% .
following germination , seedlings were transferred to opaque plastic 4 l hydroponic vessels , filled with modified quarter - strength johnson 's solution , consisting of : 5 mm ca(no3)2 , 0.5 mm nah2po4 , 0.25 mm mgso4 , 0.2 mm caso4 , 0.125 m na2moo4 , 20 m feedta , 25 m h3bo3 , 2 m znso4 , 0.5 m mnso4 , and 0.5 m cuso4 .
control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) .
potassium concentrations were adjusted to treatments of 1.5 , 5 , 10 , 20 , and 40 mm by addition of k2so4 .
solutions were replaced after 2 d. plants remained in hydroponic solutions for 4 d prior to experimentation . in select treatments ( 1.5 mm and 40 mm k , at low and high nacl ) , plants were also grown for 2 weeks ( 11 d in solution ; see fig .
compartmental analysis by tracer efflux was conducted as described in detail elsewhere ( kronzucker et al . , 1999 , 2006 ;
in brief , roots of intact seedlings were immersed for 1 h in a nutrient solution identical to the growth solution , except that it contained na or k in addition to non - radioactive na or k. roots were desorbed of radioactivity in tracer - free solutions for the monitoring of na or k efflux , by periodic washing with a timed series of non - radioactive aliquots of nutrient solution ( kronzucker et al . , 1999 , 2006 ;
the time course of aliquots was as follows : 15 s ( 4 ) , 20 s ( 3 ) , 30 s ( 2 ) , 40 s ( 1 ) , 50 s ( 1 ) , 1 min ( 5 ) , 1.25 min ( 1 ) , 1.5 min ( 1 ) , 1.75 min ( 1 ) , and 2 min ( 8 ) .
radioactivity was measured in aliquots and plant tissues by gamma counting , using a canberra - packard counter , quantum cobra series ii , model 5003 .
unidirectional na or k fluxes were determined using standard analyses ( for further details , see kronzucker et al . , 1999 , 2003 , 2006 ;
, 2001):(i )  efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) .
1).(ii )  net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux.(iii )  cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where  is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume .
activity coefficients (  ) for cytosolic concentrations were estimated using the debye  hckel 
onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) .
( i )  efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) .
net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux .
( iii )  cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where  is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume .
activity coefficients (  ) for cytosolic concentrations were estimated using the debye  hckel  onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) .
representative plots of k and na ( inset ) efflux from roots of intact barley seedlings , under varying ionic conditions .
roots had been preloaded for 60 min in radioactive solution , then eluted of radioactivity in a timed series of non - radioactive growth - solution aliquots .
dashed lines represent the slowest exchanging compartment ( cytosolic ) , with a minimum of 12 time points used for linear regression .
roots of barley seedlings were desorbed for 5 min in 10 mm caso4 to remove extracellular k and na . roots and shoots were then separated , weighed , and oven dried for a minimum of 72 h at 8085 c , then pulverized and digested with 30% hno3 for a minimum of 72 h. k and na concentration was determined using a single - channel flame photometer ( digital flame analyzer model 2655 - 00 ; cole - parmer , anjou , qubec ) .
statistical analyses were conducted using one - way analysis of variance ( anova ) with the statistical package spss ( ver .
figure 1 shows representative plots of k ( main plot ) and na ( inset ) release from roots of intact barley seedlings , previously labelled with respective tracers for 60 min .
steady - state tracer efflux curves of this type were analysed under five external potassium conditions ( 1.5 , 5 , 10 , 20 , and 40 mm ) in control ( 1 mm nacl ) or salt - treated ( 100 mm nacl ) plants , to determine unidirectional fluxes of na and k across the plasma membrane of root cells , kinetic constants for cytosolic exchange of the two ions , and estimates of the ions ' cytosolic activities . in fig .
