Keywords

11.1 Introduction

Drought, salinity, and extreme temperatures are the major abiotic stress factors that adversely affect plant growth, development, and crop productivity. They alleviate the photosynthetic activity and induce nutrient scarcity and ionic and osmotic stress conditions in plants (Munns and Tester 2008; Rehman et al. 2005; Ashraf et al. 2008).

Salinity leads to degradation of soil fertility as a result of both natural and anthropogenic activities such as irrigation in arid and semiarid regions. Approximately 20% of the irrigated lands, i.e., 45 million hectares, is affected by soil salinization worldwide (Yeo 1999; Munns and Tester 2008). Moreover the change in global climate made rainfall less predictable and caused a drastic shift in the general rainfall pattern. This is of serious concern as there is much decrease in rainfed farm lands which produce one third of the world’s food supply.

Under high salinity, plants experience both osmotic and ionic stress. The salt concentrations outside the roots rise rapidly, thereby leading to inhibition of water uptake by the roots, cell expansion, and lateral bud development (Munns and Tester 2008). Ionic stress develops when excess Na+ accumulates particularly in leaves leading to increase in leaf mortality with chlorosis and necrosis and subsequently decrease in essential cellular metabolism activities such as photosynthesis (Yeo and Flowers 1986; Glenn et al. 1999). As NaCl is the most soluble and widespread salt, all plants have evolved mechanisms to regulate its accumulation.

Under salinity stress, plant cells need to maintain low cytosolic Na+ level and high K+ levels, resulting in a high cytosolic K+/Na+ ratio that is crucial for vital cellular metabolisms (Jeschke 1984; Blumwald 2000). The strategies generally employed by plants for the maintenance of a high K+/Na+ ratio in the cytosol include Na+ extrusion and/or the intracellular compartmentalization of Na+ (mainly in the plant vacuole). These mechanisms are vital for detoxification of cellular Na+ levels and cellular osmotic adjustment which are needed to tolerate salt stress and plant survival (Blumwald 2000; Gaxiola et al. 2001; Li et al. 2010; Wei et al. 2011). The compartmentalization of Na+ into vacuoles prevents the deleterious effects of Na+ in the cytosol and allows the plants to use NaCl as an osmoticum. NaCl generates an osmotic potential that drives water into the cells (Gutiérrez-Luna et al. 2018).

The plant cell vacuole performs important biological functions such as recycling of cell components, regulation of turgor pressure, detoxification of xenobiotics, and accumulation of many useful substances. A large number of vacuolar proteins are known to be involved in support of the above multifaceted functions (Ohnishi et al. 2018). They include active pumps, carriers, ion channels, receptors, and structural proteins. Several major proteins of the tonoplast have been extensively investigated, and it was found that the three most abundant proteins of the tonoplast are vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) (Maeshima 2000, 2001; Meng et al. 2017), and water channels (aquaporins) (King et al. 2004).

V-ATPase and VPPase coexist on the plant vacuolar membrane and use ATP and inorganic pyrophosphate (PPi), respectively, as energy sources for generating an electrochemical gradient of protons across the tonoplast. This facilitates the functioning of the Na+/H+-antiporter. The V-ATPase enzyme is a multisubunit proton pump found in all eukaryotes consisting of the peripheral (V1) complex responsible for ATP hydrolysis and the membrane-integral (Vo) complex responsible for proton translocation. V-ATPase is the largest complex in the tonoplast, with a total molecular size of about 750 kDa.

VPPase is a heat stable single polypeptide found in plants, algae, photosynthetic bacteria, protozoa, and archaebacteria (Rea et al. 1992; Maeshima 2000). It functions as a tonoplast proton pump and helps in Na+ compartmentation. In plants, two isoforms of VPPase have been identified; one is potassium-dependent, while the other is potassium-independent (Belogurov and Lahti 2002; Schilling et al. 2017). Aquaporins are referred to as intercellular water channels imbedded in the membranes, and they facilitate transport of water, small solutes, and ions across membranes (Aharon et al. 2003; Porcel et al. 2005).

In this chapter, the vacuolar transporter VPPase has been reviewed with respect to its structure, function, phylogeny, and mode of action. This provides us with an understanding how plants tolerate and survive under salt-stressed environments.

