Potassium transport and plant salt tolerance

Physiologia Plantarum 133: 651–669. 2007 Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317

REVIEW

Potassium transport and plant salt toleranceSergey Shabala* and Tracey A. Cuin

School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania 7001, Australia

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 8 October 2007

doi: 10.1111/j.1399-3054.2007.01008.x

Salinity is a major abiotic stress affecting approximately 7% of the world’s total

land area resulting in billion dollar losses in crop production around the globe.

Recent progress in molecular genetics and plant electrophysiology suggests

that the ability of a plant to maintain a high cytosolic K1/Na1 ratio appears to

be critical to plant salt tolerance. So far, the major efforts of plant breeders havebeen aimed at improving this ratio by minimizing Na1 uptake and transport to

shoot. In this paper, we discuss an alternative approach, reviewing the

molecular and ionic mechanisms contributing to potassium homeostasis in

salinized plant tissues and discussing prospects for breeding for salt tolerance

by targeting this trait. Major K1 transporters and their functional expression

under saline conditions are reviewed and the multiple modes of their control

are evaluated, including ameliorative effects of compatible solutes, poly-

amines and supplemental calcium. Subsequently, the genetic aspects ofinheritance of K1 transport ‘markers’ are discussed in the general context of salt

tolerance as a polygenic trait. The molecular identity of ‘salt tolerance’ genes is

analysed, and prospects for future research and breeding are examined.

Salinity as an issue

The extent of the problem

Global food production will need to increase by ap-

proximately 50% by 2050 to match the projectedpopulation growth (Flowers 2004, Rengasamy 2006). At

the same time, the most suitable land has already been

cultivated, implying a need for either expansion into new

areas to meet the above target or a dramatic increase in

crop production on existing cultivated lands. Given the

fact that about 15% of the total land area of the world has

already been degraded by various factors, and the other

half is ‘perennial desert’ or ‘drylands’ which can only bemade more productive by irrigation (Flowers 2004), both

tasks appear to be rather challenging. One of the major

threats to this is soil salinity.

Over 800 million hectares of land worldwide is

affected by salinity (Munns 2005), comprising nearly

7% of the world’s total land area. Irrigation systems are

particularly prone to salinization, with nearly one third of

irrigated land being severely affected. Despite persistent

questioning of the sustainability of irrigation as a method

to increase food production (e.g. Flowers and Yeo 1995

and references within), it is highly unlikely that suchpractices will be stopped in the near future. Taking

Australia as an example, the overall profit from irrigated

crops is about $6 billion annually, which comprises 20–

25% of Australia’s gross value for agriculture, although

irrigated land accounts for only 0.4% the country’s

farmland (Haw et al. 2000). Thus, it can be predicted

that such short-term economic benefits will prevail,

despite the above concerns.Most crops are glycophytes, thus are not capable of

growing in high concentrations of salt in the soil. Given

the above trends, improving crop salt tolerance is fast

becoming one of the key aspects of plant breeding in the

future. However, all attempts so far towards improving

crop salt tolerance through conventional breeding

Abbreviations – CPA2, cation:proton antiporter family 2; CNGC, cyclic nucleotide-gated channels; iGluRs, glutamate receptors;

NSCC, non-selective cation channels; KOR, outward-rectifying K1 channel

Physiol. Plant. 133, 2007 651

programmes have met with very limited success, primar-

ily because of the physiological and genetic complexity of

this trait (Flowers 2004). In this review, we discuss some

of the reasons behind this, and suggest a new approach

of breeding plants for salt tolerance by targeting K1

homeostasis in plant tissues.

Physiological constraints and adaptation to salineenvironment

Salinity affects plant growth by imposing both ionic and

osmotic stresses. Because of the osmotic gradient

generated, elevated Na1 levels in the soil solution drives

water out of the cell (Fig. 1). To maintain the cytosolicwater activity (required for normal cell metabolism),

water moves from the vacuole into the cytosol to

compensate for such depletion. As a result, the cell turgor

is reduced almost instantaneously, and a substantial

reduction in leaf and root elongation rate is observed

within minutes after the onset of salt stress (Yeo et al.

1991). The growth is restored, albeit at a slower rate,after plant cells adjust to new osmotic conditions

(typically, approximately 1 h after stress onset; Munns

2002). Not only the root, but leaf tissues are also exposed

to this osmotic challenge as a result of the accumulation

of high Na1 concentrations in the leaf apoplast (Flowers

et al. 1991). Cell osmotic adjustment is, therefore, critical

to plant salt tolerance. Surprisingly, such osmotic

adjustment is usually taken for granted, and the essenti-ality of this trait is typically ignored by plant physiologists

who focus predominantly on Na1 exclusion from uptake

(Munns 2005). The specific mechanisms of cell osmotic

adjustment will be considered in detail in the section

Potassium and cell osmotic adjustment.

The second major constraint is the specific Na1

toxicity. Because of the similarity in physicochemical

properties between Na1 and K1 (i.e. ionic radius and ionhydration energy), the former competes with K1 for major

binding sites in key metabolic processes in the cytoplasm,

such as enzymatic reactions, protein synthesis and

ribosome functions (Marschner 1995). With over 50

cytoplasmic enzymes being activated by K1 (Marschner

1995), the disruption to metabolism is severe, both in root

and leaf tissues. Several mechanisms may reduce specific

Na1 toxicity. At the cellular level, restricted Na1 uptake,active Na1 exclusion back to the soil solution (via the

plasma membrane salt overly sensitive (SOS1) Na1/H1

antiporter; Zhu 2003) and compartmentation of excessive

Na1 in the vacuole (by the tonoplast Na1/H1 exchanger;

Zhang and Blumwald 2001) are considered to be central

to salt tolerance. There is also growing evidence that

a cell’s ability to repair damage, scavenge reactive oxygen

species (ROS) and protect sensitive cellular structures byincreased the synthesis of a wide variety of chaperone-

acting proteins, may be at least as important as the above

traits. At the whole-plant level, plant adaptation to salinity

may be achieved by traits such as reduced Na1 delivery to

the shoot, Na1 recirculation in the phloem, tissue- and

organ-specific compartmentation (such as preferential

Na1 loading into physiologically ‘unimportant’ struc-

tures, e.g. senescing leaves, leaf sheath or epidermis),and, in some species, by Na1 secretion from the leaf

through, e.g. specialized structures such as salt glands or

bladders (see Tester and Davenport 2003 for review).

Another important mechanism is the recirculation of Na1

from the photosynthetic organs back to the roots. There

are strong suggestions that this process is mediated

by high affinity K1 transporter (HKT) transporters

K+

K+,Na+

SKOR

NORC

KEK+

H+

H+

ATP

K+

H+

K+

HUK

NSCC

HKT

KIR

KOR

ATP

H+

Na+

K+, Na+

K+, Na+

HKT

KAT2

AKT2/3

GluR2

K+

K+, Na+

K+

Na+

K+, Na+

Na+

K+

Water

water

Na+

K+

STOP

STOP

Leaf

Root

Stele Epidermis

Fig. 1. Adverse effects of salinity on K1 homeostasis and water relations

in plants. Elevated Na1 levels in the soil solution drive water out of cell

following the osmotic gradient (1). Not only root, but also leaf tissues are

exposed to this osmotic challenge as a result of accumulation of high Na1

concentrations in the leaf apoplast (6). Salinity also substantially reduces

K1 availability to roots as a result of reduced K1 activity in the soil solution

(2). Na1 also competes with K1 for uptake sites at the plasmamembrane,

including both low-affinity (3) and high-affinity (4) transporters. Finally,

salinity dramatically increases K1 leak through depolarization-activated

outward-rectifying (KOR) K1 channels (5).

652 Physiol. Plant. 133, 2007

(Davenport et al. 2007, Rus et al. 2004). Given the fact

that these transporters are also involved in K1 uptake by

plant tissues (Rubio et al. 1995), it is not surprising that

HKT transporters have recently become a major target for

strategies in breeding for salt tolerance (Byrt et al. 2007).

Another major constraint is salinity-induced nutritionaldisorders and, specifically, K1 deficiency. First, high

concentrations of Na1 in the soil substantially reduce the

activity of many essential nutrients (including K1),

making them less available for plants (Fig. 1). Secondly,

Na1 competes with K1 for uptake sites at the plasma

membrane, including both low-affinity [e.g. non-selec-

tive cation channels (NSCC)] and high-affinity (e.g. HKT)

affinity transporters (Fig. 1; see also section Major targetsfor genetic improvement). Moreover, when positively

charged Na1 crosses the plasma membrane, a significant

membrane depolarization (by 60–80 mV for 100 mM

NaCl treatment; Shabala et al. 2003, 2005a) is observed.

Such depolarization makes passive K1 uptake through

inward-rectifying K1 channels thermodynamically im-

possible and, at the same time, dramatically increases K1

leak through depolarization-activated outward-rectifyingK1 channels (KOR) (Fig. 1). Finally, increased de novo

synthesis of various compatible solutes used for osmo-

protection under saline conditions (see section Potassium

and cell osmotic adjustment for details) severely reduces

the available ATP pool, making high-affinity K1 uptake

even more problematic.

Surprisingly, salinity-induced perturbations in K1

homeostasis are usually considered of ‘secondary impor-tance’ (Tester and Davenport 2003) and are often ignored

by most researchers. We believe that this is wrong and

argue here that overcoming this constraint may open up

new avenues for plant breeding for salt tolerance.

Improving salt tolerance: some principles anddogmas of modern breeding

Thus far, all attempts at improving salt tolerance, either

via traditional breeding or via marker-assisted selection or

genetic engineering, has focused along two major lines:

(1) breeding for better Na1 exclusion and (2) breeding for

better osmotic adjustment. Unfortunately, neither of these

approaches has, so far, resulted in salt-tolerant cultivars in

the farmers’ field (Flowers 2004).

The first approach was specifically advocated for wheatbreeding (Munns et al. 2006). According to Munns et al.

(1999), plants grown at 200 mM NaCl should exclude

over 97% of all Na1 presented at the root surface,

regardless of whether they are glycophytes or halophytes.

As Na1 is toxic to cell metabolism (see above), high

cytosolic Na1 is detrimental for plants. Unfortunately,

exclusion of Na1 from root uptake per se does not solve

the problem of the osmotic component of salt stress. The

existing osmotic gradient will keep driving water out of

the plant, reducing cell turgor, and slowing root and leaf

expansion growth. Moreover, such massive Na1 exclu-

sion may lead to a substantial and progressive build-up of

high Na1 concentrations in cell walls or near the rootsurface, further exacerbating the problem of the osmotic

component of salt stress. This problem may be especially

severe in perennial species. Thus, it is unlikely that such

an approach of breeding for Na1 exclusion only would

result in a significant improvement of plant performance

in saline environments. At the very least, a better Na1

excluding ability in plants should be complemented by

better osmotic adjustment. Literature analysis shows thatthe latter is rarely the case.

Moreover, the central dogma of a negative correlation

between Na1 accumulation and plant salt tolerance

(Munns 2002, 2005, Tester and Davenport 2003) does not

hold in many cases. The most obvious exception is

halophytes, capable of accumulating very large amounts

of Na1 (up to 50% of shoot dry weight) without dying.

One can argue that these species live longer thenglycophytes under saline conditions, hence, accumulate

more Na1 as a result. However, this argument is rebutted

by the fact that optimal halophyte growth is observed at

soil salinities ranging between 200 and 400 mM NaCl

(Khan et al. 2005), conditions at which most crops would

rapidly die. The Chenopodium family is another example

(Jacobsen et al. 2003). In glycophytes, no correlation

between Na1 accumulation and salinity tolerance wasfound in Arabidopsis, with salt overly sensitive mutants

(such as sos1) showing lower shoot Na1 content than the

wild-type (WT) (Ding and Zhu 1997). Maize and rice are

also two notable exceptions (reviewed in Tester and

Davenport 2003). In tomato, salt-tolerant Lycopersicon

peruvianum accumulates a much higher concentration of

Na1 than salt-sensitive Lycopersicon esculentum (Santa-

Cruz et al. 1999). Finally, recent screening of over 40varieties of Australian bread wheat has also found no

relationship between Na1 exclusion and salt tolerance in

this species (Genc et al. 2007).

