K + retention in leaf mesophyll constitutes an important component of salinity tolerance

tolerance mechanism

K+ plays an important role in plant life acting as a dominant counterion to balance the negatively charged proteins and nucleic acids as well as being involved in enzyme activation, protein synthesis stabilization, membrane potential formation, maintenance of turgor pressure and cytosolic pH homeostasis, and mediating all types of plant movements (Shabala, 2003; Véry and Sentenac, 2003; Dreyer and Uozumi, 2011; Chérel et al., 2014; Anschütz et al., 2014). More than 50 enzymes are activated by K+ _which

cannot substitute by Na+; further, protein synthesis requires high K+ concentration (Tester and Davenport, 2003). Supply of K+ _{fertilizers ameliorates detrimental effects of salinity}

on plant performance (Cakmak, 2005; Shabala and Pottosin, 2014).

A strong positive correlation between root’s K+ _{retention ability and salt tolerance was}

revealed in many species (Cuin et al., 2012; Chen et al., 2007b,c,d; Smethurst et al., 2008; Sun et al., 2009). Here we provide compelling evidence that K+ retention in the photosynthetic-active mesophyll tissue may be an equally important trait contributing to the overall salinity tolerance (Fig. 3). The R2 value for group 1 plants (include tolerant and intermediate genotypes; Fig. 3C) was as high as 0.43, suggesting that over 40% of genetic variability in salinity tolerance in this cluster was attributed to just one physiological trait, namely better cytosolic K+ retention. Thus, targeting this trait in the

Fig. 6. Genetic variability in the shoot water content (SWC; % control) in barley and its relationship with overall salinity tolerance. (A) - all genotypes are ranked according to their SWC. (B) - correlation between SWC and damage index. (C) - correlation between SWC and K+ _{retention ability of leaf mesophyll estimated as net K}+ _efflux

upon 100 mM NaCl treatment. (D) - correlation between SWC and NaCl-induced steady state H+ _{flux. Each point}

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breeding programs may open additional avenues in pyramiding the overall tolerance. The reported results may also explain previous reports of higher shoot (or leaf) K+ content found under salinity stress in salt tolerant compared with sensitive varieties in barley (Liang, 1999), tomato (Taleisnik and Grunberg, 1994; Al-Karaki, 2000), soybean (Essa, 2002), and lucerne (Smethurst et al., 2008).

Fig. 7. The suggested model depicting ionic mechanisms contributing to K+ _{retention in leaf}

mesophyll in barley. Upon NaCl exposure, the plasma membrane is depolarized by the massive entry of external Na+ _{via non-selective cation channel (NSCC) and results in K}+ _{loss mediated by}

depolarization-activated K+ _{outward rectifying channel (KOR) channels. Cytosolic K}+ _{homeostasis is}

disrupted. The accompanied production of ROS has detrimental effects of leaf photochemistry in chloroplasts and, in severe cases, could trigger PCD or lead to necrosis. ROS also activate NSCC exacerbating K+ _{loss from cytosol. Compared with salt sensitive genotypes, higher vacuolar K}+ _pool

in the leaf mesophyll in salt tolerant barley genotypes allows plants to maintain cytosolic K+

homeostasis by releasing vacuolar K+ _{into cytosol; this process is mediated by tonoplast K}+_-

permeable channels (TPK in the model). It is also suggested that tolerant varieties may have higher Na+_/H+ _{NHX exchanger activity and, thus, can replace vacuolar K}+ _{by Na}+ _{to maintain its osmotic}

pressure in vacuole. As K+ _{uptake by AKT1 into leaf mesophyll is inhibited by the membrane}

depolarization, plants should rely on high affinity K+ _{uptake to restore cytosolic K}+ _{homeostasis (a}

HAK/KUP family K+_/H+ _{co-transporter in the model). A concurrent K}+ _{and H}+ _{uptake through}

HAK/KUP exchanger results in a +2 net charge transfer and may further depolarise the plasma membrane, resulting in a futile cycle of K+_{. This process is partially compensated by higher ATPase-}

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In our electrophysiological experiments, salinity stress was applied to leaf segments of plants grown under control conditions and, thus, bypassed all possible uncertainties and confounding factors related to genotypic differences in plants’ ability to exclude Na+ from uptake or load it into the xylem. Interestingly, sensitive and tolerant barley varieties showed a different extent of dependence on their ability to retain K+ _{in leaf mesophyll}

(Fig. 3C), indicated by different slope of relationship between the damage index and net K+ _{efflux. As commented above, the physiological rationale behind this observation may}

be that tolerance varieties may possess some other (additional) mechanisms to deal with salinity (such as higher SOS1 or NHX activity; Fig. 7), while sensitive varieties must rely heavily on K+ _{retention (e.g. tissue tolerance) for their survival.}

Reflecting a true tissue tolerance mechanism, mesophyll K+ retention may be targeted in the breeding programs using the protocols described in this work (Fig. 2). Varieties identified in this work as possessing superior (YWHKSL, DYSYH, Numar) and poor K+ retention under salinity stress (Aizao 3; Mundah; Keel) can be used to produce double haploid populations to fine-map QTLs conferring tissue tolerance mechanisms in barley. This trait may be then added to more traditional (previously identified) traits to create salt-tolerant varieties via “pyramiding” approach (Flowers and Yeo, 1995; Shabala, 2013). In the past, such work was done predominantly for QTLs related to Na+ exclusion traits. Selected examples include mapping Nax1 and Nax2 genes in durum wheat (Lindsay et al., 2004; James et al., 2006a; Munns et al., 2012) and HvNax3 and HvNax4 genes in barley (Shavrukov et al., 2010; Rivandi et al., 2011). Also, HKT2;1/2-like, HKT2;3/4- like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, and HKT1;5-like genes (contributing to Na+ _{accumulation in the shoot) were mapped to the wheat–barley chromosome groups 7,}

7, 2, 6, 2, and 4, respectively (Huang et al., 2008). However, until now no QTLs related to K+ _{retention trait have been reported in barley and wheat. The present work provides a}

possibility to overcome this limitation and exploit QTLs associated with K+ _{retention in}

leaf mesophyll.