From the dissertation of Mark J. Daniels (Chrispeels Lab at UCSD)

Characterization of Water Channel Proteins (Aquaporins) in Plants

Membranes and Water Transport

The invention of the microscope led to the discovery of single-celled organisms (animacules) by Antonie van Leeuwenhoek [1] and of the existence of compartments (cells) in plant tissues by Robert Hooke. Although the cells of plants are separated by cell walls, cells maintain their identity because they are delimited by semipermeable membranes that permit them to function as autonomous units. The flow of materials in and out of the cell is regulated by channels, transporters and pumps in these limiting membranes.

In plant cells, the cytoplasm is actually sandwiched between two membranes: the plasma membrane, which forms the outer boundary of the cytoplasm, and the tonoplast or vacuole membrane. The tonoplast forms the boundary between the cytoplasm and the vacuolar compartment. The fluid mosaic model of cellular membrane structure, first proposed by Singer and Nicholson [2] , presented the first coherent picture of the membrane, as a bilayer of phospholipids and glycolipids studded and spanned by proteins partially or fully solvated by the lipid matrix.

Membrane-bound proteins that cannot be removed from the lipid bilayer without disrupting the membrane are known as intrinsic membrane proteins, distinguishing them from the loosely bound extrinsic membrane proteins. Many of these intrinsic membrane proteins have been found to be responsible for either the transport of molecules or the transmission of signals across the membrane boundary.

Water is a small, slightly polar molecule to which all biological membranes show some degree of permeability as a result of diffusion across the lipid bilayer. Membrane water permeability was often ascribed to diffusion alone [3, 4] . The diffusion of water can be driven by concentration gradients of osmotically active solutes, which generate an osmotic force, or by physical pressure, which generates a hydraulic or hydrostatic force. Based upon observations that osmotically or hydraulically driven water flow in red blood cell membranes was greater than that produced by simple diffusion alone, biologists postulated that water flow through cells was facilitated by pores in the membrane [5-8] . These water-filled pores were hypothezied to enable the bulk flow of water through a membrane.

This idea was supported by subsequent results showing that certain membrane and tissue types exhibited unusually great water permeability [9, 10] . Additional evidence for the flow of water through pores came from work showing that the energy of activation- the thermodynamic work required- for the movement of water across red blood cell (erythrocyte) membranes is in the range of 4-6 kcal/mol, significantly less than that for water flow across a lipid bilayer, which is about 11-14 kcal/mol [11] . A proteinaceous route for this anomalous water flow was suspected when it was found that transmembrane water flux was inhibitable by mercury sulfhydyl reagents such as mercuric chloride and para-chloromercuribenzenesulfonate (pCMBS), which are known to bind proteins and inhibit enzymatic activities [12] . These combined results implied the existence of a facilitated route for water transport, likely mediated by transport proteins (reviewed in [13] ).

The physical size of the functional unit responsible for this facilitated transport was determined by radiation inactivation to be a molecule or molecules with a size of 30,000 dalton [14] , which corresponded with the 28,000 dalton size of some proteins in the red blood cell membrane. The identity of this putative water channel protein, or the gene encoding the protein, still remained unresolved.

Water Channel Proteins and the MIP Family of Proteins

When it was realized that the water transporting unit was likely to be a protein, investigators attempted to purify and characterize it, so that it could be cloned and characterized further. Xenopus laevis oocytes as a model system to study membrane proteins and the expression of injected messenger RNA , provides a convenient system to examine the function of membrane proteins (reviewed in [15, 16] ). Early work showed that mRNA isolated from cells with highly water permeable membranes, such as erythrocytes or kidney cells, when injected into the Xenopus oocytes, caused a water channel to be expressed [17] .

Earlier, a membrane intrinsic protein with a size of 28,000 dalton had been purified from erythrocytes and kidney cells and named CHIP28 (for channel-like membrane integral protein of 28kD) [18] . The cDNA corresponding to this CHIP28 protein was cloned and the nucleotide sequence found to encode a protein homologous to a family of putative channel proteins [19] . When CHIP28 cRNA was injected into and expressed in Xenopus oocytes, the oocytes showed a greatly increased osmotic water permeability, which was inhibited by the transmembrane water flow inhibitor mercuric chloride [20] . The CHIP28-expressing oocytes also showed a decreased activation energy for water transport, similar to that found previously with erythrocyte membranes. These results clearly indicated that the CHIP28 protein functioned as a water channel.

A number of cDNAs encoding putative intrinsic membrane proteins, including that for CHIP28, had been found in a variety of organisms and were homologous to a previously characterized membrane channel protein, the bovine lens fiber major intrinsic protein known as MIP (or MP26) [21, 22] . This growing class of proteins became known as the MIP protein family, and those members which were water channels were named aquaporins [23]. Water channel activity in MIP family proteins has been demonstrated for a number of CHIP28/AQP-1 homologs in animals [24-29] , AqpZ in the bacteria E. coli [30] , P25 from an homopteran insect [31] , and the PIP and TIP proteins from plants [32-35] . Not all MIP family proteins act as water-selective or water-conducting channels, however; GlpF from E. coli is strictly a glycerol channel [36] . Nod26 from soybean peribacteroid membranes appears to be an ion channel and a water channel [37, 38] . MP26 has been shown to conduct water and glycerol, and perhaps ions as well [22, 39, 40] , while AQP-3 from mammalian kidney conducts water, glycerol and urea [41] . CHIP28/AQP-1 and WCH-CD/AQP-2 appear to function as channels for glycerol in addition to water [42] , and CHIP28/AQP-1 may be induced to form a cation channel [43, 44] .