The second approach to improve salt tolerance in

plants is to overexpress genes responsible for biosynthesis

of the so-called ‘compatible solutes’. These are organic

osmolytes that can accumulate in the cytosol in sub-

stantial quantities without interfering with cell metabo-lism. They are involved in both cell osmotic adjustment,

hence maintenance of normal turgor pressure, and in

osmoprotection of various membrane structures and

proteins. Osmolyte accumulation has long been empha-

sized as a selection criterion in traditional crop breeding

programmes (Ludlow and Muchow 1990, Morgan 1983).

Indeed, recent progress in molecular biology has made

Physiol. Plant. 133, 2007 653

this approach central to molecular breeding programmes,

largely because of the fact that osmolyte accumulation is

often controlled by only one gene (Serraj and Sinclair

2002), making it easy to manipulate. Approximately 13

species were transformed with nearly 40 genes between

1993 and 2003 (Flowers 2004), with most of the genesrelated to the biosynthesis of compatible solutes.

However, while a large body of data indicate a positive

correlation between proline accumulation and adapta-

tion to salt or drought stress (Delauney and Verma 1993),

this is not corroborated by other studies, particularly from

transgenic plants (Chandler and Thorpe 1987, Hassan

and Wilkins 1988). Differences between WT and trans-

genic plants are usually either small, restricted to a narrowdevelopmental stage, or achieved in non-physiological

(e.g. in the absence of transpiration) conditions. Several

more drawbacks such as the high energy cost of

biosynthesis of compatible solutes (between 40 and

50 mol ATP per 1 mol of compatible solute synthesized;

Raven 1985), and increased susceptibility to fungal dis-

eases and lodging (Bohnert and Shen 1999), negate the

beneficial effect of high osmolyte content on salt toler-ance. As a result, practical outcomes of this approach are

only marginal (Bajaj et al. 1999) and do not result in an

improved salt tolerance of plants under field conditions

(Flowers 2004).

Potassium homeostasis and plant salttolerance

Intracellular K1/Na1 ratio as a key determinantof salt tolerance

With Na1 toxicity occurring as a result of its competitionwith K1 for enzyme activation and protein biosynthesis, it

is obvious that it is not the absolute quantity of Na1 per se,

but rather the cytosolic K1/Na1 ratio that determines cell

metabolic competence and ultimately, the ability of

a plant to survive in saline environments. Indeed, the

cytosolic K1/Na1 ratio has been repeatedly named as

a key determinant of plant salt tolerance (Colmer et al.

2006, Cuin et al. 2003, Dvorak et al. 1994, Gaxiola et al.1992, Gorham et al. 1991, Maathuis and Amtmann

1999). Surprisingly though, not much direct experimental

evidence has been presented supporting this. Further-

more, there is, unfortunately, a tendency in the literature

to use the ratio between shoot (or leaf) tissue K1 and Na1

content as an equivalent of cytosolic K1/Na1 ratio

(Botella et al. 1997, Cramer et al. 1991). Such ‘surrogate’

estimates are rarely justified as they fail to take intoaccount the intracellular compartmentation of each of

these two ions. Indeed, because the vacuole occupies the

majority of the intracellular volume in most mature plant

cells, changes in tissue K1 concentration are largely

a reflection of the behaviour of K1 within that compart-

ment (Walker et al. 1996). It is, after all, the cytosolic K1

homeostasis rather than the vacuolar content that is

essential for plant metabolic processes (Maathuis and

Amtmann 1999, Marschner 1995, Walker et al. 1996).The major hurdle is a lack of appropriate and convenient

techniques to enable such compartmentational analysis.

So far, several methods including NMR, energy-dispersive

X-ray microanalysis and multi-barrelled microelectrodes

have been employed (reviewed by Shabala 2006), all

supporting the concept of the cytosolic K1 homeostasis.

The optimal cytosolic K1/Na1 ratio can be maintained

by either restricting Na1 accumulation in plant tissues orby preventing K1 loss from the cell. Plant breeders (Ashraf

and Khanum 1997, Garthwaite et al. 2005) and cell

physiologists (Tester and Davenport 2003) have focussed

almost exclusively on the former mechanism. This is

surprising considering the growing bulk of evidence

demonstrating that the ability of plant ability to retain K1

in the cytosol may be crucial in achieving increased salt

tolerance. Indeed, it has been shown that the difference insalt sensitivity between bread wheat (hexaploid, ABD

genomes) and durum wheat (tetraploid, AB genomes) may

be in enhanced K1/Na1 discrimination, a feature con-

trolled by a Kna1 locus on chromosome 4D (Dubcovsky

et al. 1996, Dvorak et al. 1994, Gorham et al. 1991).

Moreover, comparisons between Arabidopsis and its

salt-tolerant relative Thellungiella halophila have shown

that the latter is capable of actually increasing mesophyllK1 content under saline conditions, while Arabidopsis

shows a ‘classical’ decline (Volkov et al. 2003). This

suggests that mechanisms are in place within mesophyll

cells to ameliorate ionic changes, protecting and main-

taining their photosynthetic and metabolic activity under

saline conditions. This is consistent with recent studies in

our laboratory showing that improving tissue K1/Na1

ratios by externally applied divalent cations enablenormal leaf photochemistry in plants, even under high

(100 mM) salinity conditions (Shabala et al. 2005b).

Potassium and cell osmotic adjustment

As mentioned above, osmotic adjustment is an essential

attribute of plant salt tolerance. Under saline conditions,

Na1 and Cl2 are cheap osmotica that can be used tomaintain normal cell turgor, assuming they are efficiently

compartmentalized in the cell vacuole. However, as salt

levels increase in the vacuole, a substantial osmotic

potential gradient is established between the vacuole and

the cytosol, reducing water activity in the latter. This issue

needs to be managed by a concurrent increase of

osmolality in the cytosol to match the accumulation of

654 Physiol. Plant. 133, 2007

vacuolar Na1. There are two major avenues by which this

can be achieved. One is de novo synthesis of compatible

solutes, and the other, an increased uptake of inorganic

ions. The first avenue was discussed briefly in the section

Improving salt tolerance: some principles and dogmas of

the modern breeding. It should be added though, that theconventional view of increased concentrations of com-

patible solutes in the cytosol being responsible for water

retention, so providing the turgor necessary for cell

expansion (Delauney and Verma 1993, Zhang et al.

1999), has been strongly challenged over the last decade

for a number of reasons. First, the absolute osmolyte

concentrations in stressed plants are too low for an overall

osmotic adjustment (Hare et al. 1998). Secondly, theenergetic cost of osmolyte production is far too high

(Raven 1985). Thirdly, synthesis of compatible solutes is

a rather slow process, operating over a timescale of hours

and days, so cannot be responsible for the observed rapid

recovery of plant growth. Thus, a direct contribution of

compatible solutes to osmotic adjustment is highly un-

likely. Instead, other roles have been proposed for these

solutes such as osmoprotection of functional componentsthrough their stabilizing or chaperoning properties

(Bohnert and Shen 1999, Hare et al. 1998).

Given the relatively minor direct contribution of

organic osmolytes to cytosolic osmotic potential (Chen

and Murata 2002), combined with their primary location

within chloroplasts (Papageorgiou and Murata 1995) plus

the fact that cytosolic Na1 concentrations rarely exceed

30 mM, even under severe salinity stress (Tester andDavenport 2003), it leaves K1 to fulfil the role of the major

osmoticum used in cell osmotic adjustment. Indeed,

onset of hypertonic stress does lead to an increased

uptake of K1 in various plant tissues (Shabala et al. 2000,

Teodoro et al. 1998, Zingarelli et al. 1999). We have

previously shown in Arabidopsis root epidermal cells that

>90% of the cell turgor is recovered by inorganic ion

uptake within 40 min after the onset of hyperosmoticstress (Shabala and Lew 2002), in good agreement with

whole-plant data (Azaizeh et al. 1992). This leads to the

question of whether such osmotic adjustment can occur

under saline conditions.

Under saline conditions, strong membrane depola-

rization caused by Na1 uptake favours K1 efflux via

depolarization-activated outward-rectifying K1 channels

(Shabala et al. 2006; Fig. 2). On the contrary, isotonicmannitol solution causes significant membrane hyperpo-

larization, resulting in increased K1 uptake as mentioned

above (Shabala et al. 2000, Shabala and Lew 2002;

Fig. 2). These two facts highlight the importance of

voltage gating and implicate Shaker-type K1 channels

(see section Regulation and functional expression of K1

transporters under saline conditions for details) as

possible downstream targets during cell osmotic adjust-

ment. This is further supported by experiments withArabidopsis K1 transport mutants. No substantial differ-

ence in NaCl-induced K1 efflux was found between WT

Columbia and the akt1 mutant, while gork roots showed

a much more attenuated response to salinity treatment

(Fig. 3A), with a strong correlation between membrane

potential changes and net K1 flux (Fig. 3B). Conversely,

hyperosmotic mannitol treatment caused similar K1

uptake in WT and gork roots, but had essentially noimpact on K1 fluxes in akt1 roots (Fig. 3C).

Earlier Palmgren (1991) suggested that the relaxation of

the stretched status of the membrane might activate the

plasma membrane H1-ATPase, thus providing hyperpo-

larization of the plasma membrane under hypertonic

conditions (as depicted in Fig. 2; see also Shabala and

Lew 2002 for experimental evidence). Depending on the

severity of salt stress, such hyperpolarization caused bythe osmotic component of salt stress, could be overcome

by the much stronger depolarization resulting from

a massive Na1 influx across the plasma membrane

(Fig. 2). Further support for this model was found in

direct experiments with isotonic NaCl and mannitol

Fig. 2. Cellular mechanisms involved in the perception of ‘ionic’ and

‘osmotic’ components of salt stress. Elevated NaCl levels in the soil

solution depolarize cell plasma membrane as a result of massive Na1

influx via NSCC channels. At the same time, changes in solution

osmolality are sensed by some putative osmosensor and are translated

into increased activity of H1-ATPase pump, resulting in membrane

hyperpolarization. The downstream targets of each of these components

are voltage-dependent hyperpolarization-activated (KIR) and depolariza-

tion-activated (KOR) Shaker-type K1 channels. Depending on severity of

the salt stress, one of the components dominate, resulting in either

increased K1 uptake (at mild salinity levels), or in a massive K1 leak (at

more severe concentrations) from the cell. Increased H1-pump activity

might also provide additional driving force for the high-affinity K1 uptake

via HAK/KUP transporters.

Physiol. Plant. 133, 2007 655

solutions in barley roots (Chen et al. 2005). Relatively low

changes in water activity (0.05 MPa) caused a slight

increase in K1 uptake, regardless of the nature ofosmoticum used (e.g. NaCl or mannitol). In contrast,

when severe osmotic stress was applied (0.2 MPa), K1

was taken up in response to mannitol treatment, but was

lost in response to NaCl treatment (Chen et al. 2005).

The above data strongly indicate that different ionic

mechanisms are involved in the perception of ‘ionic’ and

‘osmotic’ components of salt stress, invoking different

Shaker-type K1 channels as downstream targets of each ofthese components. However, given the plethora of K1

transport systems in plants (see section Salt tolerance as

a polygenic trait), it would be naive to assume that these

Shaker-type K1 channels are the only transport systems

involved in K1 homeostasis under saline conditions. K1-

permeable stretch-activated channels are also reported

(Cosgrove and Hedrich 1991), and pharmacological and

patch-clamp experiments have suggested that a substan-tial part of NaCl-induced K1 efflux may be mediated by

NSCC (Shabala et al. 2006). Hence, any strategy to

reduce NaCl-induced K1 leak through one of these

transport systems would assist in a better retention of K1

in the cell cytosol and, eventually, contribute to betterosmotic adjustment. The next section discusses possible

regulatory mechanisms of such K1 efflux systems by

a range of chemicals known to ameliorate the detrimental

effects of salinity.

