Advances in functional regulation mechanisms of plant aquaporins: Their diversity, gene expression, localization, structure and roles in plant soil-water relations (Review)

Abstract

Aquaporins are important molecules that control the moisture level of cells and water flow in plants. Plant aquaporins are present in various tissues, and play roles in water transport, cell differentiation and cell enlargement involved in plant growth and water relations. The insights into aquaporins’ diversity, structure, expression, post-translational modification, permeability properties, subcellular location, etc., from considerable studies, can lead to an understanding of basic features of the water transport mechanism and increased illumination into plant water relations. Recent important advances in determining the structure and activity of different aquaporins give further details on the mechanism of functional regulation. Therefore, the current paper mainly focuses on aquaporin structure-function relationships, in order to understand the function and regulation of aquaporins at the cellular level and in the whole plant subjected to various environmental conditions. As a result, the straightforward view is that most aquaporins in plants are to regulate water flow mainly at cellular scale, which is the most widespread general interpretation of the physiological and functional assays in plants.

• Plant aquaporins
• water channel
• stress
• biomembrane interface
• soil-water relations

Abbreviations
AQPs	=	aquaporins
MIPs	=	major intrinsic proteins
TIPs	=	tonoplast intrinsic proteins
NIPs	=	nodulin like plasma membrane intrinsic proteins
SIPs	=	small intrinsic proteins
NPA	=	asparagine-proline-alanine
ABA	=	Abscisic acid

Introduction

The discovery of aquaporins has shown a new insight into the mechanism of water-transmembrane transportation, which provides solid molecular basis for fast and reversible regulation of transmembrane water transport and gives strong support to the idea that such high water permeability might be required for certain physiological processes [1], [2]. Aquaporins, or major intrinsic proteins (MIPs), are channel-forming membrane proteins with the extraordinary ability to combine a high flux with a high specificity for water across biomembranes. They belong to a well conserved and ancient family of proteins called MIPs with molecular weights in the range of 26–34 kDa [3], and with members found in nearly all living organisms [3]. The aquaporin family in plants is large, indicating complex and regulated water transport within the plant body in order to adapt to different environmental conditions, which includes more than 150 membrane channel proteins [4]. Regulation of aquaporin-mediated water flow, through indirect or direct means, appears to be a mechanism by which plants can control cellular and tissue water movement [5]. All aquaporin isoforms probably work together in an orchestrated manner, where each individual aquaporin isoform displays a specific localization pattern, substrate specificity, and regulatory mechanism [6–8]. The structure, function and gene regulation of aquaporins as well as research methodology are reviewed in this study.

Species diversity of plant aquaporins

The physiological role of water channel proteins is particularly important in plants because of their continuous water recruitment [7], [8]. Many MIP family genes have been identified in plants, such as the members in Arabidopsis, tobacco, spinach, tomato, the ice plant (Mesembryanthemum crystallinum), radish, and snapdragon [2], [3], [9]. The related metabolite facilitator proteins or aquaporins from 38 genes of Arabidopsis thaliana were listed in Table 1. The permeability values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among plant aquaporins [10], [11].

There are two highly important aspects of plant aquaporins [2]. One aspect is their tremendous diversity in plants. In the Arabidopsis genome, 38 AQP genes (Table I) have been identified [2], [11], [12]. The other aspect is the discovery that some plant aquaporins are multifunctional channel proteins, allowing some small neutral solutes across cellular membranes, such as glycerol, CO₂, ammonia (NH3), urea, boron, and hydrogen peroxide [13–17]. The diversity of aquaporin isoforms in plants can also be explained in part by their presence in multiple subcellular compartments [7], [9], [18]. Thus, the capacity of some of these proteins to transport small non-electrolytes in addition to water, together with their broad range of sub-cellular localizations, provides new clues to explain the great diversity of aquaporins in plants.

Table I.  38 genes encoding metabolite facilitator proteins or aquaporins are encoded in the Arabidopsis thaliana genome-tremendous diversity*.

