Open access peer-reviewed chapter

Plant Plasma Membrane H+-ATPase in Adaptation of Plants to Abiotic Stresses

By Małgorzata Janicka-Russak

Submitted: December 2nd 2010Reviewed: April 29th 2011Published: August 29th 2011

DOI: 10.5772/24121

Downloaded: 5459

1. Introduction

H+-ATPase is a major enzyme protein of the plant plasma membrane (PM). This protein belongs to a large superfamily of pumps termed P-type ATPases. A characteristic feature of P-ATPases is the formation of a phosphorylated intermediate during the catalytic cycle. P-type ATPase genes can be divided into ten phylogenetic branches. Six of them are found in plants (Axelsen & Palmgren, 2001). The plasma membrane proton pump belongs to the P3-type ATPase family. The H+-ATPase branch is subdivided into five subfamilies (Arango et al., 2003). Plasma membrane H+-ATPase is a tightly bound and integral transmembrane protein. The enzyme is a single polypeptide of ~100 kDa. By the use of the chemical energy of ATP, plasma membrane connected ATPases extrude protons from cells of plants to generate electrochemical proton gradients. The generation of this gradient has a major role in providing the energy for secondary active transport across the plasma membrane. The plant plasma membrane H+-ATPase is a proton pump which plays a central role in physiological functions such as nutrient uptake, intracellular pH regulation, stomatal opening and cell growth. Besides regulation of physiological processes, the plasma membrane proton pump also plays a role in adaptation of plants to changing conditions, especially stress conditions. Thus, H+-ATPase can be a mutual element for resistance mechanisms that are activated in various stress conditions. Many studies have shown the changes of gene expression of the plasma membrane H+-ATPase in response to a variety of environmental factors. Moreover, besides the genetic regulation of the proton pump, its activity may undergo fast post-translational modulation.


2. H+-ATPase structure and H+ transport

The enzyme is a functional single polypeptide chain with mass of about 100 kDa. The protein can oligomerize to form dimeric and hexameric complexes (Kanczewska et al., 2005). The H+-ATPase has N- and C-terminal segments, which emerge into the cytoplasm (Duby & Boutry, 2009). The structure of plasma membrane H+-ATPase consists of domains A, M, P, N, and R. The A-domain (actuator) consists of the N-terminal segment and small loop. The M-domain (membrane) corresponds to a transmembrane domain with ten helices, M1 to M10. The P-domain (phosphorylation) is located in the large loop. The N-domain (nucleotide binding) is located between two parts of the sequence forming the P-domain. The R-domain (regulatory) consists of the C-terminal fragment of the protein which acts as an autoinhibitory domain (Pedersen et al., 2007; Duby & Boutry, 2009). The plant plasma membrane H+-ATPase is kept at a low activity level by its C-terminal domain, the inhibitory function of which is thought to be mediated by two regions (regions I and II) interacting with cytoplasmic domains essential for the catalytic cycle (Speth et al., 2010).

The catalytic cycle of H+-ATPase is described by two main conformational states, E1 and E2. In the E1 conformation, the transmembrane binding site has high affinity for the proton and for ATP, whereas in E2 the same site has low affinity for both ligands. The states E1 and E2 alternate during transport (Buch-Pedersen et al., 2009). The cytoplasmically positioned N, P and A domains are in charge of ATP hydrolysis. Conformational changes in these domains during catalysis lead to simultaneous movements in the membrane-embedded part that directs the proton transport. The transporting unit of plasma membrane proton pump is defined by a centrally located proton acceptor/donor – a single protonable aspartic acid residue (Asp684), an asparagine residue (Asn106), an arginine amino acid residue (Arg655), and a large central cavity likely to be filled with water (Buch-Pedersen et al., 2009). The Asp684 is in close contact with Asn106. In the E1P structure protonation of Asp684 is believed to facilitate hydrogen bond formation between these two. Phosphorylation leads from E1P to E2P conformational changes and proton unloading from Asp684. Arg655 is suggested to play an important role in proton release and proton pumping against high membrane potentials. The positive charge of Arg655 approaching Asp684 will favour proton release from Asp684 and inhibit reprotonation of Asp684 with an extracellular proton (Buch-Pedersen et al., 2009). In plasma membrane H+-ATPases, a single proton is believed to be transported per hydrolysed ATP (Palmgren, 2001). However, partial uncoupling between ATP hydrolysis and proton transport has been suggested (Buch-Pedersen et al., 2006). The plant plasma membrane H+-ATPase is stimulated by potassium (Palmgren, 2001). K+ is bound to the proton pump in the cytoplasmic phosphorylation domain (P-domain). Binding of K+ promotes dephosphorylation of the phosphorylated E1P reaction cycle and it controls the H+/ATP coupling ratio. It was suggested that potassium acts as an intrinsic uncoupler of the plasma membrane H+-ATPase (Buch-Pedersen et al., 2006).