Potassium and amelioration of detrimental effectsof salinity

There are several possible practical ways of minimizing

the detrimental effects of salinity on crop yield. These

include (1) amelioration by supplemental Ca21, (2)

exogenous application of compatible solutes or poly-

amines and (3) use of transgenic crops expressing

elevated levels of compatible solutes or polyamines. It

appears that each of these approaches targets K1

homeostasis and transport across cell membranes,improving the cytosolic K1/Na1 ratio and assisting cell

osmotic adjustment.

R2 = 0.88

–90

–80

–70

–60

–50

–40

–30–180 –160 –140 –120 –100

–220

–170

–120

–70

–20

30

–5 0 5 10 15

WT

gork

akt1

Net

K+ fl

ux (

nmol

m–2

s–1

)

Time (min) K+ flux (nmol m–2 s–1)

Mem

bran

e po

tent

ial (

mV

)

100 mM NaCl

Time (min)

–150

–100

–50

0

50

100

150

0 10 20 30 40 50

akt1

WT

300 mM mannitol

Fig. 3. Net K1 flux responses (efflux negative) measured from mature root epidermis of 8-day-old Arabidopsis plants (WT Columbia and two K1

transport mutants, gork and akt1, lacking outward- and inward-K1-rectifying channels, respectively). (A) NaCl-induced K1 transients. Data are

mean � SE (n ¼ 6–8). For growth details and composition of the bath solution, see Shabala et al. (2006). (B) Correlation between NaCl-induced K1 flux

changes and changes in root membrane potential measured inWT plants. One (of five) representative example is shown. (C) Transient K1 flux responses

after hyperosmotic stress caused by mannitol addition to the bath solution. One (of four) representative example is shown.

656 Physiol. Plant. 133, 2007

Application of supplemental Ca21 significantly ameli-

orates salinity stress symptoms in many species (Cramer

et al. 1987, Reid and Smith 2000, Shabala et al. 2003) and

is a widely used agricultural practice. For a long time,

such amelioration was attributed to Ca21 restriction of

Na1 uptake via NSCC: a major route for Na1 uptakeunder saline conditions (Apse and Blumwald 2007,

Demidchik and Tester 2002, Tester and Davenport

2003). Recently, we have provided evidence for an

additional mechanism of Ca21 action on salt toxicity in

plants: inhibiting Na1-induced K1 efflux through out-

wardly directed, K1-permeable channels (Shabala et al.

2006). Patch-clamp experiments have shown the pres-

ence of two populations of Ca21-sensitive K1 effluxchannels, showing different voltage dependence, activa-

tion kinetics and sensitivity to Na1. We have also shown

that not only Ca21 but also other divalent cations (Mg21,

Ba21, Zn21) may prevent K1 leakage in response to

salinity (Shabala et al. 2005b), thus assisting in maintain-

ing the high K1/Na1 ratio required for optimal plant

growth and leaf photosynthesis.

It has also been shown that salt-resistant plant varietiescontain higher polyamine levels under saline conditions

(Basu and Ghosh 1991, Erdei et al. 1990) and that their

exogenous application can ameliorate detrimental salin-

ity effects (Ndayiragije and Lutts 2006). However, the

underlying cellular mechanisms remain elusive. We have

recently shown that one of the modes of action of

polyamines may be through their blockage of NSCC

(Shabala et al. 2007). This reduces the magnitude ofNaCl-induced plasma membrane depolarization and, in

turn, the depolarization-induced K1 efflux through KOR.

In addition to this mechanism, polyamines may directly

block outward K1 currents through NSCC and activate

the plasma membrane H1-ATPase, so restoring the

membrane potential (Shabala et al. 2007). Once again,

cytosolic K1/Na1 ratio will be the main beneficiary of

such polyamine regulation of membrane transportactivity.

Molecular engineering of transgenic species over-

expressing genes responsible for biosynthesis of various

compatible solutes has long been, and remains, a major

avenue for improving plant salt tolerance (Bohnert et al.

1995, Bray 1997). In addition, numerous reports have

suggested that crop performance under salinity may be

improved by exogenous application of compatiblesolutes in the form of either aerial sprays or seed priming

(Harinasut et al. 1996). It has been proposed that the role

of compatible solutes in cytosolic osmotic adjustment

may be indirect through the plethora of regulatory or

osmoprotective functions (Hare et al. 1998, Hasegawa

et al. 2000). One such regulatory function appears to be

the maintenance of cytosolic K1 homeostasis by prevent-

ing NaCl-induced K1 leakage from the cell (Cuin and

Shabala 2005), most likely achieved through the

enhanced activity of the H1-ATPase. This would control

voltage-dependent outward-rectifying K1 channels and

create the electrochemical gradient necessary for sec-

ondary ion transport processes. Indeed, the activation ofproton pumps by salt stress is positively correlated with

salt tolerance (Golldack and Dietz 2001), and the effect is

stronger in salt-tolerant than in salt-sensitive species (Kefu

et al. 2003). Furthermore, such an increase in proton

pumping would, at the same time, provide the driving

force for a plasma membrane Na1/H1 exchanger to move

Na1 from the cytoplasm into the apoplast (Ayala et al.

1996), thereby reducing the cytosolic Na1 load. This,alongside increased K1 uptake, would further improve

the K1/Na1 ratio.

The above findings were further supported by experi-

ments testing 26 protein and non-protein amino acids (a

major class of compatible solutes) applied to plant roots at

physiologically relevant exogenous concentrations.

Twenty one of these caused a significant mitigation of

NaCl-induced K1 efflux (Cuin and Shabala 2007). It wasalso shown that the potential mitigating effect of amino

acids on salt-induced K1 efflux is dependent on the type

of amino acid supplied, shows strong dose dependency,

acts from the cytosolic side and correlates with the extent

of membrane depolarization. Evidence for the additive

effects of amino acids on NaCl-induced K1 efflux was

also presented, suggesting that it is not the increase in the

total amino acid content of plants per se but the actualratio between different types of amino acids that has the

mitigating effect on K1 homeostasis in salinized plant

tissues.

Our understanding of the processes behind the

mitigating effect of these compatible solutes is severely

limited and can be attributed to literally thousands of

components of a complex signalling network within the

cell. Only by showing the specific ionic and molecularmechanisms underlying their regulation and influence

over K1 (and Na1) transport will we be able to increase

the efficiency of the mode of action, possibly through

genetic engineering of salt-tolerant crops.

Breeding for salt tolerance by targetingK1 homeostasis

Salt tolerance as a polygenic trait

There is little doubt that salt tolerance is a complex

multigenic trait showing heterosis, dominance and

additive effects (Flowers 2004, Fooland 1997). Salt

tolerance is also multifaceted physiologically, with

numerous tissue- and age-specific components involved.

Physiol. Plant. 133, 2007 657

As such, salt tolerance will be determined by a number of

subtraits (specific for each particular species), any of

which might, in turn, be determined by any number

of genes. It is estimated that salinity affects the level of

transcription of approximately 8% of all genes (Tester and

Davenport 2003). To add to this complexity, fewer than25% of the salt-regulated genes are salt stress specific

(Ma et al. 2006), with a complex set of pathways observed

in response to salinity stress. Alterations in the regulation

of gene expression and metabolic adjustment in response

to salinity share common elements tootherabiotic stresses,

and it is very difficult (or even impossible – at least to our

current knowledge) to separate these components. Using

the microarray technique in the aforementioned study,Ma et al. (2006) analysed 1000 highly upregulated and

the 500 most downregulated salt-responsive genes,

grouped in 22 clusters, of which only one (!) contained

what could be called salt-specific upregulation. The latter

is, strictly speaking, an overstatement, as some moderate

induction under drought conditions was also observed.

And even in this case, the above cluster contained 171

genes, 60 of which had no known function! Not sur-prising, improving salt tolerance has always been, and

remains a great challenge for generations of breeders

(Flowers 2004, Flowers and Yeo 1995).

As a consequence of having a large number of

component traits of comparable importance conferring

salt tolerance, several points should be raised (Garcia

et al. 1997). First, no individual trait will necessarily

correlate with overall plant performance. Secondly,tolerance could be improved by a favourable recombi-

nation of the trait. Finally, selection should be for the

component trait and not for overall performance. The last

point is especially important, because most traditional

breeding is carried out based on plant vigour or yield.

Unsurprisingly, all attempts at improving crops’ salt

tolerance through conventional breeding programmes

have met with very limited success (Flowers 2004). Theprospect of changing a phenotype through genetic

manipulation or through conventional breeding is much

greater if one or a few defined regions of chromosome are

targeted rather than by generating a desired phenotype by

changing a large number of genes, each with a small

effect, scattered all over the genome (Koyama et al. 2001).

As discussed below, there is some cautious optimism that

this may be the case if breeding for salt tolerance isachieved through targeting K1 homeostasis.

NaCl-inducedK1 leak as ameasureof salt tolerance

Using seven barley cultivars contrasting in their salt

tolerance, a comprehensive study was undertaken to

correlate whole-plant responses to salinity (changes in

growth rate, biomass, net CO2 assimilation, chlorophyll

fluorescence, root and leaf elemental and water content)

and the ability of plant roots to retain K1 in the cytosol.

The latter trait was estimated by the magnitude of NaCl-

induced K1 efflux from root epidermis measured by non-

invasive ion selective microelectrodes (Chen et al. 2005).A very strong negative correlation (r2 > 0.8) was found

between the magnitude of K1 efflux from the root and salt

tolerance of a particular cultivar. The average K1 efflux

40 min after salt application ranged between 20 and

25 nmol m22 s21 for salt-tolerant cultivars, and between

150 and 180 nmol m22 s21 for salt-sensitive cultivars

(for 80 mM NaCl treatment). The effect showed a clear

dose dependence and was retained in seedlings ofdifferent ages (Chen et al. 2005). Thus, although hundreds

of different genes may be involved in conferring salt

tolerance, it appears that over 80% of the genetic

variability may be attributed to just one physiological

trait, namely a cell’s ability to prevent NaCl-induced K1

leak. Surprising as it may be, such an unexpectedly high

correlation may be explained by the fact that the observed

K1 efflux may be an integral characteristic of manydifferent factors controlling intracellular K1 homeostasis.

In other words, NaCl-induced K1 leak can be judged as

the ‘integrated’ measurement of tissue Na1 tolerance. It

is also important to emphasize that the difference in

the magnitude of K1 efflux between salt-tolerant and

susceptible genotypes was six-fold – much wider than the

genotypic dispersion for any of the other physiological

parameters measured (root Na1 content, 1.2-fold; flagleaf sap osmolality, 1.7; plant biomass, 2.2; net CO2

assimilation, 2.3) (Chen et al. 2005).

Further validation of K1 flux measurements as a screen-

ing tool for salt tolerance in barley was performed on

crosses between tolerant and sensitive barley cultivars

(Chen et al. 2005). The lowest K1 efflux was measured

from the two tolerant cultivars and the cross between

them, while the highest K1 efflux was observed fromsensitive cultivars and the cross between them. Crosses

between tolerant and sensitive cultivars showed inter-

mediate K1 efflux. These intermediate responses of F1s

suggest additive genetic control of salinity tolerance and

indicate inheritance of the K1 retention trait. The latter

issue is discussed in detail in the section Inheritance of K1

transport ‘markers’.