Gene family	Gene name	Gene structure	Chromosome	Tissue specificity
PIP –Plasma membrane sub-family	PIP1a	3 introns	3	R, AG, FB
	PIP1b	3 introns	2	R, AG,
	TMPB	3 introns	1	GS, FB, AG, R, ripening fruit
	PIP2a	3 introns	3	R, GS, FB, AG
	MIPL	3 introns	2	R
	PIP3	3 introns	4	R, FB, GS, AG
	MIPF	3 introns	4	R, GS, FB, AG
	MIPG	3 introns	5	R
	MIPH	3 introns	2	FB, AG
	MIPK	3 introns	3	GS
	MIPI	3 introns	2	R, FB
	MIPM	3 introns	4	GS, R, FB, AG
	TMP2c	3 introns	2	R, GS
TIP – Tonoplast sub-family	TIP2c	1 introns	5	whole plant
	TIP2g	1introns	3	R, AG, GS
	TIPA	2 introns	1	DS, GS, developing embryo
	TIPB	2 introns	1	DS, rosette leaves
	TIPC	1 introns	4	R, AG
	TIPD	2 introns	3	R, FB, AG
	TIPG	1 introns	2	R, other vegetative organs
	TIPH	no introns	4	–
	TIPI	2 introns	3	–
	TIPK	2 introns	2	R
	TIPL#	1 introns small**	1	–
NLM – Nodulin-like MIP sub-family	NLM1	4 introns	4	R
	NLM2	4 introns	4	DS
	NLM3	3 introns	2	–
	NLM4	4 introns	4	rosette leaves
	NLM5	3 introns	4	R
	NLM6	4 introns	3	FB
	NLM7#	4 introns, small**	2	–
	NLM8#	4 introns	2	–
	NLM9	4 (long) introns	5	–
	NLM10	4 introns	5	–
	NLM11	2 introns	3	R
	NLM12	2 introns	5	R, AG
	NLM13	2 introns	3	R, AG
	NLM14	3 introns	1	–

Compiled data collected from The Arabidopsis Information Resource (TAIR). *ESTs, numbers rounded for the most abundant ESTs, have been collected from different ecotypes; **TIPL and NLM7 include only two transmembrane segments; For abundant ESTs, the tissues for cDNA library preparation are listed. Italic, underlined are tissues from which a high percentage of the ESTs was recovered; R, roots; GS, green siliques; AG, above ground; FB, flower bud; DS, developing seeds; # Possible pseudogenes.

Aquaporins are differentially expressed in different organs and membranes. In the plant kingdom, a single plant expresses a considerably large number of MIP homologues. These homologues can be subdivided into four groups with highly conserved amino acid sequences and intron positions in each group: The tonoplast intrinsic proteins (TIPs) [19–21], the plasma membrane intrinsic proteins (PIPs) [22], [23], the nodulin-like plasma membrane intrinsic proteins (NIP) [24], and the small intrinsic proteins (SIP) [25]. There are various isoforms of plant TIP: Alpha (seed), gamma, Rt (root), and Wsi (water-stress-induced). These proteins may allow the diffusion of water, amino acids and/or peptides from the tonoplast interior to the cytoplasm [21–26]. The plasma membrane aquaporins (PIPs) can be divided into two subfamilies: PIP1 with inactive or low water channel activity and PIP2 with high water channel activity [27]. The high diversity of aquaporins reveals novel facets of plant biomembrane functions [6], [28].

Discovery of the aquaporin family of water channel proteins has provided a molecular explanation for rapid water movements across the plasma membranes of cells while diffusion still works in parallel [9], [26], [28], [29]. This may be one reason for the abundance and diversity of aquaporins, in particular in plants. Their activity may be required for fine regulation at the gene and/or protein levels, which is influenced directly or indirectly by cell metabolism, for example, via phosphorylation of the aquaporin proteins and water stress [6], [29–33].