3. Transcriptional regulation of plant plasma membrane H+-ATPase

The molecular study of plant H+-ATPase has shown that this enzyme is encoded by a multigene family. The genes have been identified in: Arabidopsis thaliana12 genes (Palmgren, 2001), Lycopersicon esculentum10-12 genes (Kalampanayil & Wimmers, 2001), Oryza sativa10 genes (Baxter et al., 2003), Cucumis sativus10 genes (Wdowikowska, data unpublished), Nicotiana plumbaginifolia9 genes (Oufattole et al., 2000), Vicia faba5 genes (Nakajima et al., 1995), and Zea mays4 genes (Santi et al., 2003). Phylogenetic and gene structure analysis of plant H+-ATPases divided them into five subfamilies (Arango et al., 2003). Expression of H+-ATPase subfamilies I and II is not restricted to particular organs. These subfamilies are highly expressed in many cell types. In A. thalianatwo genes, AHA1and AHA2, are expressed in all tissues and organs. AHA1is predominantly expressed in shoots, AHA2in roots. In N. plumbaginifolia, pma4was highly expressed in several cell types, including meristematic tissues, root epidermis, hairs, phloem, and guard cells (Moriau et al., 1999). In maize, MHA2was expressed in the same set of cells as PMA4(Frias et al., 1996). Conversely, expression of genes belonging to subfamilies III, IV, and V is limited to specific organs or cell types (Arango et al., 2003). In A. thaliana, AHA6and AHA9transcripts are present mainly in anthers, AHA10in the endothelium of the developing seed coat, and AHA7and AHA8in pollen (Gaxiola et al., 2007). It has been shown that various genes are expressed in the same organ. Moreover, even within the same cell type at the same developmental stage, at least two H+-ATPase genes are expressed (Harms et al., 1994; Hentzen et al., 1996; Moriau et al., 1999). In N. plumbaginifoliatwo different plasma membrane H+-ATPase genes, PMA2and PMA4, are expressed in guard cells (Moriau et al., 1999).This observation suggests that isoforms with distinct kinetics might co-exist in the same cell.

In addition to tissue-specific expression, the plasma membrane H+-ATPases are differentially expressed according to environmental factors. Several studies have indicated that the H+-ATPase genes might be activated by various abiotic and biotic stresses. With such a phenomenon the amount of H+-ATPase might be increased under conditions requiring greater transport activity. The external signals result in changes in plant plasma membrane H+-ATPase gene expression, include salt (Niu et al., 1993; Binzel, 1995; Janicka-Russak & Kłobus, 2007), low temperature (Ahn et al., 1999, 2000), heavy metals (Janicka-Russak et al., 2008), dehydration (Surowy et al., 1991), light conditions (Harms et al., 1994), mechanical stress (Oufattole et al., 2000) and externally applied hormones (Frías et al., 1996).

4. Post-translational regulation of plant plasma membrane H+-ATPase

Plant plasma membrane H+-ATPase is involved in many different physiological roles and what is more its activity is changed by a large number of physiological factors. Therefore, it can be assumed that there are multiple regulatory features that integrate signals from the environment. As to post-translational regulation, the best known mechanism described to date involves the autoinhibitory action of the C-terminal domain (approximately 100 amino acids) of the enzyme protein. Deletion of the carboxyl terminus by trypsin treatment or genetic engineering results in constitutively activated enzyme. Although it is clear that the carboxyl terminus is the main regulatory domain involved in activation of the H+-ATPase, recent results suggest that the N-terminus may also play a role in modification of plasma membrane proton pump activity.

4.1. Regulation by phosphorylation

Phosphorylation and dephosphorylation of proteins is a very common example of post-translational modification that has the potential to alter protein activity. The activity of the enzyme is well known to be regulated by 14-3-3 proteins, the association of which requires phosphorylation of the penultimate H+-ATPase residues of Thr 947 (Svennelid et al., 1999). The binding of 14-3-3 regulatory protein displaces the inhibitory R-domain, activating the enzyme. R-domains contains two regions (I and II), of about 20 residues each, which contribute to keeping the enzyme at a low activity level. Mutagenesis of these regions abolishes the inhibitory effect of the C-terminus (Morssome et al., 1998; Axelsen et al., 1999;). 14-3-3 binding to H+-ATPase is stabilized by the fungal toxin fusicoccin, which decreases the dissociation rate. One 14-3-3 protein dimer binds two C-terminal polypeptides simultaneously, so a high activity state of H+-ATPase could involve formation of dimers or multimeric complexes. An analysis with cryo-electron microscopy showed that PMA2-14-3-3 complex is a wheel-like structure with 6-fold symmetry, suggesting that the activated complex consists of six H+-ATPase molecules and six 14-3-3 molecules (Kanczewska et al., 2005).

Activation of plasma membrane H+-ATPase involves protein kinase mediated phosphorylation of penultimate Thr in the C-terminus (region II) of the enzyme protein. Which specific kinases catalyse the reaction is an unresolved question. The protein kinase responsible for this phosphorylation has not yet been identified. Recently, it was reported that calcium-dependent protein kinase (CDPK) stimulated by abscisic acid leads to phosphorylation-dependent activation of H+-ATPase (Yu et al., 2006). In rice, a 55 kDa calcium-dependent protein kinase was shown to phosphorylate two Thr residues from the autoregulatory domain (region I) of the plasma membrane proton pump (Ookura et al., 2005).