A large-scale trial was conducted to follow-up thiswork (Chen et al. 2007a). Nearly 70 barley cultivars of

various agronomic and genetic traits (e.g. winter vs

spring, feed vs malt, six-rowed vs two-rowed, husked vs

huskless, awned vs awnless) were grown to harvest

under saline conditions in a glasshouse over two con-

secutive years. In a parallel set of experiments, plant salt

tolerance was evaluated by non-invasive microelectrode

658 Physiol. Plant. 133, 2007

measurements of net K1 flux from roots of 3-day-old

seedlings of each cultivar following 1-h treatment in

80 mM NaCl. Sixty two of 69 cultivars followed an

inverse relationship between K1 efflux and salt tolerance

(Chen et al. 2007), with r2 ¼ 0.71 found between the

magnitude of K1 efflux and the overall ranking score(judged by six physiological traits such as relative grain

yield, shoot biomass, plant height, CO2 assimilation,

survival rate and thousand seed weight). Interestingly, the

few remaining ‘exceptions’ had a superior ability to

prevent Na1 accumulation in plant leaves, thus main-

taining a higher K1/Na1 ratio by this means. Therefore, it

appears that in 100% of plants, salt tolerance was

determined by the ability to maintain a higher K1/Na1

ratio; 90% of genotypes (62 of 69) achieved this by

retaining cytosolic K1, while about 10% (7 of 69)

achieved this by efficiently excluding Na1 from the

shoot. Taken together, our results show that a plant’s

ability to maintain high K1/Na1 ratio (either through

retention of K1 or by preventing Na1 from accumulating

in leaves) is a key feature for salt tolerance in barley, and

that in most genotypes this is achieved by K1 retentionrather then Na1 exclusion. This questions the conven-

tional view of Na1 exclusion being the major avenue to

optimize intracellular K1/Na1 ratio (Munns 2005, Tester

and Davenport 2003). Furthermore, it is consistent with

reports of Dvorak et al. (1994) who found that a novel

tetraploid wheat germplasm with enhanced K1/Na1

discrimination (via Kna1 locus) showed leaf Na1 con-

centrations not significantly different from its durumparents, whereas K1/Na1 ratios were substantially higher.

It also appears that similar patterns are characteristic in

other species (e.g. lucerne, unpublished results from our

laboratory). A large-scale experiment on other cereals

(durum and bread wheat) is currently in progress to

validate this concept.

Inheritance of K1 transport ‘markers’

Wide hybridization remains a false hope in terms of

practical breeding, as the damage to the crop genome by

alien genetic material almost always outweighs any

advantage (if there was one) of introducing alien genes

in the first place (Flowers and Yeo 1995). Therefore,

pyramiding component physiological traits remains the

most efficient avenue to improve plant salt tolerance,assuming such traits are inheritable.

Unexpectedly, there have been only a limited number

of studies in which the heritability of salt tolerance have

been reported, as well as very few measurements of the

heritability of particular traits for which the underlying

physiological or anatomical basis is understood (Garcia

et al. 1997). Indeed, most breeders focus on such

unspecific characteristics as seed germination (Mano

and Takeda 1997) or shoot or root growth (Ashraf et al.

1986). Nonetheless, high heritability of salt tolerance

based on Na1 and K1 uptake and the K1/Na1 ratio has

been reported (Gregorio and Senadhira 1993). K1/Na1

discrimination has also been a subject of QTL analysis forsalt tolerance in some cereals (Koyama et al. 2001,

Lindsay et al. 2004), and these reports have suggested that

the overall K1/Na1 ratio is heritable, although not in all

species (Garcia et al. 1997).

The genetics of salt tolerance is rather complex, with

additive, dominance and overdominance gene effects

reported (Flowers 2004). For example, Gregorio and

Senadhira (1993) found that the Na1/K1 ratio in rice wasgoverned by both additive and dominance gene effects.

In addition, Mano and Takeda (1997) found that salt

tolerance of barley at the germination stage was con-

trolled by overdominant alleles, and non-additive genetic

variance was larger than additive genetic variance.

In contrast, at the seedling stage, tolerance was pre-

dominantly controlled by additive genes, with also some

effects of dominance. The extent to which K1 transport‘markers’ are inherited, and which of above patterns are

attributed to this trait, remains to be answered.

A half diallel cross was made in our laboratory between

six barley cultivars contrasting in salt tolerance to test the

genetic model and also to study the inheritance of the K1

efflux trait in plant roots under saline conditions.

Significant differences were found between different

parents or F1 populations. The average values of root K1

flux of F1 progenies were very close to the midparent

values, while those of F2 populations were significantly

lower (i.e. greater efflux) than the midparent values,

indicating the dominance of salt tolerance (Z. Chen,

M. Zhou, N. Mendham, I. Newman and S. Shabala,

University of Tasmania, Hobart, unpublished data).

Variance and covariance analysis showed the existence

of epistatic effects, which was confirmed by further testsusing six different populations (parents, F1, F2, BC1 and

BC2) from two different crosses. However, the tolerance

was controlled mainly by additive effects, with relatively

smaller contributions from dominant and epistatic effects.

Based on a single MIFE (UTas Innovation, Hobart,

Australia) measurement, the estimated heritability was

relatively low (between 0.73 and 0.82). Using the average

values from around 10 measurements, the heritability ofthe trait increased dramatically, being 0.97, 0.98 and

0.97, respectively, from ANOVA of parents and F1s, diallel

analysis of F1s and diallel analysis of F2s (Z. Chen,

M. Zhou, N. Mendham, I. Newman and S. Shabala,

unpublished data). Such extremely high heritability of

NaCl-induced root K1 flux may also be the result of the

relatively smaller environmental effects of the MIFE

Physiol. Plant. 133, 2007 659

measurement compared with the many factors affecting

field experiments. Overall, our results confirm a high

heritability for salt tolerance in barley and show that

plants could be effectively selected and bred for salt

tolerance based on the NaCl-induced root K1 flux

measurements.

Molecular identity of ‘salt tolerant’ genescontrolling potassium homeostasis

Regulation and functional expression of K1

transporters under saline conditions

Potassium transport across plant membranes is mediated

by at least seven major families of cation transporters (75

genes in total in Arabidopsis). As this topic has been

extensively reviewed over the last few years (Cherel 2004,

Cuin and Shabala 2006, Gierth and Maser 2007, Lebaudyet al. 2007, Maser et al. 2001, Very and Sentenac 2002,

2003), only a brief summary is given here; the major

emphasis is on their functional expression under saline

conditions.

In general, two major groups are distinguished:

(1) three families of potassium permeable channels and

(2) three families of potassium transporters. The first group

includes (a) Shaker-type potassium channels (9 genesin Arabidopsis), (b) ‘two-pore’ potassium channels

(6 genes), as well as (c) NSCCs. The latter are further split

into (i) cyclic nucleotide-gated channels (CNGC)

(20 genes) and (ii) glutamate receptors (iGluRs) (20 genes).

The second group (potassium transporters) consists of

(d) KUP/HAK/KT transporters (13 genes), (e) HKT trans-

porters (1 gene), and (f) K1/H1 antiporters (6 genes). All

these show rather complex tissue- and organ-specific

expression (Fig. 4).

Shaker-type potassium channels

Shaker-type potassium channels are located at the plasma

membrane and mediate most of the K1-selective voltage-

gated currents at both hyperpolarized and depolarized

membrane potentials (Very and Sentenac 2002). These

channels are expressed in various cell types and operatein the millimolar range (low-affinity passive transport).

Based on their voltage dependency, the channels are

grouped into three functional subfamilies: (1) hyper-

polarization-activated inward-rectifying channels (AKT1,

KAT1, KAT2 and SPIK), (2) weakly inward-rectifying

channels (AKT2/3) and (3) depolarization-activated out-

ward-rectifying channels (SKOR and GORK). The phys-

iological roles and functional expression of the Shakerfamily channels are diverse. Both AKT1 and AtKC1 are

expressed in roots (Bertl et al. 1994, Ivashikina et al.

2001, Pilot et al. 2003), with AKT1 being a major player in

K1 acquisition by the plant (Hirsch et al. 1998). The

function of AKT2/AKT3 (which enables bi-directional K1

transport) is attributed to phloem loading and/or unload-

ing (Lacombe et al. 2000, Marten et al. 1999) and K1

Fig. 4. Tissue-specific expression of K1 transporters in plants. Most of the data come from experiments with Arabidopsis, although results from other

plant species are also included. With the exception of KCO channels, all other transporters shown are likely to be expressed at cell plasma membrane.

Reproduced from Cuin and Shabala (2006), with kind permission of Springer Science and Business Media.

660 Physiol. Plant. 133, 2007

uptake into leaf mesophyll cells (Dennison et al. 2001).

KAT1 and KAT2 are guard cell-specific channels mediat-

ing K1 influx for turgor-dependent regulation of stomatal

aperture (Szyroki et al. 2001). Potassium release from

most cells (including guard cells, root hairs and root

epidermis) is mediated by the depolarization-activatedGORK channel (Ivashikina et al. 2001), while K1 release

into the xylem occur through the SKOR channel

(Gaymard et al. 1998).

There are a number of reports of adverse effects of Na1

on the functioning of Shaker channels. Sodium may have

both direct (reducing a channel’s open probability) and

indirect (reducing the total number of expressed chan-

nels) effects on the AKT channel (Qi and Spalding 2004).Reports have shown that the AKT2/3 K1 channel is

upregulated during salinity (Marten et al. 1999), suggest-

ing that AKT2/3 may be involved in the recirculation of K1

through the phloem. Similarly the stelar-root tissue-

located SKOR is also upregulated by salinity (Maathuis

2006). The substantial salt-induced upregulation of both

SKOR in roots and AKT2/3 in shoots would result in

increased rates of K1 circulation through the vasculartissue, pointing towards a long distance redistribution of

K1 between the roots and shoots. Interestingly, the

opposite, i.e. a downregulation of SKOR and AKT2/3

occurs during K1 deprivation (Pilot et al. 2003).

Other Shaker-type channels though may have a much

larger role in K1homeostasis under saline conditions. The

large Na1 influx that occurs upon the subjection of plants

to salinity results in membrane depolarization (Babourinaet al. 2000, Horie et al. 2001, Shabala et al. 2003). Such

a voltage change would result in the activation of

depolarization-activated outward-rectifying K1 chan-

nels. Indeed, we have found, using K1-specific channel

blockers, that the massive K1 efflux resulting from salt

application is mediated through such channels in both

Arabidopsis (Shabala et al. 2006) and barley (Z. Chen,

S. Shabala and I. Pottosin, unpublished data). Further-more, measurements on a variety of K1 transporter

mutants have shown that this efflux is strongly reduced

in Arabidopsis gork1 mutants (Fig. 3A). As this NaCl-

induced efflux corresponds to decreases in cellular K1

content (Shabala et al. 2006), it strongly implicates a role

for GORK in plant salt tolerance.

‘Two-pore’ potassium channels

‘Two-pore’ potassium channels have two families in

Arabidopsis (KCO-1P and KCO2-P), with one and five

members in each, respectively (Czempinski et al. 2002).

These channels appear to be located predominantly at

tonoplast and show strong Ca21 dependence and out-

ward rectification (Schonknecht et al. 2002). Although

the involvement of KCO channels in plant adaptive

responses to salinity is mostly unknown, a recent report

has indicated that the transcript level of the K1-selective,

largely voltage-independent, vacuolar-located TPK5

(KCO5) decreases under saline conditions, possibly

signifying its involvement in maintaining cytoplasmicK1 levels and/or the exchange of vacuolar K1 for Na1

(Maathuis 2006).

