Multifaceted roles of aquaporins as molecular conduits in plant responses to abiotic stresses.

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.973367

REVIEW ARTICLE

Multifaceted roles of aquaporins as molecular conduits in plant responses to abiotic stresses Ashish Kumar Srivastava1, Suprasanna Penna1, Dong Van Nguyen2, and Lam-Son Phan Tran3

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Laval on 12/01/14 For personal use only.

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Plant Stress Physiology and Biotechnology Section, Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India, National Key Laboratory for Plant Cell Technology, Agricultural Genetics Institute, Vietnamese Academy of Agricultural Science, Hanoi, Vietnam, and 3Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan

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Abstract

Keywords

Abiotic stress has become a challenge to food security due to occurrences of climate change and environmental degradation. Plants initiate molecular, cellular and physiological changes to respond and adapt to various types of abiotic stress. Understanding of plant response mechanisms will aid in strategies aimed at improving stress tolerance in crop plants. One of the most common and early symptoms associated with these stresses is the disturbance in plant– water homeostasis, which is regulated by a group of proteins called ‘‘aquaporins’’. Aquaporins constitute a small family of proteins which are classified further on the basis of their localization, such as plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nodulin26-like intrinsic proteins (initially identified in symbiosomes of legumes but also found in the plasma membrane and endoplasmic reticulum), small basic intrinsic proteins localized in ER (endoplasmic reticulum) and X intrinsic proteins present in plasma membrane. Apart from water, aquaporins are also known to transport CO2, H2O2, urea, ammonia, silicic acid, arsenite and wide range of small uncharged solutes. Besides, aquaporins also function to modulate abiotic stress-induced signaling. Such kind of versatile functions has made aquaporins a suitable candidate for development of transgenic plants with increased tolerance toward different abiotic stress. Toward this endeavor, the present review describes the versatile functions of aquaporins in water uptake, nutrient balancing, long-distance signal transfer, nutrient/heavy metal acquisition and seed development. Various functional genomic studies showing the potential of specific aquaporin isoforms for enhancing plant abiotic stress tolerance are summarized and future research directions are given to design stress-tolerant crops.

Abiotic stress, aquaporins, plant, regulations, stress tolerance, transport

Introduction The world population is continuously on the rise and, by 2050, it is expected to reach nine billion people (http:// www.fao.org/wsfs/world-summit/en/). In order to meet this ever-increasing demand of feeding the population, plant scientists are striving to facilitate global food production which is estimated to be around approximately 44 million metric tons per year (Tester & Langridge, 2010). Although, a steady progress has been seen for major crops in the USA; for example, the production of maize (Zea mays), wheat (Triticum spp.) and soybean (Glycine max) has shown positive linear increase in average yield from 1930 to 2012 (USDA-NASS, 2013); however, it has become difficult to maintain this trend because of decrease in arable land area due to climatic

Address for correspondence: Ashish Kumar Srivastava, Plant Stress Physiology and Biotechnology Section, Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India. E-mail: [email protected]; [email protected] or Lam-Son Phan Tran, Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan. E-mail: [email protected]

History Received 1 August 2014 Revised 2 October 2014 Accepted 2 October 2014 Published online 26 November 2014

change, urbanization, industrialization and increased episodes of various abiotic stresses, such as cold, drought, salinity, heavy metal and nutritional imbalances. Thus, a clear challenge to maintain sustainable agriculture is to reduce the crop loss due to these stresses. To achieve this, it is essential to understand detailed molecular mechanisms of how plants try to cope with these stresses (Ha & Tran, 2014; Jogaiah et al., 2013; Osakabe et al., 2014; Thao & Tran, 2012). A common point of toxicity associated with majority of abiotic stresses is the disturbance in plant–water homeostasis which is regulated by a large super-family of major intrinsic proteins called ‘‘aquaporins’’ or ‘‘water-channel proteins’’. In plants, so far more than 100 aquaporins have been discovered. These are sub-divided into the following five sub-families: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26 (Nod26)-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and uncategorized X intrinsic proteins (XIPs) (Ishibashi et al., 2011). A new-sub-family of aquaporins called ‘‘large intrinsic proteins’’ is recently discovered from several algae species of phylum Heterokontophyta (Khabudaev et al., 2014). All these

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A. K. Srivastava et al.

Crit Rev Biotechnol, Early Online: 1–10

Table 1. Summarized distribution of major aquaporin isoforms in Arabidopsis and other crop plants. Number of homologues Organism

PIP1 PIP2 NIP1 NIP2 NIP3 NIP4 NIP5 NIP6 TIP1 TIP2 TIP3 TIP4 TIP5 SIP1 SIP2 XIP1 XIP2 Reference


A. thaliana 5 Rice (O. sativa) 3 Barley (H. vulgare) 5 Cabbage (Brassica rapa) 8 Maize (Z. mays) 6 Poplus (Populus 5 trichocarpa) Potato (Solanum tuberosum) 5 Tomato (S. lycopersicum) 5 Soybean (G. max L.) 8 Wheat (Triticum aestivum) 2 Cotton (G. hirsutum) 15