11.2 Molecular Phylogeny of VPPase

VPPases have been reported to be highly conserved among land plants and less among archaeon, protozoan, and bacteria (Suneetha et al. 2016). VPPase from R. rubrum (Baltscheffsky et al. 1998), Acetabularia acetabulum (marine algae) (Ikeda et al. 1999), and Chara coralline (green algae) (Nakanishi et al. 1999) predicted the overall identities of amino acid sequences among these three phylogenetically separated organisms. It was reported that R. rubrum PPase synthase (660 residues) exhibited 36–39% with V-PPases of land plants and 40% with A. acetabulum V-PPase. Moreover A. acetabulum V-PPase shared 47% identity with land plant VPPases. However, the highest identity was observed in case of C. corallina (71%) with respect to land plants. These observations of sequence similarity suggest that C. corallina is evolutionarily closer to land plant than R. rubrum and A. acetabulum. Phylogeny with respect to other land plants revealed that VPPase of A. thaliana (AtVPP), H. vulgare (HvVPP), B. vulgaris (BvVPP), N. tabacum (NtVPP), and O. sativa (OsVPP) ranged from 761 to 771 amino acids in length. The amino acid sequences were found to be highly conserved with 86–91% sequence similarity among the land plants.

Phylogeny is used in establishing the origin and evolution pattern of a gene of particular species with respect to the other species. Generally phylogenetic tree is constructed using neighbor-joining (NJ) or maximum parsimony (MP) or maximum likelihood (ML) method (Saitou and Nei 1987). Suneetha et al. (2016) carried out phylogenetic studies on land plants, archaea, and bacterial V-PPases (Fig. 11.1).

Fig. 11.1
figure 1

Relationship of 28 VPPases among land plants, land plant precursor, and bacteria as represented in a phylogenetic tree. (Source: Suneetha et al. 2016)

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Suneetha (2015) generated three phylogenetic trees in land plants using NJ, MP, and ML which showed similar topologies in both distance and character methods but differed in their branching order. Topological similarity of the trees obtained by different methods (NJ, MP, and ML) indicates that these clusters are not incidental and branching order reflected the expected pattern in all plants. The MP tree was constructed from 772 characters, out of which 515 were observed as conserved and 255 were variable, and of these 183 were parsimony informative. The tree length (L), consistency index (CI), and retention index (RI) in land plants were found to be 677, 0.61, and 0.77. The ML tree has a significant maximum likelihood tree length (−6594.00) (Fig. 11.2).

Fig. 11.2
figure 2

Relationship of VPPases among land plants. The phylogenetic tree was generated using maximum likelihood method. (Source: Suneetha 2015)

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Similarly, Liu et al. (2011) reported that VPPase isolated from Suaeda corniculata showed highest similarity with Kalidium foliatum (96%), Suaeda salsa (94%), Chenopodium rubrum (89%), Beta vulgaris (89%), Chenopodium glaucum (88%), and Arabidopsis thaliana (87%). Dong et al. (2011) reported that apple VPPase (MdVHP1) shared highest similarity with peach VPPase (94%) followed by 87% similarity with VPPases of tobacco, grapevine, and Arabidopsis. Similarly, VPPase of H. caspica showed high sequence similarity with VPPases from Chenopodiaceae family and shared 95% sequence identity with VPPase of K. foliadum. All the studies reported the evolutionary history and relationship of VPPase gene among bacteria, land plants, and its precursor. The studies also provided enough evidence to conclude that VPPase gene is highly conserved among plant family members.

11.3 Motifs of VPPase

The structural model of VPPase showing N- and C-terminals in vacuolar end, transmembrane helices, and three conserved regions (CS1, CS2, and CS3) was reported by Maeshima (2001). Immunochemical analysis confirmed that these conserved sequences are located in the cytosolic loops (Takasu et al. 1997).

Comparison of all VPPase genes from C. coralline, A. acetabulum, R. rubrum, and land plants reported with three highly conserved regions called motifs. The conserved motifs have been designated as CS1, CS2, and CS3 motifs (Rea and Poole 1993; Baltscheffsky et al. 1999; Maeshima 2000; Mimura et al. 2004; Suneetha 2015). Plant VPPase are characterized by the presence of cytosolic loops (CLs), vacuolar loops (VLs), and transmembrane domains (TMDs) besides the N- and C-terminals residues (Zhen et al. 1997). Site-directed mutagenesis and immunochemical analysis revealed that the cytosolic domains are more conversed than the vacuolar domains and thus are crucial for VPPase enzyme activity.