Non-selective cation channels

NSCCs are ubiquitous in both plasma and endomem-

branes (specifically, tonoplast) of plant cells, with 40

putative NSCCs shown in the Arabidopsis genomesequence (Maser et al. 2001, Very and Sentenac 2002,

2003). NSCCs show a high selectivity for cations over

anions but, as a rule, do not discriminate strongly

between cations, and have K1/Na1 selectivity ratios

between 0.3 and 3 (Demidchik and Maathuis 2007,

Demidchik et al. 2002). The physiological role of NSCCs

is to function in low-affinity uptake. NSCCs may be gated

by a large number of factors and may be classified asdepolarization-activated, hyperpolarization-activated,

voltage-insensitive, calcium-activated, mechanosensi-

tive, cyclic nucleotide-gated and glutamate-gated chan-

nels (Demidchik et al. 2002). The last two groups are

considered in more detail below.

It is well accepted that NSCC are the prime mediators of

Na1 influx into plant roots (Demidchik and Maathuis

2007, Demidchik et al. 2002, Maathuis and Sanders2001, Tyerman et al. 1997), and transcript levels of a

number of members of this family are modified by salinity

(Maathuis 2006). This may be beneficial during salt stress

because it will help decrease the osmotic potential of

tissues, thereby alleviating water stress. However, if the

excess Na1 is not compartmentalized into the vacuole,

the result can be severe toxicity in the cytosol.

Recently, work in our laboratory has implicated NSCCin mediating at least a part of the NaCl-induced K1 efflux

from the cell, alongside K1 outward-rectifying channels

(Shabala et al. 2006). However, the full contribution of

NSCC towards K1 acquisition and transport within plants

under saline conditions remains to be quantified.

CNGCs are ligand-gated channels that are activated by

either cAMP or cGMP (Leng et al. 2002), resulting in an

influx of cations into the cell (Balague et al. 2003). Thesechannels are widely present across the plant kingdom,

with 20 members of the CNGC family found in

Arabidopsis genome (Maser et al. 2001). In contrast to

animal CNGCs, plant CNGCs are also gated by calcium

and calmodulin (Maser et al. 2001, Very and Sentenac

2002). Being classified as non-selective, they have almost

equal permeability for K1 and Na1 (Balague et al. 2003).

Physiol. Plant. 133, 2007 661

CNGCs are expressed ubiquitously in all tissues (Talke

et al. 2003) and are localized to the plasma membrane

(Arazi et al. 1999).

The large size of the plant CNGC family indicates

a wide diversity of physiological functions, ranging from

cell signalling (Very and Sentenac 2002) to root cationuptake (Maathuis and Sanders 2001). Their contribution

towards K1 acquisition and transport within plants under

saline conditions remains to be quantified. Notwith-

standing, for several members of the CNGC family,

functional data are available (Balague et al. 2003, Leng

et al. 2002). All characterized CNGCs are capable of

conducting K1 and, apart from CNGC2, Na1 as well.

Transcriptome analysis has revealed that transcript levelsfor CNGC1, CNGC19 and CNGC20 increased under

saline conditions in Arabidopsis roots, while in shoots,

CNG19, CNGC3 and CNGC8 transcript levels were

increased (Maathuis 2006). However, the significance of

this is yet to be revealed.

iGluRs are expressed ubiquitously throughout the

plants, with some of them being most abundant in root

tissues (Davenport 2002). The precise physiological roleof the plant glutamate receptor family is unknown

(Lacombe et al. 2001), and it remains to be proven

whether GluRs are actually capable of forming functional

ion channels. GluRs3.2 overexpression creates hyper-

sensitivity to K1 and Na1 (Kim et al. 2001). Thus,

glutamate receptors (GLR) activity during salt stress may

not only influence Na1 fluxes per se, but also affect K1

nutrition. In contrast to GLR2.3, GLR2.5 is downregu-lated in root tissue, possibly pointing towards a function

in the redistribution of ions such as K1 (Maathuis 2006).

KUP/HAK/KT transporters

KUP/HAK/KT transporters are involved in both high- and

low-affinity K1 uptake (Santa-Marıa et al. 1997, Vallejo

et al. 2005). They form a large family, with 13 members inArabidopsis (Maser et al. 2001), at least 17 members in

rice (Banuelos et al. 2002) and 5 in barley (Santa-Marıa

et al. 1997). They are ubiquitously expressed in plant

tissues, functioning both at the plasma membrane and the

tonoplast (Banuelos et al. 2002, Serrano and Rodrıguez-

Navarro 2001).

While the KUP/HAK/KT family of K1 transporters might

mediate some low-affinity Na1 influx at high externalNa1 concentration, the full extent of this is not known.

Nevertheless, the transcript levels and activity of a num-

ber of these transporters from this group are modified

under saline conditions, thus could be targets for genetic

improvement. When expressed in yeast, HvHAK1 from

barley mediates low-affinity Na1 transport in addition to

high-affinity K1 uptake (Santa-Marıa et al. 1997).

Elevated Na1 also inhibits K1 transport through heterol-

ogously expressed AtKUP1 (Fu and Luan 1998) and

AtHAK5 (Rubio et al. 2000) and transcript expression of

KUP2 is decreased in shoots of NaCl-treated plants

(Maathuis 2006). Also reported is the upregulation

of Mesembryanthemum crystallinum McHAK1 andMcHAK2 under both K1 starvation and NaCl stress (Su

et al. 2002). These isoforms are proposed to be involved in

maintaining cytoplasmic K1 levels and/or turgor regula-

tion under conditions where external Na1 inhibits K1

uptake and cellular Na1 replaces K1.

HKT transporters

The main function of plant HKT transporters is to facilitate

K1/Na1 symport under high-affinity conditions (Rubio

et al. 1995, Schachtman and Schroeder 1994). Further

studies have suggested that this transporter may also

mediate low-affinity Na1 transport into roots (Laurie et al.

2002), especially under low K1 to Na1 ratios (Rubio et al.

1995). The HKT transporters are proposed to play a major

role in Na1 uptake and recirculation within salt-stressedplants (Apse and Blumwald 2007, Rus et al. 2001).

The HKT family of proteins has been implicated in Na1

transport in a number of species. Members of this family

have been shown to function as Na1/K1 symporters and

as Na1-selective transporters at both high and low affinity

(Garciadeblas et al. 2003, Rubio et al. 1995). Certainly,

decreased expression of HKT under salt stress often

correlates with plant salt tolerance (Golldack et al. 1997),suggesting that this transporter may be a determinant of

salt sensitivity in plants (Rubio et al. 1995). However, its

role in salt tolerance is far from resolved. The rice

OsHKT1:5 functions as a Na1-selective transporter in

oocytes and is hypothesized to control shoot Na1 and

influence shoot K1 by withdrawing Na1 from the xylem

stream into the xylem parenchyma cells (Ren et al. 2005).

A similar recirculating role has been proposed inArabidopsis for AtHKT1:1 in mediating the loading of

Na1 from the shoot into the phloem and then unloading it

into the roots (Berthomieu et al. 2003, Sunarpi et al.

2005). HKT has been also implicated in the regulation of

K1 transport (Uozumi et al. 2000) and homeostasis (Ren

et al. 2005, Sunarpi et al. 2005) under saline conditions

in several species.

K1/H1 antiporters

The Arabidopsis genome contains six putative K1 efflux

antiporters (named KEA1 through KEA6) that belong to

the monovalent cation:proton antiporter family 2 (CPA2

family). Overall, three major families of such antiporters

are known: CPA1 (a monovalent cation:proton antiporter

662 Physiol. Plant. 133, 2007

family; eight members in Arabidopsis), CPA2 or cation/

proton exchanger family [(CHX), 28 members] and NhaD

(two members) (Maser et al. 2001). CPA1 transporters

appear to be crucial for regulating cytosolic sodium level.

The role of these transporters in K1 homeostasis is poorly

understood, although there are suggestions that thetonoplast-located Na1/H1 antiporter also mediates K1

transport (Apse et al. 2003, Zhang and Blumwald 2001).

Cellier et al. (2004) suggested that at least one member

of CPA2 family: the root and leaf-expressed AtCHX17,

may contribute towards K1 acquisition and homeostasis

under saline conditions, as its transcript level increases

under salinity (Maathuis 2006). However, in chx17

mutants, no difference in Na1 content was observedfrom WT plants when grown under saline conditions,

while the K1 levels decreased in the mutant. This

indicates that CHX17 transports K1 rather than Na1

(Cellier et al. 2004), suggesting its involvement in root K1

homeostasis, particularly during NaCl stress.

Members of the CHX family also play a well-known

and significant role in cytosolic Na1 extrusion from

the cell. These transporters display Na1/H1 antiporteractivity, both at the plasma (AtSOS1; Shi et al. 2000) and

vacuolar (AtNHX1; Apse et al. 1999, 2003) membranes.

Overexpression of both these transporters has been

shown to improve salt tolerance in several plant species

(Blumwald et al. 2000, Shi et al. 2003). In addition,

CHX21 has been functionally characterized as a Na1

transporter in the xylem parenchyma (Hall et al. 2006).

Other transporters

Two other types of transporters may potentially contribute

to regulation of cellular ionic and osmotic homeostasis

under saline conditions. These include the wheat LCT1

low-affinity transporter mediating uptake of a wide range

of monovalent cations, including K1 and Na1 (Amtmann

et al. 2001) and a putative members of the cation chlo-ride co-transporter family (Very and Sentenac 2003,

Colmenero-Flores et al. 2007).

Major targets for genetic improvement

Given the fact that plant salt tolerance correlates with its

ability to retain K1 in the cell and that NaCl-induced K1

efflux is mediated predominantly by GORK channels (seeabove), it would be very tempting to suggest that knocking

outGORK genes would increase salt tolerance. However,

given the multiple roles of these channels in plants, any

benefits of such modification may be outweighed by the

potential numerous physiological disturbances caused by

such mutation. Consistent with this notion, no substantial

difference in the K1-permeable outward-rectifying

channel (e.g. GORK) density was found between two

contrasting barley cultivars, CM72 and Gairdner, show-

ing a six-fold difference in their ability to retain K1 after

salt imposition (Z. Chen, S. Shabala, I. Pottosin, unpub-

lished results). Therefore, it appears to be the ability of

a cell to regulate a channel’s activity as opposed to thenumber of expressed channels per se that determines

plant salt tolerance. Hence, factors contributing to post-

transcriptional regulation of GORK channels should be

targeted. Recent work in our laboratory has explicitly

proven that multiple mechanisms determine salt toler-

ance in barley. These mechanisms include (Chen et al.

2007b): (1) better control of membrane voltage, so re-

taining a more negative membrane potential, (2) intrin-sically higher H1 pump activity, (3) enhanced ability of

root cells to pump Na1 from the cytosol to the external

medium and (4) higher sensitivity to supplemental Ca21.

The first two mechanisms appear to be closely linked

and explain most of genetic variation in the ability to

retain K1 in barley roots. Importantly, plasma membrane

H1 ATPase activity is under control of relatively few genes

(Gaxiola et al. 2007), making this trait relatively easy tomanipulate by molecular means. The potential drawback

of increased intrinsic high H1 pump activity would be an

unproportionally high amount of energy drawn from

other metabolic process, resulting in possible penalties

in terms of reduced growth rate and/or yield. Theoretically,

this problem may be overcome by introducing stress-

specific promoters, causing post-translational activation of

H1-ATPase activity only under stress conditions.It would be naive and misleading to assume that the

above modification of H1-ATPase activity leading to an

improved ability of plant to retain K1, as the only possible

way of improving salt tolerance. Without any doubt, the

latter trait should be complemented by other mechanisms

proven to also significantly contribute to tolerance.

Among these, overexpression of the tonoplast NHX1

H1/Na1 exchanger is probably the most important. Thiswould allow plants to use Na1 as a cheap osmoticum,

pumping it into vacuole, thus maintaining cell turgor. At

the same time, increased vacuolar Na1 would allow the

available K1 to be used in the cytosol rather than stored in

the vacuole for osmotic purposes. The importance of Na1

compartmentation to the vacuole by overexpressing

NHX1 has been shown by improved performance in

salt-stressed transgenic tomato (Zhang and Blumwald2001), Brassica (Zhang et al. 2001), rice (Fukuda et al.