8 8 7 12 7 10

2 4 2 2 1 5

1 2 1 2 2 1

1 3 – 2 1 5

2 1 – 2 – –

1 – – 2 – –

1 – – 2 – –

3 2 2 4 2 8

3 2 3 4 3 4

2 2 1 4 1 2

1 3 0 1 4 1

1 1 1 1 1 2

2 1 1 3 2 4

1 1 1 3 1 2

– – – – – 5

– – – – – 1

10 9 14 4 13

3 2 5 2 3

– 2 2 1 1

2 2 – – –

2 3 1 1 –

1 1 1 – 2

1 1 2 – 6

3 3 9

4 4 7 – 7

2 2 4 – –

1 1 2 1 2

1 1 1 – –

3 3 6 1 7

– 1 – 1 –

3 6 2 – 1

3 – – – –

– 14

sub-families show great diversity in terms of protein localization. PIPs, XIPs and some NIPs are located in plasma membranes, TIPs in the tonoplast, SIPs in the endoplasmic reticulum and remaining aquaporins in other sub-cellular compartments. Based on amino acid sequence similarity, these sub-families are further divided into different groups. However, inspite of these differences, all the aquaporins exhibit a typically conserved structure with a-helical bundle forming six transmembrane (TM) helices (H1–H6) with two cytoplasmic termini. These helices are connected to each other by five loops (denoted LA to LE). The LB and LE loops each consists of a short a-helix connected by highly conserved Asn-Pro-Ala (NPA) motif. These loops are partly located within the membrane to form the pore (Tajkhorshid et al., 2002). Four residues of TM helices H2, H5 and LE, ˚ from situated toward extracellular side approximately 8 A NPA region, constitute the so-called aromatic/arginine (ar/R) filter which determines the substrate specificity. Any substitution of amino acid in ar/R-filter can result in change in substrate specificity or loss of function (Mitani-Ueno et al., 2011). Aquaporins form tetramers in cell membrane, although each monomer can act as functional pore (Chaumont & Tyerman, 2014). There is a great diversity in terms of the number of aquaporin isoforms present in each sub-group in different plant species (Table 1). While the model plant Arabidopsis thaliana has only 35 aquaporin genes (Johanson et al., 2001), in cotton (Gossypium hirsutum) more than 70 isoforms have been identified (Park et al., 2010). The current line of research is to assign the substrate-specific function to these isoforms in different plants. This involves the functional expression of aquaporins in heterologous systems, such as Xenopus oocytes or yeast cells and then evaluates the transport efficiency of different substrates (Chaumont & Tyerman, 2014). A vast range of substrates have already been identified for aquaporins which include water and related molecule such as H2O2, solutes (urea and glycerol), metalloids (boron, silica and arsenic) and gases (CO2 and ammonia). Li et al. (2014) have recently summarized these functions for plant aquaporins. The present review deals with functional genomics-based research on different aquaporin isoforms to the broader context of their role in modulating abiotic stress tolerance. Some recently discovered modes of regulation for aquaporin functions are discussed and future research directions are

Johanson et al. (2001) Sakurai et al. (2005) Besse et al. (2011) Tao et al. (2014) Chaumont et al. (2001) Gupta & Sankararamakrishnan (2009) Venkatesh et al. (2013) Reuscher et al. (2013) Zhang et al. (2013) Pandey et al. (2013) Park et al. (2010)

provided to utilize aquaporins as an important tool for boosting plant’s stress tolerance.

Brief information about different modes of regulation for aquaporin function There are many different modes by which the transport efficiency of aquaporins is regulated (recently reviewed by Chaumont & Tyerman, 2014; Li et al., 2014). In brief, these mechanisms include post-transcriptional modifications (such as phosphorylation, methylation, deamidation and acetylation), gating and heteromerization. The phosphorylation is prevalent mode of regulation for PIPs. The quantitative proteomic analyses have shown that light-dependent diphosphorylation of PIP2;1 at Ser-280 and Ser-283 is necessary for increase in hydraulic conductivity of Arabidopsis rosette under dark condition (Prado et al., 2013). Similar studies have also identified the in vivo deamidated forms of aquaporins and their abundance changes in response to various stresses, such as salt, mannitol and H2O2. This might be a new mode of regulation of aquaporin functions (di Pietro et al., 2013). The interaction between PIP1 and PIP2 isoforms to form heterotetramers also alters the aquaporin efficiency. The recent study in garden strawberry (Fragaria x ananassa) using an experimental design combined with a mathematical modeling approach has dissected the individual contribution of each PIP. The results have shown that (i) PIP1 has a high water transport capacity when its encoding gene is coexpressed alongwith PIP2 (ii) PIP2 water permeability is enhanced if it physically interacts with PIP1 and (iii) the PIP1–PIP2 interaction results in the formation of heterotetramers with random stoichiometric arrangement. Among PIP1 and PIP2, FaPIP1;1 plays the key determinant role in regulating water movement (Yaneff et al., 2014). Another type of regulation is the trafficking of PIPs and TIPs. This plays an important role during root responses under salt and oxidative stresses (Hachez et al., 2013). The fluorescence recovery after photobleaching has been shown to be a useful approach for studying the cycling dynamics of plasma membrane aquaporins between intracellular compartments and the cell surface, as well as to discover the components controlling the cycling (Luu et al., 2012). In maize, a LxxxA motif has been identified in third TM of ZmPIP2;5 (highly conserved in plant PIP2s), which is important for routing