The numerous aquaporin isoforms of plants have specific expression patterns throughout plant growth-development and in response to environmental stimuli. Once a protein is involved, the cell has the ability to regulate its abundance (transcriptional or post-transcriptional) or to modulate its activity [2], [32], [34]. Water permeability of the plasma membrane and tonoplast through the aquaporins depends on their quantity, activity, and substrate specificity [33]. Partly because of this very high diversity, the knowledge about plant aquaporin expression and subcellular localization is far from complete [34].

Recent studies have shown that their primary structure, transport substrate, functional regulation, gene expression profile, protein amount, and intracellular localization are diversified. Thus, future investigations on the aquaporin family of proteins will provide important information not only on the physiology of membrane transport processes in many cell types, but also on the targeting and trafficking signals that allow proteins to enter distinct intracellular vesicular pathways in cells [6], [32], [34], [35].

Aquaporin gene expression and diurnal fluctuations in plants

Because of the potentially important roles of aquaporins in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important [35–37]. The high level of expression of ZmTIP1 in maize tissues (root epidermis, small parenchyma cells surrounding mature xylem vessels in the root, etc.) facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required. Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells [37] Barley HvPIP2:1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [38].

The abundance of water channel proteins is a critical parameter to understand their function at the tissue, cell, or subcellular levels. Studies regarding the aquaporin mRNA abundance have led to the following conclusions: (i) Many aquaporins are highly expressed in the vascular tissues, (ii) some tonoplast aquaporins are highly expressed in meristems, where vacuolar biogenesis takes place, and (iii) aquaporins are highly expressed in the tissues that can experience high water or metabolite flux [39–42]. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport [6], [18], [19].

Under water stress conditions, the expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells [43]. Therefore, the expression of aquaporin genes may be independently regulated in an organ- and stage-specific manner, while the amount of aquaporin proteins can be regulated at the translational level and by the rate of protein turnover [13], [33], [43], [44]. Meanwhile, there are numerous reports providing clear evidence that the abundance of aquaporins can be regulated by developmental and environmental factors [6], [13], [32]. Monitoring aquaporin gene expression patterns in many plant species in specific tissues, cell types or in response to phytohormones or environmental factors has highlighted the putative roles of water channels [13], [32], [44–46]. Currently, modulation of aquaporin expression in plants is considered the strategy of choice for elucidating the role of aquaporins in plant biology and membrane biology [13].

In Lotus japonicus roots, the PIP1-type water channel shows diurnal fluctuations that are correlated with the diurnal variation in root hydraulic conductivity [46]. Harmer et al. (2000) found that the mRNA level of an Arabidopsis tonoplast aquaporin, GTIP, cycles in a circadian manner [47]. The plasma membrane aquaporin in the motor cells of Samanea saman also shows a diurnal and a circadian regulation [48], [49]. There is a diurnal rhythm in PsPIP2-1 expression in lateral roots that is strongly correlated with diurnal changes in Lpr. Taproots also display a rhythm in PsPIP2-1 expression, but this is offset from that of Lpr [128].

Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understand the molecular basis and evolution of complex traits. Similarly, the comprehensive expression profile of the members of the aquaporin gene family provides a novel basis to allocate the stress-related biological function to each aquaporin gene.

Aquaporin localization in plant cells

Regardless of whether all or only the majorities of the plant MIPs are aquaporins, it is clear that a large number of aquaporins are present in plants. Some are localized in the tonoplast, some in the plasma membrane and some possibly localized in endomembranes [4], [6], [11], [18], [20], [21], [26], [32], [45]. MIP-B has been found in fractions containing tonoplast proteins, distinct from both plasma membrane and tonoplast, and also distinct from the fractions in which MIP-A is located [19], [34], [50]. Only a small amount of MIP-B is located in the plasma membrane fractions, as predicted by sequence alignments. Based on the distribution of PIP proteins within plant cells, Kirch et al. (2000) suggested that the control of plant water channel activity might appear through an analogous vesicle shuttle mechanism [51]. It is possible that the subcellular localization (in the plasma membrane or intracellular vesicles) of aquaporin tetrarners is dependent on the level of phosphorylation of the subunits [5], [52], [53].