2004), wheat (Saqib et al. 2005, Xue et al. 2004) and

cotton (Wu et al. 2004). Additional evidence supporting

the role of vacuolar transport in salt tolerance has been

provided by Arabidopsis plants overexpressing a vacuolar

H1-PPase (Gaxiola et al. 2001). In this work, transgenic

plants overexpressing AVP1 coding for the vacuolar

Physiol. Plant. 133, 2007 663

H1-PPase showed enhanced salt tolerance that was cor-

related with the ionic K1 content of the plants.

Importantly, the tonoplast-located Na1/H1 antiporter

also mediates K1 transport (Apse et al. 2003, Zhang and

Blumwald 2001), so might assist in increasing the

vacuolar K1 pool under control conditions, making itavailable when plants are hit by salt stress.

Another possible target could be the homologue of

the yeast HAL genes. Overexpressing HAL1 and HAL3

genes improved salt tolerance by increasing the cellular

K1/Na1 ratio (Gaxiola et al. 1992, Serrano 1996). Both

a reduction in K1 loss and a decrease in intracellular Na1

accumulation were observed. Genes homologous to the

yeast HAL could also be present in higher plants and maybe relevant to salt tolerance (Bordas et al. 1997, Espinosa-

Ruiz et al. 1999). However, the relationship between

these genes and K1 homeostasis in plants is unknown and

needs further study.

Conclusions and prospects

Our understanding of the importance of K1 transportsystems and its cytosolic homeostasis under saline

conditions has increased considerably in recent years.

Indeed, it is becoming more and more accepted that the

ability of a plant to maintain a high cytosolic K1/Na1 ratio

is crucial in plant salt tolerance mechanisms. In spite of

this, only a small number of genes responsible for K1 as

well as for Na1 transport have been fully characterized

physiologically. Furthermore, the majority of thesestudies on plants have been mostly carried out on the

model Arabidopsis species. The extension of some of this

work to the salt-tolerant Arabidopsis-related species

Thellungiella halophila opens up prospects for extending

such work onto a halophytic species, although, neither

this species nor Arabidopsis are useful agriculturally.

Research in our laboratory conducted over the last few

years has significantly contributed to understanding thegenetic aspects of the inheritability of cytosolic K1

homeostasis on the agriculturally important barley crop

and have highlighted the crucial importance of main-

taining an optimal cytosolic K1/Na1 ratio in salt tolerance

mechanisms (Chen et al. 2005, 2007). This research is

currently being extended to wheat. The recent sequenc-

ing of the rice genome and a progressive build-up of

genomic information regarding other crop species willundoubtedly facilitate our understanding of potential

transport mechanisms for K1 within plants, enabling the

application of information concerning K1 and Na1

transport gained from Arabidopsis to economically

important plant species. This will allow the pinpointing

of particular genes in the control of K1 homeostasis under

adverse environmental conditions. Although much is still

to be discovered concerning the mechanisms of the

maintenance of optimal K1/Na1 ratios under saline

conditions, the research outlined in this review does

allow us a certain amount of optimism regarding the

prospect of developing plants with increased salt toler-

ance that are useful to the farmer, enabling an increase inarable production under salt-stressed conditions.

Acknowledgements – This work was supported by ARS

Discovery and GRDC grants to Dr S.Shabala.

References

Amtmann A, Fischer M, Marsh EL, Stefanovic A, Sanders D,

Schachtman DP (2001) The wheat cDNA LCT1 generates

hypersensitivity to sodium in a salt sensitive yeast strain.

Plant Physiol 126: 1061–1071

Apse MP, Blumwald E (2007) Na1 transport in plants. FEBS

Lett 581: 2247–2254

Apse MP, Aharon GS, Snedden WA, Blumwald E (1999)

Salt tolerance conferred by overexpression of a vacuolar

Na1/H1 antiport in Arabidopsis. Science 285: 1256–1258

Apse MP, Sottosanto JB, Blumwald E (2003) Vacuolar cation/

H1 exchange, ion homeostasis, and leaf development are

altered in a T-DNA insertional mutant of AtNHX1, the

Arabidopsis vacuolar Na1/H1 antiporter. Plant J 36:

229–239

Arazi T, Sunkar R, Kaplan B, Fromm H (1999) A tobacco

plasma membrane calmodulin-binding transporter confers

Ni21 tolerance and Pb21 hypersensitivity in transgenic

plants. Plant J 20: 171–182

Ashraf M, Khanum A (1997) Relationship between ion

accumulation and growth in two spring wheat lines

differing in salt tolerance at different growth stages. J Agron

Crop Sci 178: 39–51

Ashraf M, McNeilly T, Bradshaw AD (1986) Heritability of

NaCl tolerance in seven grass species. Euphytica 35:

935–940

Ayala F, Oleary JW, Schumaker KS (1996) Increased vacuolar

and plasma membrane H1-ATPase activities in Salicornia

bigelovii Torr in response to NaCl. J Exp Bot 47: 25–32

Azaizeh H, Gunne B, Steudle E (1992) Effects of NaCl and

CaCl2 on water transport across root cells of maize

(Zea mays L.) seedlings. Plant Physiol 99: 886–894

Babourina O, Leonova T, Shabala S, Newman I (2000) Effect

of sudden salt stress on ion fluxes in intact wheat

suspension cells. Ann Bot 85: 759–767

Bajaj S, Targolli J, Liu LF, Ho THD, Wu R (1999) Transgenic

approaches to increase dehydration-stress tolerance in

plants. Mol Breed 5: 493–503

Balague C, Lin BQ, Alcon C, Flottes G, Malmstrom S, Kohler

C, Neuhaus G, Pelletier G, Gaymard F, Roby D (2003)

HLM1, an essential signaling component in the

hypersensitive response, is a member of the cyclic

664 Physiol. Plant. 133, 2007

nucleotide-gated channel ion channel family. Plant Cell

15: 365–379

Banuelos MA, Garciadeblas B, Cubero B, Rodrıguez-Navarro

A (2002) Inventory and functional characterization of the

HAK potassium transporters of rice. Plant Physiol 130:

784–795

Basu R, Ghosh B (1991) Polyamines in various rice (Oryza

sativa) genotypes with respect to sodium-chloride salinity.

Physiol Plant 82: 575–581

Berthomieu P, Conejero G, Nublat A, Brackenbury WJ,

Lambert C, Savio C, Uozumi N, Oiki S, Yamada K, Cellier

F, Gosti F, Simonneau T, Essah PA, Tester M, Very A-A,

Sentenac H, Casse F (2003) Functional analysis of AtHKT1

in Arabidopsis shows that Na1 recirculation by the phloem

is crucial for salt tolerance. EMBO J 22: 2004–2014

Bertl A, Anderson JA, Slayman CL, Sentenac H, Gaber RF

(1994) Inward and outward rectifying potassium currents

in Saccharomyces cerevisiae mediated by endogenous and

heterologously expressed ion channels. Folia Microbiol

(Praha) 39: 507–509

Blumwald E, Aharon GS, Apse MP (2000) Sodium transport

in plant cells. Biochim Biophys Acta 1465: 140–151

Bohnert HJ, Shen B (1999) Transformation and compatible

solutes. Sci Hortic 78: 237–260

Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptation to

environmental stresses. Plant Cell 7: 1099–1111

Bordas M, Montesinos C, Dabauza M, Salvador A, Roig LA,

Serrano R, Moreno V (1997) Transfer of the yeast salt

tolerance gene HAL1 to Cumcumis melo L. cultivats and in

vitro evaluation of salt tolerance. Transgenic Res 6: 41–50

Botella MA, Martinez V, Pardines J, Cerda A (1997) Salinity

induced potassium deficiency in maize plants. J Plant

Physiol 150: 200–205

Bray EA (1997) Plant responses to water deficit. Trends Plant

Sci 2: 48–54

Byrt CS, Platten JD, Spielmeyer W, James RA, Lagudah ES,

Dennis ES, Tester M, Munns R (2007) HKT1;5-like cation

transporters linked to Na1 exclusion loci in wheat, Nax2

and Kna1. Plant Physiol 143: 1918–1928

Cellier F, Conejero G, Ricaud L, Luu DT, Lepetit M, Gosti F,

Casse F (2004) Characterization of AtCHX17, a member of

the cation/H1 exchangers, CHX family, from Arabidopsis

thaliana suggests a role in K1 homeostasis. Plant J 39:

834–846

Chandler SF, Thorpe TA (1987) Characterization of growth,

water relations, and proline accumulation in

sodium-sulfate tolerant callus of Brassica napus L cv

Westar (Canola). Plant Physiol 84: 106–111

Chen THH, Murata N (2002) Enhancement of tolerance of

abiotic stress by metabolic engineering of betaines and

other compatible solutes. Curr Opin Plant Biol 5: 250–257

Chen Z, Newman I, Zhou M, Mendham N, Zhang G, Shabala

S (2005) Screening plants for salt tolerance by measuring

K1 flux: a case study for barley. Plant Cell Environ 28:

1230–1246

Chen ZH, Zhou MX, Newman IA, Mendham NJ, Zhang GP,

Shabala S (2007a) Potassium and sodium relations in

salinised barley tissues as a basis of differential salt

tolerance. Funct Plant Biol 34: 150–162

Chen ZH, Pottosin II, Cuin TA, Fuglsang AT, Tester M, Jha D,

Zepeda-Jazo I, Zhou MX, Palmgren MG, Newman IA,

Shabala S. 2007b).Root plasma membrane transporters

controlling K1/Na1 homeostasis in salt stressed barley.

Plant Physiol Doi:10.1104/pp.107.110262

Cherel I (2004) Regulation of K1 channel activities in plants:

from physiological to molecular aspects. J Exp Bot 55:

337–351

Colmenero-Flores JM, Martinez G, Gamba G, Vazquez N,

Iglesias DJ, Brumos J, Talon M (2007) Identification and

functional characterization of cation-chloride

cotransporters in plants. Plant J 50: 278–292

Colmer TD, Flowers TJ, Munns R (2006) Use of wild relatives

to improve salt tolerance in wheat. J Exp Bot 57:

1059–1078

Cosgrove DJ, Hedrich R (1991) Stretch-activated chloride,

potassium, and calcium channels coexisting in plasma

membranes of guard cells of Vicia faba L. Planta 186:

143–153

Cramer GR, Lynch J, Lauchli A, Epstein E (1987) Influx of

Na1, K1, and Ca21 into roots of salt-stressed cotton

seedlings. Effects of supplemental Ca21. Plant Physiol 83:

510–516

Cramer GR, Epstein E, Lauchli A (1991) Effects of sodium,

potassium and calcium on salt-stressed barley. 2.

Elemental analysis. Physiol Plant 81: 197–202

Cuin TA, Shabala S (2005) Exogenously supplied compatible

solutes rapidly ameliorate NaCl-induced potassium efflux

from barley roots. Plant Cell Physiol 46: 1924–1933

Cuin TA, Shabala S (2006) Potassium homeostasis in salinised

plant tissues. In: Volkov A (ed) Plant Electrophysiology –

Theory and Methods. Springer, Heidelberg, Germany,

pp 287–317

Cuin TA, Shabala S (2007) Amino acids regulate

salinity-induced potassium efflux in barley root epidermis.

Planta 225: 753–761

Cuin TA, Miller AJ, Laurie SA, Leigh RA (2003) Potassium

activities in cell compartments of salt-grown barley leaves.