DOI: 10.3109/07388551.2014.973367

PIP2s to plasmamembrane (Chevalier et al., 2014). The gating mechanism is also common for aquaporin regulation, and various cations such as mercury (Hg) and calcium (Ca) are identified which modulates the transport efficiency of aquaporins. Recently, in grapevine (Vitis vinifera), pHmediated gating of tonoplast aquaporin VvTnTIP2;1 is demonstrated. In yeast assay, the acidification of cytosol resulted in loss of VvTnTIP2;1 transport activity. Sequence analysis revealed the presence of His131 residue, which is unusual in TIPs and which was shown to be responsible for the loss of activity at acidic pH (Leitao et al., 2012). The aquaporin-mediated regulation of root hydraulic conductance is also dependent upon the arbuscular mycorhizal (AM) symbiosis. The ameliorative effect of AM against drought (Ba´rzana et al., 2014) and flooding (Calvo-Polanco et al., 2014a) is found to be associated with the higher expression aquaporins. In case of flooding, higher abundance of the PIP2 phosphorylation at Ser280 was also observed (Calvo-Polanco et al., 2014a). The links between the roles of plant hormones and aquaporines (ethylene, abscisic acid and auxin) in regulation of root hydraulic properties were also studied (Calvo-Polanco et al., 2014a). While ethylene was found to have secondary effect, auxin was established as a key hormone that allows the enhancement of root hydraulic conductivity under flooding conditions in AM-infected tomato (Solanum lycopersicum) plants, which was correlated with up-regulation of the SlPIP1;7 and the fungal aquaporin GintAQP1 (Calvo-Polanco et al., 2014a). Interestingly, when applied to non-mycorrhizal Arabidopsis, auxin was shown to directly implicate in reduction of root hydraulic conductivity under anoxic conditions, potentially through repression of most aquaporin genes (Pe´ret et al., 2012). Apart from these indirect modes of regulation, direct transcriptional regulators of aquaporins have been identified, such as TRANSLUCENT GREEN (TG) in Arabidopsis. TG is an AP2/EREBP family transcription factor that directly binds to the promoters of three aquaporin genes (AtTIP1;1, AtTIP2;3 and AtPIP2;2). TG-mediated control of water balance was also evident as the A. thaliana overexpressor lines showed considerable increase in drought tolerance (Zhu et al., 2014). Thus, the overall regulation of aquaporin functions is controlled at the level of transcriptional, post-transcriptional, cytosolic environment and external environmental conditions.

Role of aquaporin in regulating various plant processes The aquaporins regulate multiple plant processes which range from regulating the uptake of water, nutrients and toxic heavy metals in roots, water and CO2 transport in shoot and supply of water and nutrients in developing seeds (Figure 1A). The broad overview of these mechanisms with reference to specific aquaporin isoform is discussed below. Water transport A majority of abiotic stresses, including salt, drought and heavy metal, disturb plant–water homeostasis which is attributed to non-coordinated regulation between expression and activity of different aquaporin isoforms. In general, during early stress, plants reduce the expression/activity of aquaporins

Multifaceted roles of aquaporins as molecular conduits

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to conserve its water content. This is very important for the efficient induction of various defense responses. Contrary to this, long-term stress exposure leads to higher expression/ activity of aquaporins to meet daily water requirement of plant (Chaumont & Tyerman, 2014). In recent years, considerable progress has been made to understand the role of specific aquaporin isoforms under short- and long-term stress exposure; however, the mode of regulation is highly variable as it is not only dependent upon the crop and nature of stress but it is also specific to particular organ and developmental stage. In cucumber (Cucumis sativus), NaCl and PEG (polyethylene glycol) treatments for 2 h did not alter aquaporin expression but reduced aquaporin activity to lower down the root hydraulic conductivity. At 24 h, under both NaCl and PEG treatments, leaf hydraulic conductivity was reduced which was attributed to down-regulation of two highly expressed aquaporin isoforms, CsPIP1;2 and CsPIP2;4. At the same time point, reduction in root hydraulic conductivity was observed only under PEG stress, while NaCl treatment showed the recovery which was consistent with changes in the expression level of the two CsPIPs (Qian et al., 2014). This clearly suggested that aquaporin regulation is organ and stress specific. Apart from time-scale, aquaporin localization is also important from the view point of water transport. From both model plants like Arabidopsis and woody perennial crops like grapevine, the aquaporins are mainly present at the fine roots rather than the secondary growth zones, suggesting that aquaporin-mediated water regulation is mainly important for root-tip region. In the radial orientation, expression is always greatest in interior tissues, such as stele, endodermis and/or vascular tissues for all root zones (Gambetta et al., 2013). In plants, like common bean (Phaseolus vulgaris), under mild NaCl stress, aquaporin redistribution has also been demonstrated which increase the aquaporin abundance in cells close to xylem vessels. This helps the plants to recover from stress (Calvo-Polanco et al., 2014b). The aquaporins, especially PIP1-type ones, also function to avoid embolism (a state wherein the xylem transport capacity is temporarily reduced) in trees and perennial plants like poplar. Transgenic Populus tremula X Populus alba plants characterized by strong downregulation of multiple isoforms belonging to PIP1 sub-family were found to be more vulnerable to embolism, with 50% loss of conductance occurring at 0.3 MPa earlier than in wildtype plants. In addition to this, and the transgenic plants also showed reduced capacity to restore xylem conductance during recovery (Secchi & Zwieniecki, 2014). Considering the significance of PIPs in mediating water uptake, various bioregulators such as silicon and thiourea have also been evaluated under stress. Thiourea supplementation under short-term NaCl stress (30–90 min) down-regulated the expression of most of PIP isoforms in Brassica juncea (Srivastava et al., 2010), while silicon treatment under longterm drought stress (24 h) up-regulated their expression in sorghum (Sorghum bicolor) (Liu et al., 2014). The overall effect of these changes was manifested in the form of increased water content. Aquaporin-mediated regulation of water uptake is also essential in cold acclimation that is important for the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. Rice (Oryza sativa) plants subjected to low root temperature treatment (roots at 10 C; shoots at 25 C)