Multiple aquaporin genes could allow for variable expression in different tissues, cells and intracellular biomembranes during development, under changing external conditions and in response to rapidly changing demands for water movement [4], [6], [11], [18], [20], [21], [26], [32], [45], [50], [52]. Hufte et al. (1992) found that a presumptive tonoplast aquaporin (Phaseollus a-TIP) is located in multivesicular bodies in transgenic tobaccos that express Phaseollus a-TIP [53]. Aquaporins have been also localized in plasmalemmasomes, invaginating domains of the plasma membrane that protrude into the vacuole [54], [55]. As suggested by Robinson et al. (1996), clustering of aquaporins in the plasmalemmasomes may provide the means for achieving a rapid osmotic balance, and therefore, turgor maintenance in mesophyll cells [54]. A recent study reported the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells [129].

Repeated experiments have produced the same distribution, and clearly indicate that putatively plasma membrane-located aquaporins show a more complex pattern, and might be localized to different subcellular biomembranes or compartments. Thus, direct determination of the location of each aquaporin within tissues is still required to understand its function in plants.

Aquaporin structure and selectivity in plants

The structures of various aquaporins have provided significant insights into the molecular mechanisms of water permeation. The structure of aquaporins is highly conserved in animals, plants, yeast, and bacteria [4]. All MIP family proteins share six putative transmembrane domains with the N- and C-termini facing the cytosol (Figure 1A).

Figure 1.  Schematic illustration: The atomic structure of the water pore and the mechanism of its selectivity. (A) Share six putative transmembrane domains with the N- and C-termini facing the cytosol; (B) Water molecules passing the channel are forced by the protein's electrostatic forces to flip at the center of the channel.

The six transmembrane domains were predicted to be a-helices, packed together with the pore-forming domains outside and towards the center of an aquaporin tetramer [4], [26], [28], [56]. There are five loops (A–E) joining the transmembrane helices. The first cytosolic loop and the third extracytosolic loop are hydrophobic and contain the conserved asparagine-proline-alanine (NPA) sequence [8], [57], [58]. The two halves of the protein show obverse symmetry, with the hydrophobic loops containing the NPA motif (Figure 1B) overlapping in the middle of the lipid bilayer to form two hemipores that together create a narrow channel proposed to be similar in shape to an hour-glass [57–59].

Aquaporin polypeptides generally form homotetramers in the membrane [60–62], with each monomer forming a single water pore [59]. Water molecules passing the channel are forced by the protein's electrostatic forces to flip at the center of the channel. Water is thought to flow across the water channel pore in a direction down its potential gradient [1], [63].

To account for the molecular selectivity of MIP family channels, the pore so formed was hypothesized to function via a size-exclusion mechanism [1]. Inhibition of the water channel by mercuric chloride is thought to be the result of the sulfhydryl reagent binding a cysteine residue located in a close proximity to the pore, resulting in the physical blockage of the molecular flow through the pore [5], [63], [64]. The gating mechanism successfully unifies a significant body of biochemical and genetic evidence that has identified specific amino-acid residues governing plant aquaporin gating, and suggests how the closed structure might be stabilized or destabilized [5], [65], [66].

Nevertheless, plant aquaporin structures have been reported only at low resolution. Further investigation is necessary to determine the atomic structure of the water pore and the mechanism of its selectivity [5], [65], [67], [130]. Moreover, the specificity of more plant aquaporins also needs to be determined, and plausible plant metabolites need to be tested in order to identify regions important to specificity [5], [9], [20], [32].

The availability of a large body of high resolution structural data along with numerous atomic-scale simulation studies have resulted in an unprecedented level of understanding of the mechanism of function and selectivity in aquaporin. Meanwhile these results clearly demonstrate that modification of the expression patterns or gating properties of selected aquaporins might represent possible targets for manipulating the control of water fluxes within plants.