J Exp Bot 54: 657–661

Czempinski K, Frachisse JM, Maurel C, Barbier-Brygoo H,

Muller-Rober B (2002) Vacuolar membrane localization of

the Arabidopsis ‘two-pore’ K1 channel KCO1. Plant J 29:

809–820

Davenport R (2002) Glutamate receptors in plants. Ann Bot

90: 549–557

Davenport RJ, Munoz-Mayor A, Jha D, Essah PA, Rus A,

Tester M (2007) The Na1 transporter AtHKT1;1 controls

retrieval of Na1 from the xylem in Arabidopsis. Plant Cell

Environ 30: 497–507

Delauney AJ, Verma DPS (1993) Proline biosynthesis and

osmoregulation in plants. Plant J 4: 215–223

Physiol. Plant. 133, 2007 665

Demidchik V, Tester M (2002) Sodium fluxes through

nonselective cation channels in the plasma membrane of

protoplasts from Arabidopsis roots. Plant Physiol 128:

379–387

Demidchik V, Maathuis FJM (2007) Physiological roles of

nonselective cation channels in plants: from salt stress to

signalling and development. New Phytol 175: 387–404

Demidchik V, Davenport RJ, Tester M (2002) Nonselective

cation channels in plants. Annu Rev Plant Biol 53: 67–107

Dennison KL, Robertson WR, Lewis BD, Hirsch RE, Sussman

MR, Spalding EP (2001) Functions of AKT1 and AKT2

potassium channels determined by studies of single

and double mutants of Arabidopsis. Plant Physiol

127: 1012–1019

Ding L, Zhu JK (1997) Reduced Na1 uptake in the

NaCl-hypersensitive sos1 mutant of Arabidopsis thaliana.

Plant Physiol 113: 795–799

Dubcovsky J, Maria GS, Epstein E, Luo MC, Dvorak J (1996)

Mapping of the K1/Na1 discrimination locus Kna1 in

wheat. Theor Appl Genet 92: 448–454

Dvorak J, Noaman MM, Goyal S, Gorham J (1994)

Enhancement of the salt tolerance of Triticum turgidum L.

by the kna1 locus transferred from the Triticum aestivum L.

chromosome 4D by homoeologous recombination. Theor

Appl Genet 87: 872–877

Erdei L, Trivedi S, Takeda K, Matsumoto H (1990) Effects of

osmotic and salt stresses on the accumulation of

polyamines in leaf segments from wheat-varieties differing

in salt and drought tolerance. J Plant Physiol 137: 165–168

Espinosa-Ruiz A, Belles JM, Serrano R, Culianez-Macia FA

(1999) Arabidopsis thaliana AAtHAL3: a flavoprotein

related to salt and osmotic tolerance and plant growth.

Plant J 20: 529–539

Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:

307–319

Flowers TJ, Yeo AR (1995) Breeding for salinity resistance in

crop plants – where next? Aust J Plant Physiol 22: 875–884

Flowers TJ, Hajibagheri MA, Yeo AR (1991) Ion

accumulation in the cell walls of rice plants growing under

saline conditions: evidence for the Oertli hypothesis. Plant

Cell Environ 14: 319–325

Fooland MR (1997) Genetic basis of physiological traits

related to salt tolerance in tomato, Lycopersicon

esculentum Mill. Plant Breed 116: 53–58

Fu HH, Luan S (1998) AtKUP1: a dual-affinity K1 transporter

from Arabidopsis. Plant Cell 10: 63–73

Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A,

Hirochika H, Tanaka Y (2004) Function, intracellular

localization and the importance in salt tolerance of

a vacuolar Na1/H1 antiporter from rice. Plant Cell Physiol

45: 146–159

Garcia A, Rizzo CA, UdDin J, Bartos SL, Senadhira D,

Flowers TJ, Yeo AR (1997) Sodium and potassium

transport to the xylem are inherited independently in rice,

and the mechanism of sodium: potassium selectivity

differs between rice and wheat. Plant Cell Environ 20:

1167–1174

Garciadeblas B, Senn ME, Banuelos MA, Rodrıguez-Navarro

A (2003) Sodium transport and HKT transporters: the rice

model. Plant J 34: 788–801

Garthwaite AJ, von Bothmer R, Colmer TD (2005) Salt

tolerance in wild Hordeum species is associated with

restricted entry of Na1 and Cl2 into the shoots. J Exp Bot

56: 2365–2378

Gaxiola R, Delarrinoa IF, Villalba JM, Serrano R (1992) A

novel and conserved salt-induced protein is an important

determinant of salt tolerance in yeast. EMBO J 11:

3157–3164

Gaxiola RA, Li JS, Undurranga S, Dang LM, Allen GJ, Alper

SL, Fink GR (2001) Drought- and salt-tolerant plants results

from overexpression of the AVP1 H1-pump. Proc Natl

Acad Sci USA 98: 11444–11449

Gaxiola RA, Palmgren MG, Schumacher K (2007) Plant

proton pumps. FEBS Lett 571: 2204–2214

Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D,

Boucherez J, Michaux-Ferriere N, Thibaud JB, Sentenac H

(1998) Identification and disruption of a plant shaker-like

outward channel involved in K1 release into the xylem

sap. Cell 94: 647–655

Genc Y, McDonald GK, Tester M (2007) Reassessment of

tissue Na1 concentration as a criterion for salinity

tolerance in bread wheat. Plant Cell Environ. doi:10.1111/

j.1365–3040.2007.01726.x

Gierth M, Maser P (2007) Potassium transporters in plant –

involvement in K1 acquisition. Redistribution and

homeostasis. FEBS Lett 581: 2348–2356

Golldack D, Dietz KJ (2001) Salt-induced expression of the

vacuolar H1-ATPase in the common ice plant is

developmentally controlled and tissue specific. Plant

Physiol 125: 1643–1654

Golldack D, Kamasani UR, Quigley F, Bennett J, Bohnert HJ

(1997) Salt stress-dependent expression of a HKT1-type

high affinity potassium transporter in rice. Plant Physiol

114: 529–529

Gorham J, Bristol A, Young EM, Jones RGW (1991) The

presence of the enhanced K/Na discrimination trait in

diploid triticum species. Theor Appl Genet 82: 729–736

Gregorio GB, Senadhira D (1993) Genetic analysis of salinity

tolerance in rice (Oryza sativa L.). Theor Appl Genet 86:

333–338

Hall D, Evans AR, Newbury HJ, Pritchard J (2006) Functional

analysis of CHX21: a putative sodium transporter in

Arabidopsis. J Exp Bot 57: 1201–1210

Hare PD, Cress WA, Van Staden J (1998) Dissecting the roles

of osmolyte accumulation during stress. Plant Cell Environ

21: 535–553

Harinasut P, Tsutsui K, Takabe T, Nomura M, Takabe T,

Kishitani S (1996) Exogenous glycinebetaine accumulation

and increased salt-tolerance in rice seedlings. Biosci

Biotechnol Biochem 60: 366–368

666 Physiol. Plant. 133, 2007

Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant

cellular and molecular responses to high salinity. Annu

Rev Plant Physiol Plant Mol Biol 51: 463–499

Hassan NS, Wilkins DA (1988) In vitro selection for salt

tolerant lines in Lycopersicon peruvianum. Plant Cell Rep

7: 463–466

Haw M, Cocklin C, Mercer D (2000) A pinch of salt:

landowner perception and adjustment to the salinity

hazard in Victoria, Australia. J Rural Stud 16: 155–169

Hirsch RE, Lewis BD, Spalding EP, Sussman MR (1998) A role

for the AKT1 potassium channel in plant nutrition. Science

280: 918–921

Horie T, Yoshida K, Nakayama H, Yamada K, Oiki S, Shinmyo

A (2001) Two types of HKT transporters with different

properties of Na1 and K1 transport in Oryza sativa. Plant J

27: 129–138

Ivashikina N, Becker D, Ache P, Meyerhoff O, Felle HH,

Hedrich R (2001) K1 channel profile and electrical

properties of Arabidopsis root hairs. FEBS Lett 508:

463–469

Jacobsen SE, Mujica A, Jensen CR (2003) The resistance of

quinoa (Chenopodium quinoa Willd.) to adverse abiotic

factors. Food Rev Int 19: 99–109

Kefu Z, Hai F, San Z, Jie S (2003) Study on the salt and

drought tolerance of Suaeda salsa and Kalanchoe

claigremontiana under iso-osmotic salt and water stress.

Plant Sci 165: 837–844

Khan MA, Ungar IA, Showalter AM (2005) Salt stimulation

and tolerance in an intertidal stem-succulent halophyte.

J Plant Nutr 28: 1365–1374

Kim SA, Kwak JM, Jae SK, Wang MH, Nam HG (2001)

Overexpression of the AtGluR2 gene encoding an

Arabidopsis homolog of mammalian glutamate

receptors impairs calcium utilization and sensitivity to

ionic stress in transgenic plants. Plant Cell Physiol 42:

74–84

Koyama ML, Levesley A, Koebner RMD, Flowers TJ, Yeo AR

(2001) Quantitative trait loci for component physiological

traits determining salt tolerance in rice. Plant Physiol 125:

406–422

Lacombe B, Pilot G, Michard E, Gaymard F, Sentenac H,

Thibaud JB (2000) A shaker-like K1 channel with weak

rectification is expressed in both source and sink phloem

tissues of Arabidopsis. Plant Cell 12: 837–851

Lacombe B, Becker D, Hedrich R, DeSalle R, Hollmann M,

Kwak JM, Schroeder JI, Le Novere N, Nam HG, Spalding

EP, Tester M, Turano FJ, Chiu J, Coruzzi G (2001) The

identity of plant glutamate receptors. Science 292:

1486–1487

Laurie S, Feeney KA, Maathuis FJM, Heard PJ, Brown SJ,

Leigh RA (2002) A role for HKT1 in sodium uptake by

wheat roots. Plant J 32: 139–149

Lebaudy A, Very A-A, Sentenac H (2007) K1 channel activity

in plants: genes, regulations and functions. FEBS Lett 581:

2357–2366

Leng Q, Mercier RW, Hua BG, Fromm H, Berkowitz GA

(2002) Electrophysiological analysis of cloned cyclic

nucleotide-gated ion channels. Plant Physiol 128:

400–410

Lindsay MP, Lagudah ES, Hare RA, Munns R (2004) A locus

for sodium exclusion (Nax1), a trait for salt tolerance,

mapped in durum wheat. Funct Plant Biol 31: 1105–1114

Ludlow MM, Muchow RC (1990) A critical-evaluation of

traits for improving crop yields in water-limited

environments. Adv Agron 43: 107–153

Ma SS, Gong QQ, Bohnert HJ (2006) Dissecting salt stress

pathways. J Exp Bot 57: 1097–1107

Maathuis FJM (2006) The role of monovalent cation

transporters in plant responses to salinity. J Exp Bot 57:

1137–1147

Maathuis FJM, Amtmann A (1999) K1 nutrition and Na1

toxicity: the basis of cellular K1/Na1 ratios. Ann Bot 84:

123–133

Maathuis FJM, Sanders D (2001) Sodium uptake in

Arabidopsis roots is regulated by cyclic nucleotides. Plant

Physiol 127: 1617–1625

Mano Y, Takeda K (1997) Diallel analysis of salt tolerance at

germination and the seedling stage in barley (Hordeum

vulgare L.). Breed Sci 47: 203–209

Marschner H (1995) The Mineral Nutrition of Higher Plants.

Academic Press, London

Marten I, Hoth S, Deeken R, Ache P, Ketchum KA,

Hoshi T, Hedrich R (1999) AKT3, a phloem-localized K1

channel, is blocked by protons. Proc Natl Acad Sci

USA 96: 7581–7586

Maser P, Thomine S, Schroeder JI, Ward JM, Hirschi K,

Sze H, Talke IN, Amtmann A, Maathuis FJM, Sanders D,

Harper JF, Tchieu J, Gribskov M, Persans MW, Salt DE,

Kim SA, Guerinot ML (2001) Phylogenetic relationships

within cation transporter families of Arabidopsis. Plant

Physiol 126: 1646–1667

Morgan JM (1983) Osmoregulation as a selection criterion

for drought tolerance in wheat. Aust J Agric Res 34:

607–614

Munns R (2002) Comparative physiology of salt and water

stress. Plant Cell Environ 25: 239–250

Munns R (2005) Genes and salt tolerance: bringing them

together. New Phytol 167: 645–663

Munns R, Hare RA, James RA, Rebetzke GJ (1999) Genetic

variation for improving the salt tolerance of durum wheat.