Many reports indicate that the phosphorylation of amino acid residues at the C-terminus of the protein leads not only to raised enzyme activity but also to its inhibition (Vera- Estrella et al., 1994; Xing et al., 1996; Lino et al., 1998; Schaller & Oecking, 1999). A number of conserved serine and threonine residues found in the C-terminal regulatory domain serve as candidate residues for negative phosphorylation (Nühse et al., 2003). Some phospho-residues are close to each other and might influence the phosphorylation status of their neighbours (Duby & Boutry, 2009). In Nicotiana tabacumtwo new phosphorylation sites, Thr931 and Ser938, in the C-terminus of H+-ATPase, were identified. Their mutation suggests that phosphorylation of Thr931 or Ser938 prevents 14-3-3 protein binding, although the penultimate Thr955 was still phosphorylated, and prevented full activation of the enzyme (Duby et al., 2009). PKS5, a Ser/Thr protein kinase, is a negative regulator of the plasma membrane proton pump. In A. thalianathis kinase phosphorylates the enzyme (AHA2) at Ser931 in the C-terminus. Phosphorylation at this site inhibits interaction between H+-ATPase and 14-3-3 protein (Fuglsang et al., 2007). PKS5 is a salt overly sensitive (SOS) 2–like protein and interacts with the calcium-binding protein ScaBP1 (Fuglsang et al., 2007). During salt stress in A. thaliana, the calcium sensor Salt Overly Sensitive 3 (SOS3) binds to and activates the Ser/Thr protein kinase SOS2. The Ca2+-SOS3-SOS2 complex phosphorylates and activates the Na+/H+ antiporter SOS1, resulting in regulation of Na+ homeostasis and salt tolerance (Zhu, 2002). In Arabidopsis, a chaperone, J3 (DnaJ homolog 3, heat shock protein 40-like) activates plasma membrane H+-ATPase activity by physically interacting with PKS5 kinase (Yang et al., 2010).

Two extensively expressed plasma membrane H+-ATPase isoforms of Nicotiana tabacum(PMA2 and PMA4) are differentially regulated by phosphorylation of their penultimate threonine. Cold stress reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were observed (Bobik et al., 2010a).

A phosphorylation event requires action of a protein phosphatases to make regulation reversible. Phosphatase 2A (PP2A) competes with binding of 14-3-3 protein to the C-terminus of H+-ATPase (Fuglsang et al., 2006). Inhibition of phosphatase activity by administration of okadaic acid (inhibitor of phosphatase PP2A) to plants (maize roots) increased the bound level of 14-3-3 proteins and activity of H+-ATPase (Camoni et al., 2000).

A novel interactor of the plant plasma membrane proton pump was identified. This protein is named PPI1 (proton pump interactor, isoform 1). This interactor is able to modulate the plasma membrane H+-ATPase activity by binding to a site different from the 14-3-3 binding site (Morandini et al., 2002). PPI1 can only hyper-activate H+-ATPase molecules whose C-terminus has been displaced by other factor such as 14-3-3 protein (Viotti et al., 2005).

Recent studies have shown that the N-terminus of the plant plasma membrane H+-ATPase directly participates in pump regulation (Morsomme et al., 1998; Ekberg et al., 2010). It was suggested that transformation from low to high activity state of enzyme protein involves a structural rearrangement of both the C- and the N-terminus.

4.2. Regulation by membrane environment

The plant plasma membrane H+-ATPase requires lipids for activity. This lipid dependency suggests a possible mode of regulation of the plasma membrane proton pump via modification of its lipid environment (Kasamo, 2003). Abiotic stresses lead to changes in the plasma membrane lipid composition altering the fluidity of the membrane. The modulation of the phospholipid environment of the plasma membrane regulates the activity of H+-ATPase. This enzyme protein activity was abolished upon the removal of membrane lipids by detergents, but it was restored by exogenous addition of phospholipids (Kasamo & Nouchi, 1987, Kasamo 1990). The activation of H+-ATPase is dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with an increase in the length of the fatty acyl chain and in the degree of unsaturation of fatty acid (Hernandez et al., 2002; Kasamo, 2003, Martz et al., 2006).

Lysophosphatidylcholine (a natural detergent produced from phosphatidylcholine by phospholipase A2) increased the plasma membrane H+-ATPase activity (Pedechenko et al., 1990, Regenberg et al., 1995). It was found that lysophosphatidylcholine binds to the C-terminal region of the protein and by displacing the autoinhibitory domain leads to increase of ATPase activity.

5. Physiological roles

All living plant cells are thought to express a plasma membrane H+-ATPase. However, it has been found that the abundance of this protein varies in different cell types and tissues. The main role of roots is uptake of nutrients from soil and translocation of those nutrients to the shoots of plants. In roots high amounts of immunodetectable H+-ATPase antibodies were observed in epidermal cells, endodermis and phloem (Parets-Soler et al., 1990; Jahn et al., 1998). Because the plasma membrane H+-ATPase is responsible for establishing the proton gradient involved in the membrane energization used for solute transport, this enzyme controls the major transport processes in the plant: root nutrient uptake, xylem and phloem loading.