The first conserved segment (CS1) has consensus sequence of DVGADLVGKVE and functions as the catalytic domain for substrate hydrolysis (Rea and Poole 1993; Schocke and Schink 1998). In addition to the catalytic site, there are binding sites for Mg2+, K+, and reagents, such as N,N-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole (NBDCl), and N-ethylmaleimide (NEM) (Maeshima 2000; Sanders et al. 1999). Fukuda et al. (2004) validated the presence of NEM binding site at Cys-635 position, and Glu-306, Asp-505, and Glu-752 positions were identified as DCCD binding residues in barley. Zhen et al. (1997) conducted mutation and biochemical assays and revealed that Glu305 and Asp504 of A. thaliana V-PPase directly participate in DCCD binding and are presumably critical for catalysis.

The second conserved segment (CS2) is highly conserved and is located in a hydrophilic loop in the cytosol end. Suneetha (2015) reported that the CS2 motif has consensus sequence GSAALVSL and is approximately located at amino acid positions 543–550 in Sorghum bicolor. Suneetha (2015) reported that CS2 motif has function similar to rhodopsin like G-protein-coupled receptors (GPCRs) and is equipped with unique calcium signaling signature property that senses the high cytosolic Ca2+ levels and initiates V-PPase activity.

The third conserved segment (CS3) is located in the carboxyl-terminal part and contains 12 charged residues. It has consensus sequence GDTIGD exposed to the cytosol and plays a critical role in catalytic function in association with CS1 and CS2 segments (Liu et al. 2011; Rea et al. 1992). The position of these conserved regions change from one plant VPPase to others. For example, CS1 functional motifs DDPR and VGDN are located at 271 and 285 amino acid positions in mung bean, whereas in S. corniculata they are located at 266 and 280 amino acid positions, and in S. bicolor they occupy the 266 and 281 amino acid positions (Fig. 11.3). Similarly, the other conserved sequences CS2 and CS3 motifs are also highlighted in amino acid sequence alignment.

Fig. 11.3
figure 3

Three conserved motifs CS1, CS2, and CS3 highlighted in (a) amino acid alignment are generated for sequences of VPPases; (b) region of conserved sequences of CS1, CS2, and CS3 are highlighted taking S. bicolor VPPase (meta-analysis of motifs was carried out)

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11.4 Structure of VPPase

Vacuolar H+-pyrophosphatase (VPPase) catalyzes electrogenic H+-translocation from the cytosol to the vacuolar lumen at the expense of hydrolysis of inorganic pyrophosphate (PPi). PPi is produced as a by-product of several metabolic processes, such as polymerization of DNA and RNA and synthesis of aminoacyl-tRNA (protein synthesis), ADP-glucose (starch synthesis), UDP-glucose (cellulose synthesis), and fatty acyl- CoA (L-oxidation of fatty acid).

11.4.1 Topology

VPPase consists of a single polypeptide, and its substrate, inorganic pyrophosphate (PPi), is one of the simplest high-energy compounds (Baltscheffsky et al. 1999; Maeshima 2000; Rea and Poole 1993). V-PPase gene encodes a polypeptide with 761–771 amino acids. Various V-PPase genes have been analyzed from different plant and bacterial species (Table 11.1). It was reported that VPPase gene isolated from H. capsica encodes 764 amino acids, apple VPPase gene encodes 771 amino acids, S. corniculata encodes 764, and S. bicolor encodes 763 amino acids.

Table 11.1 List of VPPase genes and corresponding transmembrane helices
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Hydropathic and membrane topological analyses indicated that VPPase in general consists of 4–17 transmembrane domains (Table 11.2). Suneetha (2015) predicted that S. bicolor VPPase has 16 transmembrane regions using TMpred and TMHMM. The results obtained showed that the sequences has 16 inside to outside helices orientations and 16 outside to inside helices orientations of the transmembranes (Fig. 11.4a, b).

Table 11.2 Sequence positions of possible transmembrane helices from inside to outside and vice versa in VPPase of S. bicolor
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Fig. 11.4
figure 4

Transmembrane helices of VPPase in S. bicolor (a) TM pred, (b) TMHMM. (Source: Suneetha 2015)