Regulation of aquaporin water permeability in plants

Land plants have evolved to cope with rapid changes in the availability of water by regulating all aquaporins that lie within the plasma membrane [1]. Regulation of aquaporin trafficking may also represent a way to modulate membrane water permeability, and the factors affecting and regulating aquaporin behaviors possibly include phosphorylation, heteromerization, pH, Ca^2 +, pressure, solute gradients, temperature drought, flooding, etc., which suggests plant aquaporins are involved in a versatile and dynamic regulation of water movement [1–3], [5], [9], [20], [32], [50], [57], [58]. The abundance and activity of aquaporins in the plasma membrane and tonoplast may be regulated, hence enabling the plant to tightly control water fluxes into and out of its cells, as well as within the cells [5], [6], [19], [50]. Regulation of aquaporin trafficking may also represent a way to modulate membrane water permeability (Figure 2).

Figure 2.  Schematic three-dimensional structure: Water molecules passing through the aquaporin in the plant biomembrane. This Figure is reproduced in colour in Molecular Membrane Biology online.

Here, our aim is to integrate recent molecular and biophysical data on the mechanisms regulating aquaporin activity in plant membranes and to relate them to putative changes in protein structure.

Functional regulation of aquaporin activity by phosphorylation

Aquaporin activity may also be directly regulated. Phosphorylation is a major mechanism used by cells as a molecular ‘switch’ to regulate protein activity [5], [19], [20], [30]. The kinases responsible for protein phosphorylation are induced in response to a number of signals, including drought or water stress [19], [64], attacks by pathogens [68], the plant hormones auxin and abscisic acid [19], [69], and light [6], [20]. So we aim to discover how the regulation of aquaporin phosphorylation in plants may be controlled according to developmental stages or environmental cues.

Mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduce the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity [29], [35], [70]. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5°C, suggesting the inactivation of the water channel [71].

As demonstrated by Johansson et al. (2002), species- and stress-specific changes in cell hydraulic conductivity may be caused by phosphorylation of aquaporins [26], [35]. The phosphorylation of soybean nodulin 26 on Ser-262, which is catalyzed by a symbiosome membrane-associated calcium-dependent protein kinase, stimulates its intrinsic water transport rate. Soybean nodulin 26 phosphorylation is enhanced further by osmotic stresses (for instance, water deprivation and salinity) [72].

Phosphorylation of the plasma membrane aquaporin PM28A is carried out by a Ca^2 +-dependent membrane-bound protein kinase and depends also on the apoplastic water potential [26], [35]. Three PIP2 isoforms (ZmPIP2;1, ZmPIP2;3 and ZmPIP2;4) were detected in the phosphorylated band. The water channel activity of these isoforms was partially inhibited by H7, a PKC inhibitor, suggesting an important effect of phosphorylation on channel function [73]. Both tonoplast and plasma membrane aquaporins have putative phosphorylation sites that are conserved among different members of this family [24].

From current research results, the crucial physiological role of aquaporin phosphorylation and dephosphorylation in plants can be seen, even if the molecular consequence of this modification remains unknown.

Effects of turgor and osmotic pressure on aquaporin activity in plants

Aquaporins are intrinsic membrane proteins characterized by six transmembrane helices that selectively allow water or other small uncharged molecules to pass along the osmotic gradient. However, there is no direct evidence for mechanosensitivity of aquaporin proteins, although some studies have illustrated the potential for MIPS to be involved in volume or turgor homeostasis in plants [3], [74], [75].A different mechanism of mechanical inhibition was recently reported in young maize roots [76]. The dependence of mercury-induced closure of water channels on tissue turgor indicates that changes in turgor can induce changes in the conformation of the plasma membrane, or even in the aquaporins themselves [77]. In more recent experiments, membrane permeability parameters (hydraulic conductivity, permeability and reflection coefficient) of Chara cells were measured using a cell pressure probe when the concentration of variously sized osmolytes was increased [78]. Water-channel activity, inferred from the membrane permeability parameters, was seen to decrease with increasing osmotic pressure and with increasing osmolyte size. Acohesion/tension mechanism was proposed to account for the gating of Chara water channels, which suggests that the osmotic tension generated within the channel by a high osmotic potential leads to the structural collapse and closure of the protein channel. Similar effects of high salinity on membrane water permeability as reported in several species could result, at least partly, from the same mechano-sensitive mechanism [79–81].