In addition to its fundamental role in nutrient uptake, H+-ATPase plays a major role in cell growth. The so-called “acid growth” theory suggests that protons extruded by an activated H+-ATPase decrease the apoplastic pH and activate enzymes involved in cell wall loosening (Hager, 2003). It is well established that auxin activates the proton pump, resulting in loosening of the cell wall (Hager, 2003). In plants, an auxin increases membrane flow from the endoplasmic reticulum to the plasma membrane. Therefore it was assumed that this membrane flow could carry newly synthesized H+-ATPase molecules to the PM. It was reported that IAA increased the amount of antibody-detectable H+-ATPases in the PM (Hager et al., 1991). Besides, treatment of maize coleoptile segments with auxin resulted in increase of mRNA levels of plasma membrane H+-ATPase, MHA2(Frias, et al., 1996). Recent evidence has shown that the PM H+-ATPase appears to be a target of NO-mediated auxin action (Kolbert et al., 2008; Zandonadi et al., 2010).

This pump is also involved in intracellular pH regulation. Acidification of the cytosol activates the plasma membrane proton pump and enhances the extrusion of protons from the cytosol to the apoplast. This contributes to alkalinization of the cytosol (Sanders et al., 1981). In Nicotiana tabacumacidification of the cytosol increased PMA2 phosphorylation at the penultimate Thr in the C-terminus (Bobik et al., 2010b).

Moreover, the plasma membrane proton pump is involved in other important physiological functions, such as stomatal aperture. Cell turgor changes promote modification of the stomatal aperture. Activation of H+-ATPase leads to plasma membrane hyperpolarization and subsequent opening of K+ channels. K+ and anion influx lead to water uptake, turgor increase, and cell swelling. On the other hand, an elevation of cytosolic Ca2+, inhibition of plasma membrane H+-ATPase, an increase in pH, a reduction in K+,, Cl-, and organic solute contents in both guard cells surrounding the stomatal pore, are downstream elements of ABA-induced stomatal closure (Zhang et al., 2001). The plant hormone abscisic acid (ABA) is a key signal molecule, mediating responses to various environmental stresses, and has been demonstrated to induce stomatal closure, thereby preventing water loss (Assmann & Shimazaki, 1999). The opening of stomata is mediated by an accumulation of K+ in guard cells, and K+ accumulation is driven by an inside-negative electrical potential across the plasma membrane. The electrical potential is created by the plasma membrane H+-ATPase in response to blue light (Schroeder et al., 2001). Blue light activates the H+-ATPase through the phosphorylation of Thr residues in the C-terminus. Next, phosphorylation induces the binding of 14-3-3 to the penultimate residue of Thr, which acts as a positive regulator for the H+-ATPase (Kinoshita & Shimazaki, 2002). Blue light receptors that mediate activation of the plasma membrane proton pump in stomatal guard cells are phototropins (Kinoshita et al., 2001). Phototropins are autophosphorylating Ser/Thr protein kinases (Briggs & Christie, 2002). The activated phototropins transmit the signal to plasma membrane H+-ATPase for its activation (Christie, 2007). In addition, it was demonstrated that phosphatase 1 (PP1, Ser/Thr protein phosphatase) mediates the signal between phototropins and H+-ATPase in guard cells (Takemiya et al., 2006). It is known that ABA inhibits blue light dependent proton pumping by plasma membrane H+-ATPase (Zhang et al., 2004). Biochemical and genetic studies have demonstrated that H2O2 mediates inhibition of the plasma membrane proton pump by ABA (Zhang et al., 2001). In guard cells, ABA induces production of phosphatidic acid (PA). Because PA also interacts with PP1 and decreases its phosphatase activity, it seems that PA suppresses the blue light signalling of guard cells (Takemiya & Shimazaki, 2010). Studies have demonstrated that ABA induces NO synthesis through H2O2 (Bright et al., 2006) and that NO causes PA production in guard cells (Distéfano et al., 2008).


6. H+-ATPase in abiotic stress

The plant plasma membrane H+-ATPase is an important functional protein, which plays a central role in plant physiology. PM H+-ATPase couples ATP hydrolysis to proton transport out of the cell, and so establishes an electrochemical gradient across the plasma membrane, which is dissipated by secondary transporters using protons in symport or antiport. This enzyme controls the major transport processes in the plant, such as root nutrient uptake and xylem or phloem loading. Moreover, this pump has been proposed to be involved in other important physiological functions, such as stomata aperture, cell elongation, or cellular pH regulation. Generation of an electrochemical gradient across the membrane results in a proton-motive force that is used by secondary transport for assimilation of various nutrients, and also for releasing ions and toxic substances from cells. The plasma membrane proton pump is an enzyme whose activity is altered significantly in response to a number of factors, such as light, temperature, hormones and presence of salt or heavy metals in the environment. Besides regulation of growth and development processes, the plasma membrane proton pump also plays a role in plastic adaptation of plants to changing conditions, especially conditions of stress. Adaptation is a complex process. Some of the modifications in plants subjected to abiotic stress are indicated to be adaptive. Physiological modifications caused by environmental stress and allowing continued plant functions are ascribed by plant physiologists as being adaptive.