Austr J Agric Res 51: 69–74

Munns R, James RA, Lauchli A (2006) Approaches to

increasing the salt tolerance of wheat and other cereals.

J Exp Bot 57: 1025–1043

Ndayiragije A, Lutts S (2006) Exogenous putrescine reduces

sodium and chloride accumulation in NaCl-treated calli of

the salt-sensitive rice cultivar I Kong Pao. Plant Growth

Regul 48: 51–63

Palmgren MG (1991) Regulation of plasma membrane

H1-ATPase activity. Physiol Plant 83: 314–323

Physiol. Plant. 133, 2007 667

Papageorgiou GC, Murata N (1995) The unusually strong

stabilizing effects of glycine betaine on the structure and

function of the oxygen-evolving photosystem II complex.

Photosynth Res 44: 243–252

Pilot G, Gaymard F, Mouline K, Cherel I, Sentenac H (2003)

Regulated expression of Arabidopsis Shaker K1 channel

genes involved in K1 uptake and distribution in the plant.

Plant Mol Biol 51: 773–787

Qi Z, Spalding EP (2004) Protection of plasma membrane K1

transport by the salt overly sensitive1 Na1/H1 antiporter

during salinity stress. Plant Physiol 136: 2548–2555

Raven JA (1985) Regulation of pH and generation of

osmolarity in vascular plants: a cost-benefit analysis in

relation to efficiency of use of energy, nitrogen and water.

New Phytol 101: 25–77

Reid RJ, Smith FA (2000) The limits of sodium/calcium

interactions in plant growth. Aust J Plant Physiol 27:

709–715

Ren ZH, Gao JP, Li LG, Cai XL, Huang W, Chao DY, Zhu MZ,

Wang ZY, Luan S, Lin HX (2005) A rice quantitative trait

locus for salt tolerance encodes a sodium transporter.

Nat Genet 37: 1141–1146

Rengasamy P (2006) World salinization with emphasis on

Australia. J Exp Bot 57: 1017–1023

Rubio F, Gassmann W, Schroeder JI (1995) Sodium-driven

potassium uptake by the plant potassium transporter HKT1

and mutations conferring salt tolerance. Science 270:

1660–1663

Rubio F, Santa-Marıa GE, Rodrıguez-Navarro A (2000)

Cloning of Arabidopsis and barley cDNAs encoding HAK

potassium transporters in root and shoot cells. Physiol

Plant 109: 34–43

Rus A, Yokoi S, Sharkhuu A, Reddy M, Lee BH,

Matsumomoto TK, Koiwa H, Zhu JK, Bressan RA,

Hasegawa PM (2001) AtHKT1 is a salt tolerance

determinant that controls Na1 entry into plant roots.

Proc Natl Acad Sci USA 98: 14150–14155

Rus A, Lee BH, Munoz-Mayor A, Sharkhuu A, Miura K, Zhu

JK, Bressan RA, Hasegawa PM (2004) AtHKT1 facilitates

Na1 homeostasis and K1 nutrition in planta. Plant Physiol

136: 2500–2511

Santa-Cruz A, Acosta M, Rus A, Bolarin MC (1999)

Short-term salt tolerance mechanisms in differentially salt

tolerant tomato species. Plant Physiol Biochem 37: 65–71

Santa-Marıa GE, Rubio F, Dubcovsky J, Rodriguez-Navarro A

(1997) The HAK1 gene of barley is a member of a large

gene family and encodes a high-affinity potassium

transporter. Plant Cell 9: 2281–2289

Saqib M, Zorb C, Rengel Z, Schubert S (2005) The expression

of the endogenous vacuolar Na1/H1 antiporters in wheat

(Triticum aestivum L.) Plant Sci 169: 959–965

Schachtman DP, Schroeder JI (1994) Structure and transport

mechanism of a high-affinity potassium uptake transporter

from higher plants. Nature 370: 655–658

Schonknecht G, Spoormaker P, Steinmeyer R, Bruggeman L,

Ache P, Dutta R, Reintanz B, Godde M, Hedrich R, Palme

K (2002) KCO1 is a component of the slow-vacuolar (SV)

ion channel. FEBS Lett 511: 28–32

Serraj R, Sinclair TR (2002) Osmolyte accumulation: can it

really help increase crop yield under drought conditions?

Plant Cell Environ 25: 333–341

Serrano R (1996) Salt tolerance in plants and

microorganisms: toxicity targets and defense responses. Int

Rev Cytol 165: 1–52

Serrano R, Rodrıguez-Navarro A (2001) Ion homeostasis

during salt stress in plants. Curr Opin Cell Biol 13:

399–404

Shabala S (2006) Non-invasive microelectrode ion flux

measurements in plant stress physiology. In: Volkov A (ed)

Plant Electrophysiology – Theory and Methods.

Springer-Verlag, Berlin, pp 35–71

Shabala S, Lew RR (2002) Turgor regulation in osmotically

stressed Arabidopsis epidermal root cells. Direct support

for the role of inorganic ion uptake as revealed by

concurrent flux and cell turgor measurements. Plant

Physiol 129: 290–299

Shabala S, Babourina O, Newman I (2000) Ion-specific

mechanisms of osmoregulation in bean mesophyll cells.

J Exp Bot 51: 1243–1253

Shabala S, Shabala L, Van Volkenburgh E (2003) Effect of

calcium on root development and root ion fluxes in

salinised barley seedlings. Funct Plant Biol 30: 507–514

Shabala L, Cuin TA, Newman IA, Shabala S (2005a)

Salinity-induced ion flux patterns from the excised roots of

Arabidopsis sos mutants. Planta 222: 1041–1050

Shabala S, Shabala L, Van Volkenburgh E, Newman I (2005b)

Effect of divalent cations on ion fluxes and leaf

photochemistry in salinised barley leaves. J Exp Bot 56:

1369–1378

Shabala S, Demidchik V, Shabala L, Cuin TA, Smith SJ, Miller

AJ, Davies JM, Newman IA (2006) Extracellular Ca21

ameliorates NaCl-induced K1 loss from Arabidopsis root

and leaf cells by controlling plasma membrane

K1-permeable channels. Plant Physiol 141: 1653–1665

Shabala S, Cuin TA, Pottosin I (2007) Polyamines prevent

NaCl-induced K1 efflux from pea mesophyll by blocking

non-selective cation channels. FEBS Lett 581: 1993–1999

Shi HZ, Ishitani M, Kim CS, Zhu JK (2000) The Arabidopsis

thaliana salt tolerance gene SOS1 encodes a putative Na1/

H1 antiporter. Proc Natl Acad Sci USA 97: 6896–6901

Shi HZ, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of

a plasma membrane Na1/H1 antiporter gene improves salt

tolerance in Arabidopsis thaliana. Nat Biotechnol 21:

81–85

Su H, Golldack D, Zhao CS, Bohnert HJ (2002) The

expression of HAK-type K1 transporters is regulated in

response to salinity stress in common ice plant. Plant

Physiol 129: 1482–1493

668 Physiol. Plant. 133, 2007

Sunarpi, Horie T, Motoda J, Kubo M, Yang H, Yoda K, Horie

R, Chan WY, Leung HY, Hattori K, Konomi M, Osumi M,

Yamagami M, Schroeder JI, Uozumi N (2005) Enhanced

salt tolerance mediated by AtHKT1 transporter-induced

Na1 unloading from xylem vessels to xylem parenchyma

cells. Plant J 44: 928–938

Szyroki A, Ivashikina N, Dietrich P, Roelfsema MRG, Ache P,

Reintanz B, Deeken R, Godde M, Felle H, Steinmeyer R,

Palme K, Hedrich R (2001) KAT1 is not essential for stomatal

opening. Proc Natl Acad Sci USA 98: 2917–2921

Talke IN, Blaudez D, Maathuis FJM, Sanders D (2003)

CNGCs: prime targets of plant cyclic nucleotide

signalling? Trends Plant Sci 8: 286–293

Teodoro AE, Zingarelli L, Lado P (1998) Early changes of Cl2

efflux and H1 extrusion induced by osmotic stress in

Arabidopsis thaliana cells. Physiol Plant 102: 29–37

Tester M, Davenport R (2003) Na1 tolerance and Na1

transport in higher plants. Ann Bot 91: 503–527

Tyerman SD, Skerrett M, Garrill A, Findlay GP, Leigh RA

(1997) Pathways for the permeation of Na1 and Cl2 into

protoplasts derived from the cortex of wheat roots. J Exp

Bot 48: 459–480

Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A,

Bakker EP, Nakamura T, Schroeder JI (2000) The

Arabidopsis HKT1 gene homolog mediates inward Na1

currents in Xenopus laevis oocytes and Na1 uptake in

Saccharomyces cerevisiae. Plant Physiol 122: 1249–1259

Vallejo AJ, Peralta ML, Santa-Marıa GE (2005) Expression of

potassium-transporter coding genes, and kinetics of

rubidium uptake, along a longitudinal root axis. Plant Cell

Environ 28: 850–862

Very A-A, Sentenac H (2002) Cation channels in the

Arabidopsis plasma membrane. Trends Plant Sci 7:

168–175

Very A-A, Sentenac H (2003) Molecular mechanisms and

regulation of K1 transport in higher plants. Annu Rev Plant

Biol 54: 575–603

Volkov V, Wang B, Dominy PJ, Fricke W, Amtmann A (2003)

Thellungiella halophila, a salt-tolerant relative of

Arabidopsis thaliana, possesses effective mechanisms to

discriminate between potassium and sodium. Plant Cell

Environ 27: 1–14

Walker DJ, Leigh RA, Miller AJ (1996) Potassium homeostasis

in vacuolate plant cells. Proc Natl Acad Sci USA 93:

10510–10514

Wu YY, Chen GD, Meng QW, Cheng CC (2004) The cotton

GhNHX1 gene encoding a novel putative tonoplast Na1/

H1 antiporter plays an important role in salt stress. Plant

Cell Physiol 45: 600–607

Xue ZY, Zhi DY, Xue GL, Zhang H, Zhao YX, Xia GM (2004)

Enhanced salt tolerance of transgenic wheat (Triticum

sativum L.) expressing a vacuolar Na1/H1 antiporter gene

with improved grain yields in saline soils in the field and

a reduced level of leaf Na1. Plant Sci 167: 849–859

Yeo AR, Lee KS, Izard P, Boursier PJ, Flowers TJ (1991)

Short-term and long-term effects of salinity on leaf growth

in rice (Oryza sativa L). J Exp Bot 42: 881–889

Zhang HX, Blumwald E (2001) Transgenic salt-tolerant

tomato plants accumulate salt in foliage but not in fruit.

Nat Biotechnol 19: 765–768

Zhang JX, Nguyen HT, Blum A (1999) Genetic analysis of

osmotic adjustment in crop plants. J Exp Bot 50: 291–302

Zhang HX, Hodson JN, Williams JP, Blumwald E (2001)

Engineering salt tolerant Brassica plants: characterization

of yield and seed oil quality in transgenic plants with

increased vacuolar sodium accumulation. Proc Natl Acad

Sci USA 98: 12832–12836

Zhu JK (2003) Regulation of ion homeostasis under salt stress.

Curr Opin Plant Biol 6: 441–445

Zingarelli L, Marre MT, Massardi F, Lado P (1999) Effects of

hyper-osmotic stress on K1 fluxes, H1 extrusion,

transmembrane electric potential difference and

comparison with the effects of fusicoccin. Physiol Plant

106: 287–295

Edited by J. K. Schjørring

Physiol. Plant. 133, 2007 669