1 , the main plot shows the potent reduction , by elevated na provision , of the efflux of k from barley roots ( note that the y - axis is logarithmic ) , a phenomenon paralleled in reductions in the influx and net flux of k ( see fig .
2 ) . by contrast , the inset of this figure shows the lack of a reciprocal effect : a 27-fold difference in k supply had no influence on the efflux of na under saline conditions .
components of unidirectional k influx in roots of intact barley seedlings , grown and monitored with or without 100 mm na .
error bars refer to sem of 414 replicates , with asterisks indicating significant differences in influx values within a given k treatment ( p  <  0.05 ) . where differences in influx between na treatments were observed , significant differences in efflux and net flux were also found ( p  <  0.05 ) .
figure 2 shows the steady - state unidirectional and net fluxes of k into barley roots , obtained using compartmental tracer analysis under the 10 conditions examined . over the range of k supply ,
the influx of potassium in salt - treated plants was lower than in controls ( by 2060% ) , significantly so in all cases except for 40 mm [ k]ext ( p  <  0.05 ) .
( 1985 ) showed that application of 3 mm na resulted in a 50% suppression of k influx in maize , but this effect was limited to the low - affinity transport range for k , and was only seen in  low - salt plants .
this result contrasts with the present study and with previous work in barley , which showed the suppression , by na , of k influx at the high - affinity supply provision of 0.1 mm ( kronzucker et al . , 2006 ) , as well as in the low - affinity range in the  high - salt plants ( grown at up to 40 mm k ) ; two possible explanations for this disagreement include the use of a much higher na provision ( 100 mm ) , and the examination of plants grown and tested under steady - state nutritional conditions .
consistent with the present work , effects of na on k uptake have also been observed in both high- and low - affinity transport ranges in a wide range of other studies ( rains and epstein , 1967 ; santa - mara et al . , 1997 ; fu and luan , 1998 ; rubio et al . , 2000 , 2003
net fluxes and unidirectional effluxes of k were also reduced under elevated nacl , significantly so in all cases except for the 40 mm k treatment ( fig .
the reduction of k efflux with high nacl provision ( figs 1 , 2 ) may appear , on the surface , to contradict the finding that na stimulated a net efflux of k from roots of several plant species ( see , for instance , chen et al .
, 2005 ; cuin and shabala , 2007 ) , but it must be pointed out that such demonstrations of na - stimulated k efflux involve short - term changes following nacl shock , whereas the present study involves measurements made under steady - state conditions . influx and efflux of na at 100 mm [ na]ext were high under all k conditions ( fig .
3 ) , and indicated substantial futile cycling of the ion at the plasma membrane .
rapid unidirectional influxes of na in glycophytes under salinity conditions have been observed by others ( essah et al . , 2003 ; wang et al . , 2006 ; davenport et al . , 2007 ;
2007 ) ; in particular , the influx values reported here are in excellent agreement with short - term na labelling data in a recent study by chen et al .
. the high ratios of efflux to influx seen consistently throughout the present treatments are also supported by previous studies ( cheeseman , 1982 ; mills et al . , 1985 ; essah et al . ,
in sharp contrast to the suppression of unidirectional and net k fluxes by na , a reciprocal influence was not seen . at 100
mm [ na]ext , the 27-fold variation in k supply resulted in no significant differences in influx , efflux , or net flux of sodium .
previous work on the interactions between these ions in the high - affinity k transport range ( kronzucker et al . ,
2006 ) also showed no significant differences in na between 0.1 mm and 1.5 mm external [ k ] .
the two studies together therefore show that a 400-fold range of k provision fails to modify na fluxes , while k fluxes across this range are generally suppressed by elevated sodium .
this lack of reciprocity was further confirmed by experiments conducted in 2-week - old seedlings of barley , indicating that the pattern was not limited to one developmental stage ( data not shown ) .
this conclusion , however , was not drawn in a classic study , also with barley , by rains and epstein ( 1967 ) : a strong reciprocal suppression of the flux of one ion ( k or na ) by the co - presence of the other was observed in the low - affinity  mechanism 2 of alkali cation transport. again , however , it is important to note that the present study differed from that of rains and epstein ( 1967 ) in that the fluxes in the present study were measured under steady - state nutritional supply conditions , which may be more relevant to plant performance in the field than perturbational conditions might be .