6.1. Salt stress

Salt stress is a complex abiotic stress in which both ionic and osmotic components are involved (Alvarez et al., 2003). Under conditions of elevated NaCl levels outside the cell, sodium passively enters into the roots through non-selective cation channels and the low-affinity sodium transporter HKT1. Salinity tolerance of plants is a complex trait involving adaptation at the level of cells, organs and the whole plant. The key factor of salinity tolerance, beside osmotic adjustment, is the control of intracellular ion homeostasis (Niu et al., 1993). The excess of Cl- is passively effluxed across the membrane down the ΔμH+ generated by plasma membrane H+-ATPase. To prevent accumulation of toxic Na+ amounts in the cytosol, active sodium efflux into the apoplast and its compartmentalization inside the vacuole occur. Since Na+ pumps responsible for sodium extrusion in animals and microorganisms are absent in higher plant cells, secondary sodium/proton antiporters in both the plasma membrane and the tonoplast are needed to translocate sodium ions against their electrochemical gradients (Apse and Blumwald 2007). Molecular analyses in Arabidopsisled to the identification of a plasma membrane SOS1 (Salt Overly Sensitive 1) and vacuolar NHX1, Na+/H+ antiporters upregulated at the genetic level in response to NaCl (Apse et al. 1999; Gaxiola et al. 1999; Shi et al. 2000, 2003). These proton-coupled sodium transporters use the proton-motive force created by the specific proton pumps. The only pump which generates an electrochemical proton gradient across the plasma membrane is H+-ATPase (Palmgren, 2001). For this reason, it is believed that plant plasma membrane H+-ATPase plays a major role in salt stress tolerance. The importance of plant plasma membrane H+-ATPase in salt tolerance is demonstrated by observations such as increase in its activity in halophytes (Braun et al., 1986; Niu et al., 1993; Vera-Estrella et al., 1994, Sahu & Shaw, 2009). In halophytes and salt-tolerant cultivars significantly greater activity of the enzyme is observed in normal conditions, without NaCl (Sahu & Shaw, 2009). Moreover, it has been well documented that salt treatment of plants induces the activities of the plasma membrane proton pumps both in halophytes and glycophytes (Niu et al., 1993; Perez-Prat et al., 1994; Binzel, 1995; Kłobus and Janicka-Russak, 2004, Sahu & Shaw, 2009; Lopez-Perez et al., 2009; Shen et al., 2011). However, there are few reports about the inhibition or no effect of NaCl on the plasma membrane H+-ATPase in leaves (Chelysheva et al., 2001; Zörb et al., 2005; Pitann et al., 2009; Wakeel et al., 2010). The authors observed an increase of apoplastic pH in salt stress conditions. The salinity would thus limit leaf elongation.

The aforementioned salt-dependent activation of the plasma membrane proton pump encompasses the transcriptional as well as post-translational level. Accumulation of mRNAs of PM H+-ATPase under NaCl stress and the positive correlation with salt tolerance are well documented (Niu et al., 1993; Perez-Prat et al., 1994; Janicka-Russak & Kłobus 2007; Sahu and Shaw, 2009). In Suaedamaritima(a natural halophyte) and Oryza sativasalt-tolerant cultivar accumulation of PM H+-ATPase gene transcript was greater than that in a non-tolerant cultivar of rice treated with NaCl (Sahu & Shaw, 2009). As mentioned earlier (subsection 11.3), plant plasma membrane ATPase is encoded by a large gene family. The existence of multiple isoforms of the enzyme creates the opportunity of their role in abiotic stress tolerance, particularly salt stress tolerance. In rice a new isoform of the enzyme (finding maximum homology with OSA7) in response to salt treatment was discovered. In the halophyte Suaedamaritimethe related gene (SM2, which has 89% homology with the new isoform from rice) does not require the presence of salt for its expression. So the salt-inducible isoform of the plasma membrane H+-ATPase gene in rice remains constitutively expressed in the halophyte S. maritime(Sahu & Shaw, 2009). The isoform of PM H+-ATPase from tomato LHA8is most closely related to Nicotiana plumbaginifoliagene PMA6. Both of these isoforms are specially induced by stress: LHA8by salt stress, PMA6by mechanical stress (Oufattole et al., 2000; Kalampanayil & Wimmers, 2001).

A direct role of H+-ATPase in salt tolerance was confirmed by studies with transgenic tobacco, using a PMA4 mutant, lacking the autoinhibitory domain (ΔPMA4). In the mutant a constitutively activated PMA4 H+-ATPase isoform was present. The ΔPMA4 plant roots showed better growth in saline conditions than those of untransformed plants (Gévaudant et al., 2007).

In rice phospholipase Dα (PLDα) is involved in salt tolerance by the mediation of H+-ATPase activity and transcription (Shen et al., 2011). When rice suspension-cultured cells were treated with 100 mM NaCl, PLDα activity increased. The knockdown of OsPLDα1 prevented NaCl-induced increase in transcript levels of OSA2 (which encodes PM H+-ATPase) as well as ATPase activity.