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Figure 1. Multiple functions of aquaporins in relation to different plant parts. The panel-A describes the substrates transported through aquaporins in different plant cells. In root cell, aquaporins mediate the uptake of water, nutrients and toxic heavy metals from soil solution, while in leaf cell their major function is to facilitate water and CO2 transport. In both these two organs, aquaporins also transport H2O2 to modulate ROS/ redox-mediated signaling. In reproductive cell, the major function of aquaporins is to supply water and nutrients to facilitate fertilization and seed formation. The panel-B explains the multiple effects of single aquaporin isoform on the basis of its sub-cellular localization. The same aquaporin at proximal end (facing external environment) can mediate uptake/loss of substrate, at distal end (facing vasculature) can mediate rootto-shoot translocation and at tonoplast can facilitate vacuolar sequestration of a particular substrate.

dramatically reduced root osmotic hydraulic conductivity within 1 h of stress. However, the conductivity reached 10fold higher values at 5 d after treatment which was concomitant with the coordinated up-regulation of OsPIP2;5 (Ahamed et al., 2012). In addition, aquaporin expression and plant–water relationship are also affected by above-ground environment, such as light and relative humidity (RH). Recently, in poplar, it has been demonstrated that aquaporin expression is mainly affected by RH rather than light. The expression of PIP1(PIP1;1, PIP1;2 and PIP1;3) and PIP2-type (PIP2;3, PIP2;4 and PIP2;5) genes and also the root water flow were increased as early as 4 h of decrease in RH, whereas increase in light intensity had similar effect only after 28 h (Laur & Hacke, 2013). In wild strawberry (Fragaria vesca), distinct diurnal regulation of aquaporin isoforms in relation to drought has been recently demonstrated (Surbanovski et al., 2013). For three PIP isoforms (FvPIP1;1, FvPIP2;1 and FvPIP2;2), clear daily fluctuation in expression was observed in leaf tissues with a peak at 2 h after sunrise, a reduction thereafter and recovery toward the end of night. Even under drought, although their expression was reduced, the diurnal expression remained unchanged. Such kind of regulation may help to

reduce xylem tensions during high transpiration demand (Surbanovski et al., 2013). In addition, it has been suggested that enhanced activity of leaf aquaporins during the day may favor water transport into the inner leaf tissues during maximal transpiration which would prevent very low leaf water potentials and reduce xylem tensions (Maurel et al., 2008). Gas exchange There is clear evidence that aquaporins mediate CO2 transport across cell membranes. In plants, CO2 is utilized as substrate for sugar synthesis in pentose-phosphate cycle, also known as Calvin–Benson cycle. For this process, CO2 diffuses in opposite direction from atmosphere to cells (Kaldenhoff et al., 2014). The membrane topology of aquaporins is responsible for CO2 transport. Although aquaporin monomers can function as single water channel, they tend to form tetramers. The exact reason why aquaporins tend to form tetramer is not known, but this might be to facilitate the transport of volatile substances such as CO2 or NH3. On the basis of experimental data on artificial tetramer of NtPIP1;2 and NtPIP2;1, it has been proved that tetramer formation is necessary for CO2 transport (Otto et al., 2010).


DOI: 10.3109/07388551.2014.973367

A contribution of aquaporins to CO2 transport and photosynthesis in plants has been first demonstrated in broad bean (Vicia faba) where the use of aquaporin inhibitor caused the reduction of photosynthetic activity (Terashima & Ono, 2002). NtAQP1, an aquaporin belonging to PIP1 subfamily, was the first plant aquaporin isoform which has been widely studied for CO2 transport (Uehlein et al., 2003). The reduction of NtAQP1 expression in tobacco (Nicotiana tabacum) plants resulted in reduction of cellular CO2 uptake, chloroplast CO2 concentration and photosynthetic performance. This is because under adequate light condition, CO2 availability is major rate limiting factor of photosynthesis. Thus, non-efficient CO2 transport through aquaporin in leaves and other parts reduces the photosynthesis efficiency, and hence, growth. Furthermore, the function of NtAQP1 was also found to be tissue-specific. For instance, specific expression of NtAQP1 in mesophyll tissue has been shown to play an important role in increasing both net photosynthesis and stomatal conductance. Targeting NtAQP1 to vascular envelope significantly improved the plant’s response to salt stress, while overexpression of NtAQP1 in guard cells did not have any significant effect (Sade et al., 2014). The positive role of NtAQP1 in increasing leaf mesophyll CO2 conductance has also been demonstrated by overexpressing NtAQP1 along with Arabidopsis hexokinase1 (AtHXK1) which encodes a dual-function enzyme that mediates sugar sensing and reduces the rate of photosynthesis as well as transpiration. The NtAQP1 complemented the effect of AtHXK1 and double-transgenic plants showed level of normal CO2 conductance (Kelly et al., 2014). In Arabidopsis, T-DNA insertion lines with knockout of AtPIP1;2 were analyzed for CO2 transport and results demonstrated that AtPIP1;2 can acts as CO2 transport facilitator in heterologous expression system as well as in vivo (Heckwolf et al., 2011). These lines have also been used to clearly demonstrate that cellular CO2 transport is not limited to unstirred lipid layers in membranes but is also dependent on expression of AtPIP1;2 (Uehlein et al., 2012). However, in fast growing trees like Populus, the contribution of PIP1 to the mass transfer of water and CO2 was found to be limited under control conditions only as their down-regulation did not dramatically impair any physiological needs of plant when cultivated under non-stress conditions (Secchi & Zwieniecki, 2013). In barley (Hordeum vulgare), members of PIP2 sub-family have also been evaluated for CO2 permeability using a hydrogen ionselective microelectrode method. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated the CO2 transport across the oocyte cell membrane. However, HvPIP2;4, which is highly homologous to HvPIP2;3, did not transport CO2. The protein sequence analysis revealed that isoleucine (Ile) residue at position 254 of HvPIP2;3 was conserved in PIP2 group of barley, except for HvPIP2;4, which possesses a methionine (Met) instead. This indicated that conserved Ile at the end of E-loop is crucial for CO2 selectivity (Mori et al., 2014). Thus, CO2 transport in different plants is mediated by the co-ordinated regulation of both PIP1 and PIP2 isoforms in a tissue- and condition-specific manner. This has been recently confirmed in broccoli (Brassica oleracea) plants that the expression of both PIP1 and PIP2 isoforms was upregulated under elevated CO2 environment which was found