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The 3D structure of VPPase is a vacuolar membrane-bound protein compactly folded in rosette manner in two concentric walls (Lin et al. 2012; Suneetha et al. 2016; Suneetha 2015) (Fig. 11.5). Lin et al. (2012) reported that mung bean VPPase has 16 transmembrane helices, but it exists as a homodimer, and Suneetha (2015) reported that S. bicolor VPPase exists as monomer with 16 transmembrane helices. The core has six transmembrane helices surrounded by ten transmembrane helices which form the inner and outer walls of the pump which is displayed in cylinders (Fig. 11.6). Two short helices are present on the cytosolic side; two helices and two antiparallel β-strands are present on the luminal side of the protein (Fig. 11.7). The core of the model has one IDP molecule surrounded by five Mg2+ ions which are essential for the activity of V-PPases and one K+ ion which acts as stimulator (Fig. 11.8). The above elements are highly conserved among the VPPases which forms a hydrophobic door to the hydrophilic surroundings of the vacuolar lumen. The hydrophobic gate prevents the reflux of H+ ions and helps in maintaining the translocation of H+ from cytosol to vacuolar lumen (Fig. 11.9). The space-fill representation of VPPase model is considered to analyze electrostatic surface potential. The surface potential is indicated by colors as in Fig. 11.10. The core of model which contains IDP binding site is represented within the circle the core of VPPase (Fig. 11.10).

Fig. 11.5
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VPPase protein compactly folded as membrane-bound protein. (Source: Suneetha et al. 2016)

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Fig. 11.6
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Sixteen transmembrane helices (blue cylinders) with six helices in the core surrounded by ten transmembrane helices to form inner and outer walls of the pump. (Source: Suneetha 2015)

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Fig. 11.7
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Ribbon structure of VPPase containing 16 transmembrane helices (colored in blue) and antiparallel β-strands (colored in red). (Source: Suneetha 2015)

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Fig. 11.8
figure 8

VPPase model of S. bicolor rotated to 600 to visualize the core with one imidodiphosphate (IDP), five Mg2+ (colored in green), and one K+ ions (colored in purple). (Source: Suneetha 2015)

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Fig. 11.9
figure 9

Working model of the VPPase showing the pumping of protons into vacuole to generate electrochemical gradient against which sodium is taken in under stress conditions. (Source: Suneetha 2015)

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Fig. 11.10
figure 10

The space-fill representation of modeled VPPase showing electrostatic surface potential. The electrostatic surface negative potential (red), positive (blue), and neutral (white) are represented. The core of the model contains IDP binding site. (Source: Suneetha 2015)

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11.4.2 Metal Geometry

V-PPase requires free Mg2+ as an essential cofactor. MgCl2 and MgSO4 are added to the buffers for solubilization and purification of the enzyme during its isolation (Maeshima and Yoshida 1989; Britten et al. 1989; Rea and Poole 1986). Binding of Mg2+ stabilizes and activates the enzyme. Baykov et al. (1993) reported the presence of high-affinity and low-affinity Mg2+ binding sites of mung bean. Binding of Mg2+ to VPPase not only activates the enzyme but also protects it from heat inactivation (Baykov et al. 1993). Suneetha (2015) reported that the core has five Mg2+ and one K+ ions along with one IDP which play an important role in activating VPPases by transphosphorylation reaction involving ATP’s. Each Mg2+ ion interacts with surrounding amino acids like aspartic acid, asparagines, and glutamic acid (Fig. 11.11). Potassium ion acts as stimulator of VPPase and is surrounded by amino acids like asparagine and glycine. K+ stimulates VPPase activity by more than threefold in most cases (Gordon-Weeks et al. 1999). The maximal activity of VPPase was obtained in the presence of more than 30 mM KCl in most cases. Suneetha (2015) also reported that there are eleven phosphate binding sites represented in yellow color balls and interacting residues with green color (Fig. 11.12).

Fig. 11.11
figure 11

The core of VPPase showing coordinating amino acids from IDP molecule to five Mg2+ ions. (Source: Suneetha 2015)

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Fig. 11.12
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Eleven phosphate binding sites of VPPase are represented in yellow colored balls and interacting residues in green color. (Source: Suneetha 2015)

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11.4.2.1 Regulation of VPPase Enzyme Activity

Studies on VPPase from various plant species revealed the relationship between the enzyme activity of the proton pump with respect to varying concentrations of cytosolic ions and chemical compounds. K+ ions have been associated with increased VPPase enzyme activity in A.thaliana type 2 VPPase (AVP2). Ca2+ reversibly inhibits VPPase activity through formation of Ca-PPi which is a strong, competitive inhibitor for the soluble PPases (Baykov et al. 1999). Changes in free cytosolic Ca2+ levels have also been associated with negative inhibition of VPPase activity in bean guard cells (Darley et al. 1998) and barley (Swanson and Jones 1996). Cytosolic Mg2+ concentration has also been reported for optimum enzyme activity in S. bicolor, mung bean, and barley. Moreover excessive Na+ concentrations have been reported to inhibit enzyme activity in red beet (Rea and Poole 1985).