However, the functional significance of water channels in maintaining water balance under osmotic stress remains unclear. Under drought or salt stress conditions, transcript or protein abundance of some aquaporins are upregulated, whereas others are downregulated.

Relationships between aquaporins and hormones in plants

Abscisic acid (ABA) is a well-recognized mediator of water stress responses. The responsiveness of each aquaporin to ABA is different, implying that the regulation of aquaporin expression involves both ABA-dependent and ABA-independent signaling pathways. Both energy-input and tension-gating mechanisms might be used by the plant to sense changes in turgor pressure and surrounding water availability, and to adapt the membrane water permeability in an ABA-dependent manner [76]. In many species including sunflower, barley, sorghum and maize [82–84], exogenous ABA enhanced root L_p. Short-term effects (i.e., within 1–3 h) of ABA on root aquaporin gene expression have also been described in different species including rice, Craterostigma plantagineum and Arabidopsis [11], [85–87]. In tobacco flowers, ABA induces expression of the aquaporin gene NtAQP1 [88]. The effect of ABA in rapid positive regulation of aquaporin gating might suggest a direct binding to the channel. However, for both water stress and ABA, comprehensive studies using macro- or micro-arrays are needed to determine whether the complement of aquaporin genes varies in a coordinated fashion, and parallels changes in root water transport [69], [88].

Expression of certain plant aquaporins is regulated by gibberellic acid, or water deprivation. Gibberellic acid is also known to activate the promoter of the aquaporin PIP1b [89], [90]. The significance of promoter regions for an abscisic acid- and gibberellic acid-induced gene expression could be restricted to a region between −1450bp and −1112 bp upstream of the transcription start point by transient transformation of a bicistronic vector into tobacco protoplasts [91]. Gibberellic acid and abscisic acid suppress the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs are scarcely changed by these phytohormones [92].

Brassinolide may control aquaporin activities in Arabidopsis thaliana, which has been shown to be involved in the modification of the water-transport properties of cell membranes [93], however, mRNA and protein levels of aquaporin isoforms in roots and shoots of radish are not affected by brassinolide treatment [93]. Ethylene significantly increases stomatal conductance, root hydraulic conductivity (L_p), and root oxygen uptake in hypoxic seedlings. Ethylene can increase plasma membrane permeability permitting more water to cross the cells [94]. At the whole plant level, increased water transport in hypoxic aspen seedlings exposed to ethylene has been explained in terms of enhanced aquaporin activity, probably due to a direct effect of ethylene on the phosphorylation of aquaporins [95]. The L_p of root cortex cells was maximally stimulated ≥30-fold after a 1 h exposure to 1 µM ABA, but these effects disappeared over the next hour. In the same studies it was also noted that in contrast to ABA, auxin (IAA) and cytokinin (kinetin) reduced the cell L_p by three- to four-fold [96]. Studies at the cell level provide more direct evidence for cell membranes and aquaporins involved in stimulus-induced regulation of root L_p [1–3], [5], [7], [97].

In addition, valuable insights into the knowledge of the molecular mechanisms by which important hormones in animals, such as vasopressin, secretin, oestrogen, glucagon and hormone peptide YY (PYY), regulate the expression and intracellular trafficking of some aquaporins will be provided in papers by authors who have been well recognized for their studies on aquaporin regulation in animal cells. Nevertheless, in plants it is expected that future studies will allow us to uncover new hormone-induced re-localization of aquaporins. Furthermore, the information will be interpreted in line with the accompanying changes in hormone and function.