( 2007 ) also showed that k supply had no effect on na uptake in the halophyte suaeda maritima , but only when the external [ na ] was below 75 mm .
above that concentration , a suppression of na fluxes was found , indicating , in suaeda , the involvement of akt - type potassium channels in na influx under some conditions .
components of unidirectional na influx in roots of intact barley seedlings , grown and monitored at 100 mm na and at varying external k provision .
no significant differences between treatments were observed . in the present study , the steady - state ,
non - reciprocal effect of one ion on the fluxes of the other is borne out in the tissue accumulation patterns for k and na ( figs 4 , 5 ) . for potassium
, there was a general reduction of tissue content with salt treatment , particularly in the shoot , where tissue k levels were seen to decline by as much as 50% ( fig .
5 ; as with na fluxes , the accumulation of this ion was not changed over the range of applied external [ k ] , either in the root or in the shoot . tissue content of k in roots and shoots of barley plants , grown with or without 100 mm na , and under varying external k provision .
asterisks denote significant differences within a given k treatment and plant organ ( p  <  0.05 ) .
tissue content of na in roots and shoots of barley plants , grown at 100 mm na , and under varying external k provision .
no significant differences between treatments were observed . on a finer scale of analysis , a steady - state , non
- reciprocal interaction was also seen in the effects of ion supply on the activities of na and k in the cytosolic compartment of root cells ( ana+,cyt and ak+,cyt ; figs 6 , 7 ) , as estimated by compartmental analysis . under salinity conditions ,
ana+,cyt proved to be resistant to variations in k provision , showing no significant changes across the entire range of external k supply ( fig .
100 mm sodium provision resulted in severe drops in ak+,cyt , relative to low - sodium controls ( fig .
this effect was somewhat alleviated with increasing k provision , but , even at the highest [ k]ext , the pool size of k was reduced by nearly one - half .
again , this pattern of no reciprocal effect on cytosolic pool sizes of na and k was confirmed in 2-week - old barley seedlings ( not shown ) .
cytosolic k activity in roots of barley seedlings grown under varying k provision , with or without 100 mm na .
asterisks denote significant differences within a given k treatment ( p  <  0.05 ) .
cytosolic na activity in roots of barley seedlings grown under 100 mm na and varying k provision .
other workers , using techniques of x - ray microanalysis , radiotracer analysis , subcellular fractionation , and ion - selective microelectrodes , have reported very similar values for cytosolic activities of sodium ( harvey and flowers , 1978 ; yeo , 1981 ; mills et al . , 1985 ; amtmann and gradmann , 1994 ; flowers and hajibagheri , 2001 ; james et al . , 2006 ) and potassium ( pitman and saddler , 1967 ; vorobiev , 1967 ; macklon , 1975 ; memon et al . , 1985 ; beilby and blatt , 1986 ; maathuis and sanders , 1993 ; walker et al . , 1996 ) .
there have also been reports of suppressed cytosolic k pool by na ( jeschke and stelter , 1976 ; harvey et al . , 1981 ; mills et al . , 1985 ;
hajibagheri et al . , 1987 , 1988 , 1989 ; flowers and hajibagheri , 2001 ; carden et al .
a similar suppression of ak+,cyt has been observed to result from high ammonium ( nh4 ) provision in the same model system ( kronzucker et al . , 2003 ) , but , unlike the effect of na , this effect was only seen in the high - affinity k transport range ( 0.1 mm [ k]ext ) . by contrast , suppression of ak+,cyt by high na
was not observed at this level of k supply in previous work ( kronzucker et al . , 2006 ) , suggesting that k homeostasis is more resilient to salt stress in the high - affinity range .
the variability of the cytosolic k pool in response to both salinity and external k supply , and , by contrast , the uniformity of the na pool with changes in external k , together result in substantial ( greater than 4-fold ) changes in the ratio of the two cytosolic pools ( fig .
this k : na ratio peaked at 40 mm [ k]ext , and was minimized at 5 mm [ k]ext , but did not correlate with the growth of the experimental plants ( fig . 9a ; also seen in 2-week - old plants , fig .