Almost all aquaporins are sensitive to inhibition by mercury sulfhydryl reagents. Of the water channel proteins characterized to date, only two, RD28 in plants [33] and MIWC in mammals [45, 46] are insensitive to inhibition by these agents. It should not be assumed that MIP-family aquaporins are the sole mechanism of faciliated water transport, as there is some evidence that sugar transporters may be co- transporters of water or be leaky for water [47, 48] and that ion channels may couple the transport of ions with that of water molecules [49] . The pore-forming antibiotic amphotericin B is known to conduct water [50] and has been used as a positive control in biophysical studies of water channel effects [51] .

Aquaporins in Plants

Plant-water relations and water flow in plant tissues have been well-characterized [4, 52-55] , but the presence of water pores or even proteinaceous water channels in plant membranes was not established until fairly recently [56, 57] . At the same time that the mammalian CHIP28 aquaporin was first characterized, a number of plant cDNAs were identified as members of the MIP protein family, including a-TIP in the common bean Phaseolus vulgaris [58] , TobRB7 in tobacco [59] , Nod26 in soybeans [60] , and clone 7a/trg-31 in the pea plant Pisum sativum [61, 62] . A cDNA for a MIP-like protein, called g-TIP, was cloned from Arabidopsis thaliana [63] and subsequently characterized as the first plant aquaporin [32] . Many more MIP family genes have since been identified in plants, with additional members in Arabidopsis [33-35, 63, 64] , tobacco [65] , spinach [66] , tomato [67] , the ice plant Mesembryanthemum crystallinum [68] , radish [69] , and snapdragon [70] .

A number of these genes have been found to encode aquaporins and ion channels, while many more are still being or have yet to be characterized. The roles that aquaporins and other MIP family proteins play in plants are still poorly understood. Water is taken up from the soil through the roots, where it flows from the cortex into vascular tissues. From there it is transported up to the leaves where it flows through the leaf mesophyll to the stomata, and is there lost to the atmosphere by evaporation. This transpiration stream of water is driven by the loss of water from the leaves.

The vascular tissue responsible for the upward flow of water is called the xylem, composed of highly elongated cylindrical tracheary elements stacked end to end. These tubular forms are the cellulosic remains of the cell walls produced by the vascular precursor cells, the membranes, protoplasm, and organelles of which have degraded and disintegrated. The xylem can therefore be imagined as columns full of water extending from the roots to the leaves. Water flowing into the xylem from the roots and out of the xylem into the leaves must pass through many cell layers of living tissue, where cell membranes will pose a resistance to water flow.

There are two possible routes for water flow in these living tissues; a circuitous apoplastic route, through the porous cell walls and intercellular space, or a more direct transcellular route, through the cell cytoplasm, vacuole, and associated membranes. Which route water flow takes in a plant appears to depend upon the species, developmental state, and tissue of that particular plant [53] . The presence of aquaporins in cellular membranes may facilitate a transcellular pathway for water flow. When plant roots are exposed to mercuric chloride, which is known to inhibit aquaporins, the flow of water is greatly reduced while the flow of ions is not [71, 72] . This supports the view that there exists a proteinaceous mechanism for the transport of water, independent of the facilitated transport of ions. In addition, protoplasts of leaf cells from transgenic plants expressing putative aquaporin antisense RNA swell at a greatly reduced rate when introduced to hypo-osmotic conditions [73] .

Coexpression of sense and antisense RNA for this gene should greatly diminish synthesis of the putative aquaporin, and this is suggested by the experimental results, which show a reduction in transmembrane water flow. Aquaporins are differentially expressed in different organs and membranes. If transcellular water flow is to be facilitated, it is necessary for aquaporins to be present in the two major membrane obstacles to water flow, the plasma membrane and the vacuole membrane. In Arabidopsis, this requirement is fufilled. The aquaporins g-TIP and d-TIP are found in the vacuolar membrane of vegetative tissues [34, 63] , while a-TIP is found in the protein storage vacuoles of seeds [63] . The PIP proteins and RD28 are found in the plasma membranes of vegetative tissues [33, 35] . Preliminary results indicate that the in vivo ectopic overexpression of Arabidopsis RD28 in tobacco leads to an increase in the hydraulic conductivity of the leaf tissue (Ted Hsiao, unpublished observations).

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. One manner of plant water channel regulation appears to be at the level of gene expression. The growth-inducing signals of light and the hormones abscisic acid and gibberellic acid cause the transcription of aquaporin and putative aquaporin genes in Arabidopsis [74, 75] . Plant MIP family genes have also been found to be expressed in response to water stress, caused by either drought [61, 67] or salinity [65, 68] . Pathogen attack may also lead to aquaporin expression; the plant MIP family gene tobRB7 is induced at the feeding site of root-knot nematodes in tobacco roots [76] ; this is the first characterized link of a putative aquaporin to a disease state in plants. In the case of infection by the symbiotic nitrogen-fixing rhizobia, leguminous plants have evolved root nodules with large cells that contain the bacteroids in small, vacuole-like structures.