Effects of water and nutrient stress on aquaporins in plants

The role of aquaporins in plant water status under water stress is a complex issue, because the expression of different aquaporin genes may be stimulated, reduced, or unchanged under abiotic stress [1], [98–100]. The expression of some genes that encode plasma membrane aquaporins, such as Arabidopsis RD28 and the NeMip2 and NeMip3 genes of Nicotiana excelsior, is stimulated under drought stress [101], [102]. Conversely, expression of the Mesembryanthemum crystallinum MIPA plasma membrane aquaporin gene is down-regulated under salt stress [103], whereas expression of the Arabidopsis PIP1a gene is not altered significantly by stress conditions [104].

Drought stress induced in rice seedlings appeared to increase the physiological functioning of water-channels by increasing the root water channel activity or by increasing the aquaporin amount compared with unstressed control seedlings [105]. Under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells [42]. PIP1b overexpression had no beneficial effect on transgenic tobacco under salt stress, whereas during drought stress it had a negative effect, causing faster wilting [106]. Since drought stress decreases the osmotic permeability of root protoplasts but does not have any influence on the amount of PIP1 and PIP2 aquaporins, it is plausible that drought affected the protein functionality [52]. The accumulation of transcripts arising from NgMIP2, NgMIP3 and NgMIP4 diminishes dramatically in drought-stressed plants. This down-regulation of MIP gene expression may result in reduced membrane water permeability and may encourage cellular water conservation during periods of dehydration stress [107].

Aquaporin functionality fits well with the overall water relation responses, since the two-phase adaptation to salinity may imply two types of aquaporin regulation. Salinity can negatively affect root water uptake. Work has shown that exposure of roots to salt induces changes in aquaporin expression at multiple levels. These changes include a coordinated transcriptional down-regulation and subcellular relocalization of both PIPs and TIPs [108]. On the other hand, salt and water stresses induce the accumulation of ZmTIP2-3 transcripts [109]. Similar findings on the dominance of symplastic water transport were obtained by using transgenic tobacco plants expressing an antisense construct of the tobacco NtAQP1 gene that encodes another PIP1b isoform [25], while Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [38]. Two aquaporin-encoding transcripts were found to be down-regulated during the first 15 and 60 min of a salt treatment, respectively. The expression level of the two transcripts then recovered and, after 7 d, expression had become higher than in plants grown in standard conditions [105], [110–116].

It is the incoming nutrient supply that is registered as deficient, not the plant's nutrient status. At some point, close to the initiation of these responses, changes in water channel activity may be involved. The reduction of root hydraulic conductivity in wheat plants affected by nutrient (N-, P- and S-) deficiencies, suggested that either the activity or the density of water channels in the root cell plasma membrane is diminished during nutrient deficiency, but the manner in which monitoring of nutrient stress is transduced into an hydraulic response is also unknown [106], [117–125].

The role of aquaporins in plant water status under water stress is a complex issue, because the expression of different aquaporin genes may be stimulated, reduced, or unchanged under abiotic stress. Indeed, this complex expression pattern suggests that maintenance of a reasonable water status under salt and drought stresses requires both increased symplastic water transport via aquaporins in some cells and tissues and reduced water transport via aquaporins in other cells and tissues. Additional evidence for in vivo function of aquaporins is required to conclusively define their contribution to osmotic adaptation under stress conditions.