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to be responsible for the increased tolerance against salt stress (Zaghdoud et al., 2013). Heavy metal and nutrient acquisition Heavy metal ions specifically block aquaporins and, consequently, this could indicate the significance of these proteins in alleviating heavy metal stress through reducing water deficit and oxidative damage (Zhang et al., 2008). Arsenic is a ubiquitously present heavy metal which exist in two forms, arsenate (AsV) or arsenite (AsIII), depending upon the pH and redox potential of the environment. Bienert et al. (2008) demonstrated that members of a sub-group of the NIP family of plant aquaporins can facilitate bidirectional diffusion of arsenite and antimonite across membranes. The expression levels of most of the PIPs were down-regulated during the course of As(III) exposure in B. juncea which led to decrease in total water content and hampered seedling growth (Srivastava et al., 2013). In rice, As(III) uptake is mediated by a member of the Nod26-like major intrinsic protein (NIP2;1) which is also known as Lsi1 (a low silicon uptake) (Ma et al., 2008). The spatial and temporal variations of OsLsi1 have been well characterized in rice (Yamaji & Ma, 2007). The OsLsi1 is normally present at the basal zone (410 mm from the root tip) of the root. Even in this zone, localization of Lsi1 is polar, with the highest abundance at distal side of both exodermis and endodermis where the casparian bands are formed. This supports the major role of Lsi1 in the uptake of arsenic from soil solution (Ma et al., 2008). Like PIP genes, the expression of OsLsi1/OsNIP2;1 also showed diurnal regulation with the maximum expression being observed during the day time (Yamaji & Ma, 2007). Although the protein sequence of the barley HvLsi1 is 81% identical to that of OsLsi1, HvLsi1 and OsLsi1 have different cell-type specificity of localization. Furthermore, HvLsi1 and OsLsi were also shown to exhibit different expression patterns (Chiba et al., 2009). Beside arsenic transporters, plasmamembrane (PALT1) and tonoplast-bound (VALT) transporters, which are responsible for aluminum (Al) uptake from soil and vacuolar sequestration, respectively, have also been found to be the members of aquaporin family. The Arabidopsis transgenics having independent overexpression of PALT1 or VALT displayed Al-sensitive and -tolerant phenotype, respectively (Negishi et al., 2012). Apart from these specific effects, the overall activity of aquaporin is also affected by heavy metals through gating effect. The maximum effect is observed under mercury followed by cadmium (Cd), lead (Pb) and zinc (Zn). Since, gating is mediated through oxidation, exogenous application of agents, like b-mercaptoethanol, reverses the effect of heavy metals (PrzedpelskaWasowicz & Wierzbicka, 2011). Owing to the metal-induced gating, the effect produced by nickel (Ni), copper (Cu) and chromium (Cr) resembles with that of drought stress. Also, their presence along with drought aggravates the effect in an additive manner (de Silva et al., 2012). The aquaporins are also important for acquiring nutrient elements such as boron (B) from soil. Boron is an essential micronutrient required for plant growth and is an important constituent of cell wall as it cross-links pectic polysaccharide rhamnogalacturonan II (Matoh, 1997). Boron deficiency is an