Recently evidence has been presented that NaCl also causes a rapid modulation of proton pumps, which is due to the reversible phosphorylation of enzyme proteins (Kerkeb et al., 2002; Kłobus and Janicka-Russak, 2004). In many plant tissues a salt-inducible shift in the cytoplasmic calcium level was observed (Heterington and Quatrano, 1991; Rock and Quatrano, 1995; Danielsson et al., 1996; Knight et al., 1997; Blumwald et al., 2000; Netting, 2000; Xiong et al., 2002), suggesting its involvement in the signalling pathway target under NaCl stress conditions. An SOS (salt overly sensitive) network exists in plants exposed to salt stress. SOS1, antiporter Na+/H+, enables Na+ efflux across the plasma membrane and controls long-distance Na+ transport between roots and leaves through loading and unloading of Na+ in the xylem (Shi et al. 2002). Sodium efflux through SOS1 is mediated by SOS3-SOS2 complex (Qiu et al. 2002). Perception of salt stress induces a cytosolic calcium signal activating the calcium sensor, myristoylated protein SOS3. After binding with Ca2+, SOS3 changes its conformation and interacts with the FISL motif of SOS2, a Ser/Thr protein kinase, and activates its substrate phosphorylation. The activated SOS3-SOS2 complex then phosphorylates SOS1 and activates its antiporter activity (Chinnusamy et al. 2005).

Evidence has also been presented that in the plasma membrane ATPase phosphorylation, as a target of activation by NaCl, a calcium/calmodulin-dependent protein kinase sensitive to staurosporine, is involved (Kłobus and Janicka-Russak, 2004).

Transient increases in cytosolic Ca2+ can induce the phosphorylation of different proteins in cells, improving the salt tolerance (Hasegawa et al. 2000). Evidence suggests that the major role in coupling the calcium signal to specific protein phosphorylation cascade(s) is played by the Ca2+/calmodulin-dependent protein kinases (CDPKs) and the SOS3 family of Ca2+ sensors (Xiong et al. 2002, Zhu 2002). The results of Urao et al. (1994) and Saijo et al. (2000) demonstrated that NaCl rapidly induced CDPK in different plant tissues. Furthermore, evidence has been presented that Ca2+/calmodulin-dependent protein kinases are responsible for the phosphorylation of the plasma membrane H+-ATPase protein (Van der Hoeven et al. 1996, Camoni et al., 2000).

Ca2+ has been identified as a possible mediator of ABA-induced stimulus-response coupling (Netting, 2000). Abscisic acid is known as a stress hormone, which mediates responses to a variety of stresses, including water and salt stress (Skriver and Mundy, 1990; Tan et al., 1994; Jia et al., 2001). The endogenous level of ABA increases when plants are stressed with drought or NaCl, and application of ABA to unstressed plants results in the induction of numerous water-deficit-related activities (La Rosa et al., 1985; 1987; Skriver and Mundy, 1990; Kefu et al., 1991; Cowan et al., 1997; Barkla, 1999). They include triggering of stomatal closure to reduce transpirational water loss by post-translational modulation of ion channels in guard cells (Grabov and Blatt, 1998), and alterations in gene expression through induction of ABA-responsive genes coding for structural, metabolic or transport proteins (Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1996; Barkla et al., 1999).

It was reported that ABA treatment of cucumber plants, as well as NaCl treatment, increased activity of plasma membrane H+-ATPases. ABA treatment of seedlings elevated the level of plasma membrane H+-ATPase transcript (Janicka-Russak & Kłobus, 2007). Thus activation of this proton pump by salt and abscisic acid seems to involve the genetic level.

Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signal molecules in plants subjected to abiotic stresses. In plants, the generation of H2O2 seems to be mediated by a plasma membrane bound NADPH oxidase complex (Yang et al., 2007; Lherminier et al., 2009). NO is synthesized via nitric oxide synthase (NOS) and nitrate reductase (Yamasaki & Sakihama, 2000). NO and H2O2 interact as signalling molecules in plants (Zhang et al., 2007). Under salt stress, both NO and H2O2 are produced. Treatment of plants with NO increased the activity of plasma membrane NADPH oxidase and thus the generation of hydrogen peroxide. Both these molecules stimulated the activity, as well as the expression of plasma membrane H+-ATPase in Populus euphratica(Zhang et al., 2007). However, the increase of proton pump activity is induced by NO possibly via the regulation of PM NADPH oxidase. Additionally, activity of PM H+-ATPase may depend on the membrane redox state. Plasma membrane oxidoreductase can modify activity of the PM H+-ATPase proton pump (Kłobus and Buczek 1995). PM oxidoreductase, by transporting electrons across the PM, simultaneously acidifies the cytoplasm (Lüthje et al. 1997). Low pH of the cytoplasm and membrane depolarization stimulate the activity of PM H+-ATPase (Hager and Moser 1985; Rubinstein and Stern 1986; Kłobus 1995).