Among the artificial substances tested, it reported that amino methylene bisphosphonate (AMBP) is a potent inhibitor of VPPase in mung bean and A. thaliana VPPase AVP2 and AVP1 (Zhen et al. 1994). The effectiveness of bisphosphonates as an inhibitor of VPPase was carried out, and it was concluded that a nitrogen atom in the carbon chain of bisphosphonates increased the inhibitory effect of the enzyme (Gordon-Weeks et al. 1999).

11.5 VPPase and Its Activity

Proton pump VPPase gets activated upon signals perceived by plants. The sequences of events occurring during the activation of proton pump are as follows:

Abiotic stress (high salinity, drought, high temperatures, etc.) in plants is perceived by root tissues and cells. The cells activate receptor-bound G-proteins to activate protein kinases by the breakdown of membrane-bound phosphatidylinositol bisphosphate (PIP2) to diacylglycerol (DAG) and inositol triphosphate (IP3) (Mahajan and Tuteja 2005; Tuteja 2007). IP3 induces endoplasmic reticulum in release of Ca2+ and other side; it also makes calcium channels to open to increase intracellular Ca2+ levels. CS1 and CS3 motifs form the core catalytic domain and are essential for hydrolyzing PPi and transport protons (Fig. 11.13). CS2 motif of VPPase, similar to rhodopsin-like G-protein-coupled receptor (GPCR) with calcium signaling signature property, senses these high cytosolic Ca2+ levels and transduces extracellular signal. The free available cytosolic Ca2+ may be phosphorylated to Ca-PPi by Ca2+-dependent membrane-bound protein kinase and PPi (Johannsen et al. 1996). The substrate PPi of Ca-PPi is exchanged with Mg2+ to form Mg-PPi at the core catalytic site from CS1 and CS3.

Fig. 11.13
figure 13

Web of events which show saline shock initiating cascade of signals to generate PMF that drives sodium into vacuole leading to salt tolerance in plants

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The above elements are highly conserved among the VPPases that form hydrophobic door to the hydrophilic surroundings of vacuolar lumen. The acidic residues in the core catalytic site help in PPi hydrolysis and proton transport into vacuole. The hydrophobic gate prevents the reflux of H+ ions and helps in maintaining the translocation of H+ from cytosol to vacuolar lumen. The pumping of H+ into vacuole builds electrochemical gradient (proton motive force, PMF) which changes its pH (2–4 pH units, equivalent to −120 to –240 mV) (Isayenkov et al. 2010). The PMF can energize various antiporters such as Na+ and K+: H+ exchanger, NO3− and Cl−: H+ exchanger, etc. resulting in influx of Na+, K+, NO3−, and Cl− from cytosol to vacuole. This influx reduces the toxicity of cytosol to protect the cell against deleterious effects thus caused due to abiotic stress. Therefore, overall signaling web plays an important role in providing stress tolerance to plants.

11.6 Conclusion

Vacuolar transporters are vital components of cellular network. They enable the plant to respond to the changing environmental conditions, store nutrients and energy during surplus production, and maintain optimal metabolic conditions in the cytosol. Plant vacuolar VPPase, a model of proton pump, is considered as integral enzyme due to its structure-function relationship.

Structural analysis using both laboratory and bioinformatic approaches revealed the functional domains along with the conserved segments (CS1, CS2, and CS3) that play an active role in the translocation of H+ ions into the vacuole from the cytosol. Phylogenetic analysis of all known VPPase across land plants, archaea, protozoan, and bacteria increased our knowledge of the tonoplast dramatically over the past decade. Studies established that during evolution of organisms, ancestral plant species obtained VPPase in addition to vacuolar-type V-ATPase.

However, more information is required on protein-ligand interactions and the molecular evolution of VPPase. It has been reported that the expression levels of VPPase change according to the physiological conditions and in response to environmental stresses. However, the regulatory mechanism and the posttranslational regulations of VPPase are yet to be studied. Thus, these analyses are extremely important toward establishing the role of VPPase as effective proton pump dedicated toward alleviating salt stress. The VPPase gene has been successfully used to engineer transgenic plants. Overexpression of the VPPase gene was able to confer effective Na+ compartmentation into the vacuole. Moreover, various VPPase from other species can be isolated to study their functional properties and development of transgenic plants. Thus enabling the plant to survive during salt stress and maintain an optimum osmoticum of the cytosol.