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important agricultural issue because it results in loss of yield quality and/or quantity in cereals and other crop plants. Besides, higher level of boron is toxic to plant and reduces its growth (Nable et al., 1997). Thus, an optimum level of boron is required for maximum plant growth. In rice, uptake of boron from roots is mediated through OsNIP2;1 (Schnurbusch et al., 2010). Thus, NIP2;1 is a multifunctional protein that transports silicon (Yamaji & Ma, 2007), arsenic (Ma et al., 2008), as well as boron (Schnurbusch et al., 2010). In Arabidopsis, two highly expressed NIP (AtNIP5;1 and AtNIP6;1) genes and their close ortholog in rice (OsNIP3;1) are responsible for boron distribution in shoots. OsNIP3;1 knockdown plants showed altered boron distribution in shoots especially under boron-deficient conditions (Hanaoka et al., 2014). OsPIP2;4 and OsPIP2;7 are two major aquaporins responsible for export of boron from roots, whose higher expression is desired to avoid toxicity under excessive boron conditions. Efflux assay of boron in roots indicated that 10B was effluxed from Arabidopsis transgenic plants overexpressing OsPIP2;4 or OsPIP2;7 during initial 1 h of assay (Kumar et al., 2014). Apart from boron, aquaporins are also important for maintaining the nitrogen (N) status of plants. Using heterologous complementation assay in a urea uptake-defective yeast, three maize aquaporin isoforms (ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4) have been shown to have the potential of urea uptake. Since, one of these (ZmTIP4;4) is tonoplast-located, aquaporins might change the urea levels in plants by altering its uptake and translocation in vacuole (Gu et al., 2012). Seed formation and germination Aquaporins supply water and nutritions to pollen, and hence are crucial for reproduction and plant development. In Arabidopsis, the pollen grains contain one large vegetative cell and two smaller sperm cells. Aquaporins of TIP category (AtTIP1;3 and AtTIP5;1) are expressed in pollen. On the basis of expression study in X. oocytes, AtTIP1;3 and AtTIP5;1 were shown to transport urea in addition to water, while exclude boric acid and glycerol (Soto et al., 2008). Later on, the bioinformatic predictions and GFP (green flourescent protein)-fusion data indicated that TIP5;1 is not targeted to the vacuole but to pollen mitochondria, where it plays a role in nitrogen remobilization (Soto et al., 2010). This is in contrast with the data reported in Arabidopsis mesophyll protoplasts where AtTIP5;1-GFP fusion was found to be expressed in vacuoles to avoid boron toxicity (Pang et al., 2010). Recently, using the native promoter system, the tonoplast localization of AtTIP5;1 was confirmed to be specific in sperm cells. Another TIP isoform, AtTIP1;3 was found to be specifically present in the vacuoles of vegetative cells. Although single mutant of either AtTIP1;3 or AtTIP5;1 had no significant defect in male transmission, the double knockout mutant displayed an abnormal rate of barren siliques. The phenotype was even more pronounced under limited water or nutrient supply. Thus, TIPs present on two distinct cells, such as vegetative and sperm cells, interact functionally to maintain the male fertility in adverse environmental conditions (Wudick et al., 2014). Apart from TIPs, the significant role of PIP (OsPIP1;1) was also proposed as an


active water channel which maintains the seed yield of rice under salt stress conditions (Liu et al., 2013). Apart from seed formation, aquaporins are also important for proper seed germination. Seeds are highly desiccated organs, and extensive and kinetically well-defined water exchanges are essential for seed imbibition and subsequent embryo growth. The differential expression profiling between dry and germinating materials of Arabidopsis indicated higher expression levels of selected TIP genes (TIP3;1, TIP3;2 and TIP5;1) and low expression of all PIP genes in dry and germinating materials. The inhibitory tests with mercury chloride and thiols, such as dithiothreitol, further suggested that aquaporin functions are not very important during early seed imbibition (phase I) rather they are associated with delayed initiation of phase III, that is, water uptake accompanying expansion and growth of the embryo (Vander Willigen et al., 2006). Similar studies on broad bean seeds have revealed that decreased expression of TIP3 genes was associated with the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homolog was closely linked to the fast cell elongation (Novikova et al., 2014). Apart from facilitating water movement, aquaporin-mediated nutrient transport is also important during seed germination. In French beans (P. vulgaris), expression of three PIP genes (PvPIP1;1, PvPIP2;2 and PvPIP2;3) was up-regulated to the mobilize nutrients from seed coat which facilitated the seed germination (Zhou et al., 2007). Calcium and reactive oxygen species/redox-mediated signaling In response to any environmental and developmental stimuli, calcium and ROS (reactive oxygen species) are two most important secondary messengers which regulate downstream signaling events and ultimately the cell fate (Steinhorst & Kudla, 2013). The tight interaction and crosstalk between these two have been proposed to regulate different plant processes, such as abscisic acid signaling in guard cells, seed germination, pollen tube growth, root growth and regulation of ion homeostasis under stress. Recently, aquaporins have been proposed as an important component of this complex signaling network. The calcium signal initiates in the form of transient increase in cytosolic free Ca2+ ([Ca2+]cyt), which arises because of the flux of Ca2+ into the cytosol, either from the external medium or from sub-cellular compartments, where the concentration of Ca2+ is higher as compared with that of cytosol. The increase in [Ca2+]cyt concentration led Webb et al. (1996) to formulate the concept of ‘‘Ca2+ signatures’’, which is defined as the repetitive oscillations or spiking of [Ca2+]cyt. The Ca2+ signatures generated at the site of stimulus also travel distantly for long-distance signal transfer. In the majority of plants, this occurs through apoplastic pathways, and hence is tightly regulated by rate of transpiration. In other words, we can conclude that plant calcium signaling, especially the long-distance transport, is tightly regulated through water movement. Besides, water movement itself is also regulated by Ca2+, both in the apoplast via effects on cell wall structure and stomatal aperture and within symplast via Ca2+-mediated gating of aquaporins (Gilliham


