WATER TRANSPORT AND AQUAPORINS IN GRAPEVINE
5. WATER TRAFFIC AND AQUAPORINS
The data summarized above underline the importance of water flow for the development and the yield of grapevine, as well as for its resistance to drought. Even if water flows mainly in the xylem, the phloem also plays a key role, at least in the ripening berry.
Whatever the pathway of long distance water movement, it is followed by a short dis- tance movement where several pathways may be envisaged for water flow between plant cells (a) the apoplastic path in the cell wall, (b) a symplastic path, which is mediated by the plasmodesmata bridging adjacent cells across the cell walls, so that a cytoplasmic continuum is formed, (c) a transcellular path, where two membranes, the plasma mem- brane bordering the cytoplasm, and the tonoplast bordering the vacuole must be crossed.
Both these membranes contain abundant proteins, aquaporins, facilitating transmem- brane water movement.
5.1. Aquaporins
During the last decade, it has become evident that water movement across biological mem- branes may be facilitated by special proteins behaving as water channels and called aq- uaporins. Aquaporins were first discovered in erythrocytes and renal tubuli (Preston et aI.,
WATER TRANSPORT AND AQUAPORINS IN GRAPE
'I'.poplul - ã 1.0 MJ'.
111-+-- --
80w
su....,.odl W_ood .. rlx>I,yd ... _
. A ... • .. .1.5Mh _oI~pIoot ---l.oMoI."....
Strm& 1DfS( dgp" d
249
Figure 10.2. A physiological model of water and sugar fluxes at two stages of ripening of the berry, pre- and post-veraison. During the pre-veraison stage, water influx in the berry is mediated mainly by the xylem. Sugars are imported by the phloem, and diluted by xylem water arriving in the apoplast. The flesh cells are turgescent and the water demand is weak. Part of the apoplastic sap, including ions and sugars, is re-exported by the xy- lem and recirculated by the phloem. After veraison, the xylem continuity is disrupted and prevent water flow to/from the berry through this tissue. Sugar and water import are thus mediated by the phloem and require the involvement of specialized membrane proteins (sugar transporters and aquaporins). At this stage, the water potential of the apoplast is low, and the water demand by the storage cells is high, to compensate for sugar accumulation. 'Pap, water potential of the apoplas!. nap: osmotic potential ofthe apoplast; 'P, water potential of the storage cell; P, turgor pressure. Designed from data contained in Findlay e/ ai. (1987), Lang and Thorpe (1989), Pomper and Breen (1995).
250 S. DEL ROT et al.
1992). These authors showed that the expression of AQPI (formerly called CHIP28) in Xenopus oocytes induced their osmotic swelling when the oocytes were placed into a hypoosmotic medium. The existence of proteinaceous components facilitating the movement of water had long been suspected from the observation that sulfhydryl re- agents reversibly affected the osmotic and hydraulic conductivitY of animal membranes (Macey and Farmer, 1970). This sensitivity is indeed found also in oocytes expressing some, but not all aquaporins. It must be stressed that heavy metals are poorly selective and may have many secondary effects (Eckert et al., 1999).
While the diffusional water permeability (Pd) across the lipid phase is low (about 10.5 m.s- I), the presence of water channels in the membrane results in a high osmotic permeability (Pos, about 10-4 to 10-3 m.s-I). As a result, the Pos/Pd ratio, which characterises the predomi- nant pathway of water movement, is increased in the presence of aquaporins (Maurel, 1997).
Because the water crossing an aquaporin does not need to surmount the high energy barrier of water partitioning into lipids, the activation energy measured by Arrhenius plots is low ô5 kcal.morl) for this type of transport, compared to that associated with diffusional transport (14-16 kcal.mor\
Aquaporins belong to a highly conserved group of membrane proteins called the ma- jor intrinsic proteins (MIPs) found in bacteria, yeasts, insects, mammals and plants, and comprising more than 150 members (Johansson et al., 2000). When expressed in Xeno- pus oocytes, most of the studied aquaporins are highly specific for water. However, some of these members have been shown to transport not only water, but also glycerol, ethylene glycol and urea.
The aquaporin polypeptides have a molecular mass of26 or 30 kDa and typically con- tain six transmembrane a-helices, with the Nand C termini located on the cytoplasmic side of the membrane (Fig. 10.2). The Nand C terminal halves of the polypeptide show significant sequence similarity to each other. The polypeptide is arranged as a tandem repeat of 3 transmembrane a-helices, suggesting that it arose from duplication; each half of the polypeptide has a smaller hydrophilic loop that includes a highly conserved AsniProl Ala motif. The overall structure of the aquaporin presents an intramolecular two-fold axis of symmetry, and it has been suggested that this accounts for the fact that they can mediate bidirectional water flow and do not show rectification, in contrast to many ion channels (Tyerman et al., 2000). Aquaporins have a propensity to aggregate in vitro, and size exclusion chromatography studies have given a stoichiometry of 4 sub- units per oligomer for AQPI (Smith and Agre, 1991). However, radiation inactivation analyses indicate that the monomer is functional and may form an independent pore.