The ion channel and aquaporin Nod26 is found in the membranes of these vacuoles [77] . Aquaporin activity may also be directly regulated. Phosphorylation is a major mechanism used by cells as a molecular 'switch' to regulate protein activity. The kinases responsible for protein phosphorylation are induced in response to a number of signals, including drought or water stress [78, 79] , attack by pathogens [80] , the plant hormones auxin and abscisic acid [81, 82] , and light [83] . a-TIP has been found to be phosphorylated by a tonoplast-bound calcium-dependent protein kinase (CDPK), and the phosphorylation state of a-TIP changes during development [84] . This phosphorylation of a-TIP increases its water channel activity [85] . The plasma membrane-specific aquaporin PM28a from spinach leaves is also phosphorylated, as a result of osmotic stress, by a plasma-membrane associated CDPK [66] . Protein kinase activity in the maize root elongation zone has been found to be stimulated by water stress [79] . These results may indicate the existence of a mechanism in plants by which aquaporins are phosphorylated, and their activity therefore modulated, in response to osmotic conditions.

The Structure of Aquaporins

Aquaporins belong to a family of integral membrane proteins that has members in animals, plants, yeast, and bacteria (reviewed in [86] ). These aquaporins share a unique feature in that the pore is highly selective for water. Several MIP proteins are exceptions to this rule and instead of, or in addition to, transporting water appear to conduct small ions or small uncharged molecules. All MIP family proteins share six putative transmembrane domains, with small hydrophilic loops connecting these regions, and two highly conserved motifs in extramembrane peptide loops thought to be involved in channel selectivity [87] . The ancestral MIP family progenitor may have arisen from the duplication of a three-membrane spanning domain protein gene, as evidenced by two repeats of amino acid sequence in the putative halves of MIP proteins [88, 89] . Analysis of different proteolytic digests of aquaporin CHIP28 supports such a membrane topology, in which the two halves of the protein are oriented 180o with respect to each other [90] .

The first physical analyses of aquaporin structure were radiation inactivation studies, in which the functional unit of the CHIP28 water channel was found to have a size of roughly 30,000 dalton [14] , corresponding to the 28,000 dalton size of the protein observed by gel electrophoresis [18] . Hydrodynamic studies have suggested that CHIP28, and its homolog MIP26, behave physically as if they are arranged in a tetrameric structure [91, 92] . This has been confirmed by electron microscopic observation of freeze-fractured membranes [93] and electron crystallography [94, 95] .

Based upon these observations, and the analysis of CHIP28 mutants, Jung et al. [87] proposed the hourglass model for CHIP28. According to this model, the water channel pore is formed by the partial insertion into the membrane and apposition of two homologous hairpin turns from extramembrane loops, both bearing the same conserved sequence motif. 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.

To account for the molecular selectivity of MIP family channels, the pore so formed was hypothesized to function via a size-exclusion mechanism. Inhibition of the water channel by mercuric chloride is thought to be the result of the sulfhydryl reagent binding a cysteine residue located in close proximity to the pore, resulting in the physical blockage of the molecular flow through the pore. Mutational analysis of amino acids flanking the pore has supported this hypothesis [96] and mercury- sensitive aquaporins have been generated from normally mercury-insensitive proteins by mutagenesis, through which pore-flanking amino acid residues were converted to cysteine [33, 46] .

MIP family proteins are predicted by computational sequence analysis [86] and circular dichroism (CD) spectroscopy [97] to consist of mostly a-helical structure. However, some have called the 'hourglass' model, with its prediction of six membrane- spanning domains, into question. Spectroscopic analysis of tryptophan residues in CHIP28 suggests that their positions in transmembrane domains or extramembrane loops does not entirely agree with the 'hourglass' model [98] . As presaged by Wistow and colleagues [89] , recent CD and fourier transform infrared (FTIR) spectroscopy indicates that CHIP28 and MIP26 have roughly equal proportions of a-helical and b-sheet structure [99, 100] . As a result, alternate models of aquaporin structure have been proposed, one in which the CHIP28 aquaporin is formed from four membrane-spanning a-helices [101] , and a second in which the water channel is folded as a 16-stranded b-barrel [102] . However, these alternate models have been disproved by recent results.

Several high-resolution projection maps of CHIP28 have been determined by cryo-electron crystallography and show the CHIP28 tetramer forming a square array, with a trapezoid-shaped subunit at each corner and a central cavity [103-106] . Each subunit appears to be composed of six to eight transmembrane a-helices; unfortunately, there is no agreement on the location or the structure of the water channeling pore. These results show that the hourglass model needs some revision; instead of standing to one side of the pore, the current structural data suggest that the six transmembrane a-helices surround the pore. Further investigation is necessary to determine the atomic structure of the water pore and the mechanism of its selectivity.

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