2006 ) , this lack of correlation calls into question the view that the cytosolic k : na ratio is a critical determinant of plant growth in response to salinity stress ( see introduction ) .
on the other hand , the ratio of k to na on the coarser , tissue level , is rather uniform among salinity - treated plants ( fig .
8) , as is the growth of plants under these conditions , indicating that this measure is indeed more of a more accurate predictor of plant performance under salt stress ( see introduction ) .
this conclusion is supported by previously observed correlations between salinity tolerance and the tissue k : na ratio in other cultivars of barley ( flowers and hajibagheri , 2001 ; chen et al . , 2007b ) .
( 2007 ) showed that even this measure did not correlate with growth in 21 cultivars of bread wheat .
the authors explained this as possibly reflecting alternative strategies of salt tolerance in different cereal species .
cytosolic and tissue k : na ratios in roots of intact barley seedlings , grown at 100 mm na and varying k provision .
( a ) fresh weights of barley seedlings ( roots+shoots ) , grown and monitored with or without 100 mm na and varying levels of k supply .
asterisks denote significant differences within a given k treatment ( p  <  0.05 ) .
( b ) fresh weights of barley seedlings ( roots+shoots ) , grown and monitored with or without 100 mm na and two different [ k]ext ( 1.5 mm and 40 mm ) for 2 weeks .
error bars refer to sem of 582 replicates . in summary , neither na fluxes nor cytosolic na pools responded to k over a wide range of k supply [ 400-fold , when results from a previous work ( kronzucker et al . , 2006 ) are included ] , and elevated k provision was unable to rescue barley plants from the primary toxic entry of na , contrary to the rescue from nh4 toxicity that is effected by increasing external provision of k ( britto and kronzucker , 2002 ; kronzucker et al . , 2003 ; szczerba et al . ,
the non - reciprocal nature of potassium  sodium interactions in barley provides insight into the mediation of primary na entry into plant roots under toxicity - inducing conditions , an unresolved issue under much current debate ( schachtman and liu , 1999 ; golldack et al . , 2003 ; tester and davenport , 2003 ; qi and spalding , 2004 ; fuchs et al . , 2005 ; kader and lindberg , 2005 ; flowers , 2006 ; davenport et al . , 2007
; obata et al . , 2007 ; takahashi et al . , 2007 ; wang et al . , 2007 ) .
the present results suggest that several ion transporters favoured as candidates in the recent literature should likely be discounted , at least in barley .
these include , on the one hand , k channels , such as less selective members of the akt family of transporters , and , on the other , nsccs and the low - affinity cation transporter lct , which have been shown to allow the passage of a variety of cations under certain conditions ( amtmann and sanders , 1999 ; maathuis and amtmann , 1999 ; uozumi et al . , 2000 ;
; kader and lindberg , 2005 ; kader et al . , 2006 ; wang et al . , 2007 ) .
were members of these families of transporters , or of high - affinity k transporters ( takahashi et al . , 2007 ) , critically involved in toxic na influx , increasing external k concentrations over an extensive range as employed in the present study
would be expected to strongly reduce na influx by virtue of ion competition effects . while background levels of ca were high in the present study ( 5 mm ) , and it is recognized that this may have minimized to some extent na influx contribution from nsccs and lct transporters that are known to be ca - sensitive ( see rains and epstein , 1967 ; schachtman and liu , 1999 ; amtmann et al . , 2001 ; essah et al . , 2003 ; wang et al . , 2006 ;
) , it should be pointed out that ca levels in soils are also typically in excess of 1 mm ( reisenauer , 1966 ; hawkesford and miller , 2004 ) .