Plants can increase salinity tolerance by modifying the biosynthesis of polyamines. In cucumber the level of polyamines decreased when the concentration of Na+ in the cytosol increased, so the action of polyamines contributes to ionic equilibrium (Janicka-Russak et al., 2010). Moreover, it was found that polyamines decreased activity of the plasma membrane proton pump in cucumber. So a decrease in their biosynthesis under salinity seems to be beneficial in stress tolerance.

Fatty acids, as the main component of membrane lipids, are considered to be important in salt tolerance of plants. Salt-tolerant plants showed an increase of unsaturated fatty acids (Lin & Wu, 1996). In broccoli plants a high degree of unsaturation in the plasma membrane of roots was observed (López-Pérez et al., 2009). The activity of plant plasma membrane H+-ATPase increased with an increase in the degree of unsaturation of fatty acid (Kasamo, 2003, Martz et al., 2006). Non-tolerant plants subjected to salt stress commonly show decreased levels of 18:3 in their membranes (Upchurch, 2008). A study with transgenic tobacco showed that overexpression of ω-3 desaturases, which increases 18:3, elevated tolerance to salt stress (Zhang et al., 2005).

In potato the mRNA level of StPPI1, a homologue of the A. thalianaPPI1 (proton pump interactor isoform 1), increased under salt and cold stress conditions. StPP1 increases PM H+-ATPase activity by hyperactivation of H+-ATPase whose C-terminus has been displaced earlier by 14-3-3 protein (García et al., 2011).

6.2. Heavy metals

An increase in permeability related to membrane damage is observed in plants that have been subjected to heavy metal stress. It is well known that metal ions are easily bound to both the sulfhydryl groups of proteins and hydroxyl groups of phospholipids (Devi and Prasad, 1999). They can also replace the calcium ions at essential sites of cell membranes (Breckle and Kahle, 1991). All these events result in disruption of membrane integrity and ionic homeostasis of cells. Maintaining ionic balance and replenishing the loss of essential substances in repair processes is an important issue under such conditions. Support of active transport of ions and organic compounds through the plasma membrane requires increased generation of a proton gradient by PM H+-ATPase. Generation of an electrochemical gradient across the membrane results in a proton-motive force that is used by passive transport for assimilation of various nutrients, as well as for releasing ions and toxic substances from cells.

To date, data concerning the action of heavy metals on plasma membrane H+-ATPase are limited. A few observations have indicated that enzyme activity was changed under heavy metal (Cd, Cu, Ni, Al) stresses (Lindberg & Wingstrand, 1985; Kennedy and Gonsalves, 1989; Ros et al., 1992 a,b; Fodor et al., 1995; Demidchik et al., 1997; Astolfi et al., 2003; Burzyński & Kolano, 2003; Astolfi et al., 2005; Shen et al., 2005; Janicka-Russak et al., 2008; Kabała et al., 2008). The effect of metals on plasma membrane H+-ATPase activity depends on time of exposure of plants to heavy metals, kind and concentration of heavy metal or plant species. In cucumber seedling roots, brief treatment of plants with Cd and Cu leads to inhibition of plasma membrane H+-ATPase activity (Janicka-Russak et al., 2008, Kabała et al., 2008). The inhibition of the enzyme was partially diminished in the presence of cantharidin, a specific inhibitor of PP2A and PP1 phosphatases. Moreover, Western blot analysis with an antibody against phosphothreonine confirmed that decreased activity of plasma membrane H+-ATPase in a short time (2 hours, 10 and 100 µM Cd or Cu) resulted from dephosphorylation of the enzyme protein (Janicka-Russak et al., 2008). However, longer time of treatment of cucumber roots with those heavy metals (6 days, 10 µM Cd or Cu) leads to increased activity of the enzyme measured both as hydrolysis of ATP and proton transport across the plasma membrane (Janicka-Russak, data unpublished). The same effect was observed in cucumber plants treated for 18 hours with 100 µM Cd and in maize with 100 µM both Cd and Cu (Burzyński & Kolano, 2003). In rice treated for 5 or 10 days with 100 and 500 µM Cd increased proton pump activity was observed (Ros et al., 1992a). On the other hand, in oat roots treated long term (7 and 21 days) with 100 µM Cd plasma membrane proton pump activity was inhibited (Astolfi et al., 2003). The same effect was observed in maize subjected for 4 days to cadmium (Astolfi et al., 2005). When plants were treated for 5 or 10 days with nickel, stimulation of H+-ATPase in rice shoots’ plasma membrane was observed both in 100 and 500 µM concentration. But in rice roots only 100 µM concentrations of Ni lead to stimulation of the enzyme protein (Ros et al., 1992a). The increase of ATPase activity in conditions of Ni and Cd metal stress was observed simultaneously with decrease in the degree of unsaturation and length of the phospholipid fatty acyl chain (Ros et al., 1992b). In contrast, Hernandez & Cook (1997) observed an increased degree of unsaturation of phospholipid-associated fatty acid in pea root plasma membrane as a result of 10-day cadmium treatment of plants. A similar effect was observed in the case of Cu in tomato membrane lipids as a result of 7 days’ stress (Ouariti et al., 1997). It is known that PM H+-ATPase is stimulated when the degree of unsaturation of fatty acids is increased (Hernandez et al., 2002). In soybean root, under aluminium stress, up-regulation of transcript, translation and threonine-oriented phosphorylation of plasma membrane H+-ATPase was observed (Shen et al., 2005).