Concluding remarks

The mechanism by which water passes through biological membranes was a matter of debating until the discovery of the aquaporin water channels. Because aquaporins are a family of membrane channels primarily responsible for conducting water across cellular membranes, the discovery of aquaporins in plants has resulted in a paradigm shift in the understanding of plant-water relations. Water flux across cell membranes has been shown to occur not only through the lipid bilayer, but also through aquaporins, which are members of the major intrinsic protein super-family of channel proteins [2], [3], [5], [9], [20], [32], [57], [117–126]. As has been found in other organisms, plant MIPs function as membrane channels permeable to water (aquaporins) and in some cases to small nonelectrolytes. Aquaporins greatly increase the membrane permeability for water, but may also be regulated, allowing cellular control over the rate of water influx/efflux [1–7]. As a result, aquaporins provide a unique molecular entry point into the water relations of plants and establish fascinating connections between water transport, plant growth and development and adaptive responses of plants to their ever-changing environment [2]. Biochemical and biophysical techniques can be employed to gather information on the basic functional characteristics of plant aquaporins. In fact, the physiological role of aquaporins in plants is much less well understood. The growing knowledge of how aquaporins regulate water passage eventually could help agricultural producers boost survivability and production of drought-stricken and flood-ridden crops [127]. Thus, a challenge for future research will be to understand how plants respond to stresses by integrating these mechanisms in time and space, to constantly adjust the water transport and solute transport properties of their membranes [128–130].

Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Aquaporins can provide spatial markers to explore the intricate flows of water and solutes that play a critical role throughout all stages of plant life-cycle [2]. The rate of transmembrane water flux may be controlled by changing the abundance or the activity of the aquaporins. Indeed, there are observations showing the alteration of water permeabilities in responses of plants to biotic or abiotic stresses such as high salinity, nutrient deprivation, or extreme temperatures [1–3], [5], [9], [20], [120–130]. In plants, aquaporins are likely to be important both at the whole plant level, for the transport of water to and from the vascular tissues, and at the cellular level, for buffering osmotic fluctuations in the cytosol [19]. Modulation of xylem hydraulic conductivity via sap effects on pit membrane structure may be as important as the regulation of aquaporin activity in leaf and root cell membranes in influencing saturated plant hydraulic conductivity.

By combining molecular biology with plant physiology, it should be possible to determine the role that aquaporins play in water transport in the plant [1–7]. There is growing evidence that suggests that aquaporins play different roles throughout plant development, including nutrient acquisition, carbon fixation, cell signaling and stress responses [115–126]. Therefore, aquaporin genomic information is important because assigning physiological function via transgenic reduction or removal of gene expression requires sequence information for precise targeting. Direct determination of the location of each aquaporin within tissues is still required to understand its function in the plant. A powerful tool in elucidating the aquaporin function is given by the reverse genetics that can also reveal unexpected function of water channel proteins, which benefit our understanding of sequence-structure and structure-function relationships in plants [2], [4], [5], [9], [117–126]. This should be done both at the transcript and at the protein level because aquaporin turnover appears to be variable, such as when comparing constitutively expressed and inducible aquaporins. The transcriptional and/or post-translational regulation of aquaporins would determine changes in membrane water permeability. Both phosphorylation and translocation to/from vesicles have been reported as post-translational mechanisms. Much isolated information has been accumulated, but their function is far from being understood in plants, and there is a long way to go to fully understand the significance of these proteins [124–130].

Aquaporins are thought to be involved in the regulation of trans-cellular water flow for long-distance transport in the root and leaf tissues. Their role is also critical for short-distance water transport and osmotic adjustments within a cell and between the cytoplasm and the cell wall space. The abundance of aquaporins is regulated developmentally in a cell-specific manner and by environmental signals. The activity of aquaporins is also regulated by different post-translational regulation mechanisms, which provide an efficient way for rapid and reversible regulation of the water membrane permeability [128–130]. Now our research project aims at understanding the function and regulation of aquaporins at the cellular and molecular level and in the whole plant subjected to various environmental conditions.

Acknowledgements

This work is jointly supported by 973 Program of China (2007CB106803), Shao Ming-An's Innovation Team Projects of Education Ministry of China and Northwest A&F University, International Cooperative Partner Plan of Chinese Academy of Sciences, Doctoral Foundation of Qingdao Agricultural University (630523) and Project of Shandong Provincial Education Department (J06K57). Special thanks are also given to three reviewers for their valuable comments. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.