thus , if a major contribution from nsccs or lct can not be realized or rationalized under such conditions , the contribution from these transporters may also not be particularly relevant in the field ( schachtman and liu , 1999 ; for a similar conclusion , also see wang et al . , 2007 ) .
two recent studies have also suggested that k - specific shaker - type channels are unlikely candidates for na influx ; in one case , a knockout mutation of akt1 in arabidopsis thaliana resulted in reduced growth under na stress , showing that toxic entry of na can still proceed in the absence of akt1 ( qi and spalding , 2004 ) . in the other case ,
overexpression of kat1 in yeast cells resulted in lowered na accumulation ( obata et al . , 2007 ) .
another group of transporters whose role in primary na influx has been rigorously discussed is the hkt family .
it has been proposed that the most well - studied member of this group , hkt1;1 ( rubio et al . ,
1995 ) is a major determinant of sodium influx in arabidopsis ( rus et al . , 2001 ) , but other reports suggest that its function is in long - distance recirculation of na ( berthomieu et al . , 2003 ;
davenport et al . , 2007 ) . the strong suppression of hkt2-mediated na influx by even low amounts of external k in grasses ( laurie et al .
, 2002 ; horie et al . , 2007 ) suggests that this transporter is not likely to play a significant role in na uptake under normal soil conditions or in the present study .
however , other transporter types may emerge as mediators of toxic na entry into the plant ( horie et al . , 2001 ;
, 2003 ; haro et al . , 2005 ; ren et al . , 2005 ; kader et al . ,
in particular , the involvement of sodium - specific influx transporters remains a possibility , even though such systems have not yet been identified through arabidopsis genomic analysis ( see hua et al . , 2003 ) .
a transporter of this kind has recently been identified in arabidopsis ( colmenero - flores et al . , 2007 ) . clearly , however , a definitive answer has not been forthcoming , and the search for the primary na influx transporter mediating toxic plasma - membrane influx continues ( flowers , 2006 ) .
the present study indicates that , in barley , this transporter ( or transport system ) appears to be k - independent , suggesting that attempts to improve salt tolerance in this species , via increased selectivity of k uptake pathways , are unlikely to be effective .
 the interaction of sodium and potassium ions in the context of the primary entry of na+ into plant cells , and the subsequent development of sodium toxicity , has been the subject of much recent attention . in the present study , 
 the technique of compartmental analysis with the radiotracers 42k+ and 24na+ was applied in intact seedlings of barley ( hordeum vulgare l. ) to test the hypothesis that elevated levels of k+ in the growth medium will reduce both rapid , futile na+ cycling at the plasma membrane , and na+ build - up in the cytosol of root cells , under saline conditions ( 100 mm nacl ) . 
 we reject this hypothesis , showing that , over a wide ( 400-fold ) range of k+ supply , k+ neither reduces the primary fluxes of na+ at the root plasma membrane nor suppresses na+ accumulation in the cytosol . 
 by contrast , 100 mm nacl suppressed the cytosolic k+ pool by 4773% , and also substantially decreased low - affinity k+ transport across the plasma membrane . 
 we confirm that the cytosolic [ k+]:[na+ ] ratio is a poor predictor of growth performance under saline conditions , while a good correlation is seen between growth and the tissue ratios of the two ions . 
 the data provide insight into the mechanisms that mediate the toxic influx of sodium across the root plasma membrane under salinity stress , demonstrating that , in the glycophyte barley , k+ and na+ are unlikely to share a common low - affinity pathway for entry into the plant cell . 
Introduction
Materials and methods
Plant growth
Flux analysis
Tissue K
Statistical analysis
Results and discussion