6.3. Low temperature

An increase in membrane permeability and a change in its viscosity and fluidity result in a decrease of cell turgor due to low temperature (Guy 1990). Membrane changes at low temperature concern positional redistribution of saturated and unsaturated fatty acids (Hughes and Dunn 1996). Probably the change in membrane fluidity in cold-sensitive species initiates a signal chain reaction that leads to acclimation to cold stress by increasing the expression of certain genes, for example genes encoding desaturases (Xiong and Zhu 2001; Chinnusamy et al. 2006). An increase in permeability related to membrane damage is observed in plants that have been subjected to low temperature. Maintaining ionic balance and replenishing the loss of essential substances in repair processes is an important issue under such conditions. Support of active transport of ions and organic compounds through the PM requires increased generation of a proton gradient by PM H+-ATPase. Generation of an electrochemical gradient across the membrane results in a proton-motive force that is used by active transport for assimilation of various nutrients. Published data indicate that activity of PM H+-ATPase is modulated under low temperature by changes in lipids associated with the PM proton pump (Lindberg et al. 2005; Martz et al. 2006). Plants capable of cold acclimation accumulate polyunsaturates during cold stress (Vega et al., 2004; Upchurch et al., 2008). In Pinus resinosathe activity of plasma membrane H+-ATPase increased more than twofold following cold acclimation (Martz et al., 2006). In winter hardy tree species seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation under natural conditions. Fatty acid-regulated plasma membrane proton pump activity is involved in the cellular response underlying cold acclimation and de-acclimation (Martz et al., 2006). Similarly, in rye roots H+-ATPase activity increases during acclimation to low temperature, following increases in fatty acid unsaturation, particularly linoleic acid (White et al., 1990). An increase in plasma membrane H+-ATPase at low temperature (5 oC) was also observed in cells of winter wheat seedlings which were hardened earlier. In non-hardened tissues such an increase of enzyme activity was not observed (Ling-Cheng et al., 1982). In Oryza sativaplasma membrane H+-ATPase activity increased at low temperature in chilling-insensitive plants whereas in chilling-sensitive plants a slight decrease in enzyme activity was observed (Kasamo, 1988). The length of plants’ exposure to the low temperature affects activity of the PM proton pump in various ways. Brief (1 or 3 days) exposure of cucumber seedlings to low temperature inhibits hydrolytic and transport activity of H+-ATPase (Lee et al., 2004, Janicka-Russak, data unpublished). However, stimulation of activity was observed after treating plants with a low temperature (10 oC) for 6 days (Janicka-Russak, data unpublished). On the other hand, Ahn and coworkers (2000), despite using the same plant species, cucumber, observed different results. The increase in H+-ATPase activity after 1 day of low temperature treatment was reversed and gradually diminished as root temperatures of 10 oC continued for the next 6 days.

It was shown that regulation of the plasma membrane proton pump may be based on the interaction of H+-ATPase and 14-3-3 protein. Rapid cooling of protoplasts derived from sugar beet cells results in activation of the proton pump. Moreover, cytoplasmic 14-3-3 protein associated with plasma membrane and thus the amount of ATPase-14-3-3 complexes increased (Chelsyheva et al., 1999).

The activity changes of PM H+-ATPase may partly result from changes in the pattern of expression of PM H+-ATPase genes (Ahn et al., 1999, 2000; Janicka-Russak, data unpublished).

7. Conclusion

According to the above information, we can conclude that plant plasma membrane H+-ATPase is an important functional protein, which plays a central role in plant physiology in normal growth conditions and under abiotic stresses. In plants exposed to different abiotic stresses an increase in permeability related to membrane damage is observed. Maintaining ionic balance and replenishing the loss of essential substances in repair processes is an important issue under such conditions. Support of active transport of ions and organic compounds through the plasma membrane requires increased generation of a proton gradient by PM H+-ATPase. In addition, stress conditions such as salinity or heavy metals leads to accumulation of a toxic excess of certain ions. Generation of an electrochemical gradient across the membrane results in a proton-motive force that is used by active transport for assimilation of various nutrients, as well as for releasing ions and toxic substances from cells. Thus, plasma membrane H+-ATPase can be a mutual element for resistance mechanisms that are activated in various stress conditions.

© 2011 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike-3.0 License, which permits use, distribution and reproduction for non-commercial purposes, provided the original is properly cited and derivative works building on this content are distributed under the same license.

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Małgorzata Janicka-Russak (August 29th 2011). Plant Plasma Membrane H+-ATPase in Adaptation of Plants to Abiotic Stresses, Abiotic Stress Response in Plants - Physiological, Biochemical and Genetic Perspectives, Arun Shanker and B. Venkateswarlu, IntechOpen, DOI: 10.5772/24121. Available from:

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