DOI: 10.3109/07388551.2014.973367

et al., 2011). Thus, both calcium signaling and water movement are mutually dependent upon each other and Ca2+ can increase its own transport by affecting aquaporin function. In support of this finding, overexpression of a wheat aquaporin (TaNIP) in Arabidopsis increased Ca2+ levels by 33% under normal conditions and almost doubled Ca2+ under salt stressed conditions. The transgenics also showed enhanced salt tolerance as compared with wild-type (Gao et al., 2010). Similarly, transgenic tobacco with higher expression of wheat aquaporin (TaAQP8) showed higher Ca2+ level, increased K+/Na+ ratio and enhanced salt tolerance (Hu et al., 2012). These results clearly indicated the direct involvement of aquaporins in mediating longdistance calcium signaling which is an important determinant of plant’s tolerance to different abiotic stresses. In contrast to calcium for which aquaporin regulation is more or less indirect, regulation of redox/ROS-mediated signaling is direct. In recent years, several aquaporin isoforms have been identified to transport ROS like H2O2 (Bienert & Chaumont, 2014). The redox signaling of the cell is mediated by its redox status which is defined as integrated ratio of all redox couples present inside the cell. At a given point of time, it is governed by the level of different ROSs and activities of various antioxidant producing and scavenging enzymes. H2O2 is one of the major ROS responsible for mediating redox signaling by either activating or deactivating the proteins of signaling cascade, such as mitogen-activated protein kinase (Petrov & Breusegem, 2012), or by directly modulating the activity of transcription factors, including NAC-, ZAT-, WRKY-, DREB-, bZIP- and MYB-type ones (Dietz, 2014). Like calcium, H2O2 is also involved in mediating cell-to-cell transfer of signal to distal parts of the plants (Steinhorst & Kudla, 2013). For a long time, it was believed that H2O2 diffuses freely crosses the membrane through passive nonprotein-facilitated diffusion. The first evidence against this came when H2O2 gradients were detected in mammalian cell lines (Makino et al., 2004), bacteria (Seaver & Imlay, 2001) and yeasts (Branco et al., 2004). Furthermore, physicochemical properties of H2O2 indicate that it has a slightly larger dipole moment or polarity than water which makes its non-facilitated diffusion through hydrophobic lipid bilayer less rapid than that of water. Since there are aquaporins to facilitate water transport, H2O2 transport across membrane by simple diffusion is highly unlikely (Bienert & Chaumont, 2014). The first evidence on aquaporin-mediated transport of H2O2 came when the aquaporin inhibitors, like mercuric chloride, silver nitrate and phloretin, were found to significantly inhibit the TM flux of H2O2 (Henzler & Steudle, 2000). Later on, the direct evidence came when the expression of the human hAQP8, Arabidopsis AtTIP1;1 and AtTIP1;2 in yeast markedly reduced its growth and cell survival on medium containing H2O2. The effect was not only due to the decreased antioxidant defense but also due to the higher level of H2O2 inside the cell (Bienert et al., 2007). Furthermore, hAQP8 and AtTIP1;1, which are highly permeable to H2O2, are also very efficient in transporting water, suggesting that aquaporins may possess the same capacity and selectivity toward water and H2O2. However, this is not true for PIPs. In Arabidopsis, most of the PIP2s are good water channels, and only AtPIP2;2, AtPIP2;4, AtPIP2;5


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and AtPIP2;7 facilitate H2O2 membrane diffusion, while AtPIP2;3, AtPIP2;6 and AtPIP2;8 do not (Hooijmaijers et al., 2012). So far, the PIP1-mediated H2O2 transport has not been demonstrated in any species. Even in monocots like maize, only PIP2 isoform (ZmPIP2;5) has been shown to transport H2O2 (Bienert et al., 2014). The major limitation of these studies is that they are limited to only yeast, bacteria or mammalian cell lines, and thus direct evidence of aquaporinmediated H2O2 transport in plants remains to be the task of future. Additionally, the involvement of other aquaporin isoform in mediating H2O2 transport has also been suggested. The heterologous expression of wheat aquaporin TaTIP2;2 compromised the abiotic stress tolerance of Arabidopsis transgenic plants (Xu et al., 2013). Although the authors have not analyzed the data in terms of H2O2, higher intracellular concentration of H2O2 (as observed in the case of AtTIP1;1 expressing yeast) might be responsible for loss in stress tolerance ability of transgenic plants. Besides, TaTIP2;2 overexpression might also disturb the H2O2 distribution in different sub-cellular compartments, which has been recently proposed as an important regulator of co-ordinated gene transcription in plants (Sewelam et al., 2014). The overexpression of another tonoplast aquaporin (TsTIP1;2) from Thellungiella salsuginea has been shown to enhance tolerance against multiple stresses, including drought, salt and oxidative stresses. Using the yeast model, H2O2 transport ability of TsTIP1;2 was also confirmed. Thus, TsTIP1;2 might improve H2O2-mediated long-distance signal transfer which led to the induction of multiple stress tolerance (Wang et al., 2014). Besides TIPs, different PIPs have also been used to enhance plant’s stress tolerance in different plants, such as banana (Musa spp.) (Sreedharan et al., 2013), tobacco (Hu et al., 2012) and rice (Liu et al., 2013). However, further studies on the role of specific aquaporin isoforms with respect to H2O2 transport is required for better understanding of their in planta functions in regulation of H2O2-mediated signaling.

Conclusions and future directions The present review summarizes the multiple functions of plant aquaporins beyond its normal role as water-channel proteins. These functions include transport of nutritional elements (boron and urea), heavy metals (arsenic), gaseous molecule (CO2) and signaling mediators (H2O2). The relative significance of these in different plant parts is depicted in Figure 1. Inspite of tremendous research based on functional genomic approaches, in planta function of most of the aquaporin isoforms has not been properly understood yet. This is mainly because most of them are multifunctional. For instance, OsNIP2;1 of rice has been demonstrated to transport silica, arsenic and boron. Additionally, the exact function of aquaporins is also dependent upon external environment, organ and developmental stage of the plant. Thus, assigning a particular function to any specific isoform requires more holistic approach. Moreover, any single isoform can also have multiple effects depending upon its sub-cellular localization (Figure 1B). At proximal side of cell (facing the external environment) aquaporins can mediate the uptake or loss of substrate, while at distal end (facing the vasculature), they enhance root-to-shoot translocation. The same aquaporin,


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if localized at tonoplast, can mediate vacuolar sequestration. Thus, aquaporin function needs to be described always in the terms of its localization. Beside individual isoform, the cumulative effect of aquaporin on root-hydraulic conductivity has a direct effect on transpiration rate which in turn modulates calcium transport in plants. Owing to this, aquaporins play an important role in mediating long-distance signal transfer in plants. Apart from calcium, aquaporins also modulate the redox signaling by transporting H2O2 which is the most important ROS produced in biological systems. In recent years, calcium and ROS are established as two important secondary messengers in plants and their complex interaction and crosstalk are responsible for deciding the downstream mode of signaling in responses to any stimulus. The aquaporin-mediated regulation of water and CO2 in aerial parts is also crucial for maintaining the photosynthetic efficiency, growth and yield of plants, especially under water deficit and nutrient-limited conditions. The multiple functions of aquaporins are also evident from genetic studies in which transgenic lines with up-regulated expression of aquaporin show enhanced stress tolerance with reduced membrane injury and improved ion homeostasis. However, these studies need more careful investigation in terms of nutritional imbalance and yield potential, especially under natural field conditions. Such data are currently lacking for most of the aquaporin isoforms. Thus, our ultimate goal of enhancing plant’s ability to tolerate abiotic stress using genes encoding aquaporins, as candidate genes, is dependent upon our understanding of relative efficiency of transport function of all the aquaporin isoforms for different possible substrates, such as water, H2O2, nutrients and toxic heavy metals. For instance, if we could identify a particular isoform that is specific only for silicon and not for arsenic, we could generate transgenics having more silicon content. Similarly, tonoplast-based aquaporins having more affinity for arsenic could be used for enhancing the vacuolar sequestration potential of plants. In the same line, an isoform having the potential to efficiently transport inward movement of H2O2 from apoplast and also the outward movement to its adjoining cell could be used for improving signal transfer ability of plants. Besides, ‘‘omics’’based research is required to compare the expression profiles of aquaporins during seed formation and seed germination. Since these processes involve loss and uptake of water, respectively, the data might be useful to pinpoint PIP(s) which specifically perform the function of water loss or uptake. Considering the redundant and multifunctional roles of aquaporin isoforms, assigning such distinct and specific function to a particular isoform is difficult; however, this is an important step whose success will decide the future of aquaporin-based research in plants. Additionally, if the native aquaporin with desired function is not identified, future research may also utilize the aromatic/arginine selectivity filter of different aquaporin isoforms to develop an engineered or man-made aquaporin having more controlled and specific transport functions. The simulation-based studies are also necessary to identify the novel substrates of plant aquaporins. Such studies have already been performed with mammalian aquaporins (for instance, AQP4) to demonstrate their nitric


oxide (NO) transport ability (Wang & Tajkhorshid, 2010). Since NO is also an important gasotransmitter, it will be interesting to find out the responsible aquaporin(s) in plants which can facilitate the transport of NO. Thus, area of aquaporin-based research is still naı¨ve and requires much more effort for better understanding in terms of isoformspecific function and various modes of regulation. A concerted effort from aquaporin research community is also required to ensure the successful use of aquaporin for boosting plant’s ability to tolerate different abiotic stresses and pave the way for sustainable agriculture.

Declaration of interest The authors report no declarations of interest. This work was supported in part by a grant (Project Code 31/2012/HÐNÐT) from the Ministry of Science and Technology of Vietnam to the Research Group of Dong Van Nguyen.

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Small RNAs in Plant Responses to Abiotic Stresses: Regulatory Roles and Study Methods.

Calcium Sensors as Key Hubs in Plant Responses to Biotic and Abiotic Stresses.

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses.

Plant aquaporins: roles in plant physiology.

Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses.

Multifunctional Roles of Plant Dehydrins in Response to Environmental Stresses.

Plant responses to environmental stresses-from gene to biotechnology.

The Unfolded Protein Response Supports Plant Development and Defense as well as Responses to Abiotic Stress.

Variability of metabolite levels is linked to differential metabolic pathways in Arabidopsis's responses to abiotic stresses.

The role of carbon monoxide signaling in the responses of plants to abiotic stresses.

Responses of rose RhACS1 and RhACS2 promoters to abiotic stresses in transgenic Arabidopsis thaliana.

Responses of grafted tomato (Solanum lycopersiocon L.) to abiotic stresses in Saudi Arabia.

G-protein Signaling Components GCR1 and GPA1 Mediate Responses to Multiple Abiotic Stresses in Arabidopsis.

Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses.

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How plants handle multiple stresses: hormonal interactions underlying responses to abiotic stress and insect herbivory.

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Editorial: ROS Regulation during Plant Abiotic Stress Responses.

The role of gibberellin signalling in plant responses to abiotic stress.

Genome-wide analysis of Dof family transcription factors and their responses to abiotic stresses in Chinese cabbage.