Water movement may occur in either direction, down the potential gradient, and due to the narrow size of the pore (0.3-0.4 nm), any molecule larger than water should be excluded.
5.2. Plant Aquaporins
The first plant aquaporin was described by Maurel et al. (1993) who showed that y-TIP, an abundant tonoplast intrinsic protein found in the elongating cells of Arabidopsis
WATER TRANSPORT AND AQUAPORlNS IN GRAPE 251 thaliana roots and shoots, facilitated the movement of water into the vacuolar membrane and into Xenopus oocytes. A homologous protein was then discovered on the plasma membrane of Arabidopsis cells (Daniels et ai., 1994; Kammerlohrer et aI., 1994). Both tonoplast aquaporins (called TIPs, for tonoplast intrinsic proteins) and plasma membrane aquaporins (called PIPs, for plasma membrane intrinsic proteins) are characterized by high abundance in their respective membrane. Although many clones homologous to aquaporins have now been isolated in various species (for review, Maurel, 1997; Johans- son et at., 2000), a relatively low number has been functionally tested. TIPs and PIPs are very similar in structure, but can be distinguished on the basis of their sequence and be- long to distinct phylogenetic group. A third phylogenetic group found in plant tissues consists in NOD26, expressed in the peribacteroid membrane of soybean nodules, NLMI (NOD26 like MIP, a recently discovered Arabidopsis nodulin-Iike protein) and a few related sequences (Johansson et at., 2000).
5.2.1. TIPs
In Arabidopsis, TIPs are encoded by a broad multigenic family, with more than 20 members (Weig et at., 1997). This diversity is much larger than that found for animal aquaporins, where only 9 members have been described so far. Several TIPs exhibit a more or less organ specific expression. For example, y-TIPs are expressed in the elongat- ing zones of shoots and roots, and 8-TIP is more strongly expressed in the parenchyma cells of vascular tissues of shoots than in roots (Daniels et al., 1996). The a-TIP from Phaseolus is specific for seeds and seedlings. Within the same cell type, the distribution of various TIPs to different types of vacuole is also controlled. For example, in seed pa- renchyma cells, y-TIP is confmed to the protein storage vacuole, and a-TIP to the nas- cent vegetative vacuole (Paris et aI., 1996). In mature motor cells of Mimosa pudica, which undergo large water fluxes in response to various stimuli, and which contain two types of vacuoles, a tannin vacuole and a colloidal vacuole, y-TIP is localized almost exclusively in the colloidal vacuole (Fleurat-Lessard et aI., 1997).
In addition to this control of targeting, TIPs may be controlled at the levels oftranscrip- tion, translation, and by post-translation events. The expression of a-TIP is developmen- tally regulated during seed maturation (Johnson et aI., 1989), and y-TIP progressively sub- stitutes for a-TIP after seed germination (Ludevid et ai., 1992). Immunogold labelling also indicates that the differentiation of the inner cortical cells of soybean nodules is accompa- nied by an increased expression ofy-TIP (Serraj et al., 1998). ZmTIP1, a maize aquaporin is most strongly expressed in meristems and expanding cells, which suggest that it is needed for vacuole biogenesis and to support the rapid influx of water into vacuoles during cell expansion (Chaumont et aI., 1998). y-TIP expression in Arabidopsis is induced by GA3 treatment (Phillips et aI., 1994), and the tonoplast aquaporin BobTIP26-1 from cauli- flower is strongly increased by dessication and osmotic stress (Barrieu et aI., 1999). In tulip, invertase and y-TIP are selectively expressed during cold-induced stalk elongation.
This is thought to increase the osmotic potential and vacuolar water uptake, thus providing the driving force needed for stalk-cell elongation (Balk and De Boer, 1999).
252 S. DELROT et at.
Detailed biochemical and mutagenesis studies have shown that some, but not all plant aquaporins may be regulated by phosphorylation. in pianta, a-TIP may be phosphorylated at Ser 7, near the N-terminus, by a tonoplast bound kinase (Johnson and Chrispeels, 1992).
In oocytes, phosphorylation of a-TIP occurs at three sites (Ser7, Ser23 and Ser99) located in consensus sequences for recognition by cAMP-dependent protein kinase (PKA); phos- phorylation stimulates its water channel activity (Maurel et aI., 1997). The fact that only Ser-7 was found to be phosphorylated in planta so far may be due do the fact that this resi- due, unlike Ser23 and Ser99 is bracketed by basic residues, which have been reported to favor the activity of some kinases, at least in animal cells. The relative physiological part played in vivo by the regulation of TIPs at the level of gene expression and protein phos- phorylation is still a matter of debate (Eckert et aI., 1999).
Although y-TIP is highly specific for water, Nt-TIPa, a TIP homologue cloned from tobacco cells, transports urea, and to a lesser extent water and glycerol. These data sug- gest that TIPs, and more generally plant aquaporins may have a dual function in water and solute transport (Gerbeau et aI., 1999).
52.2. PIPs
The importance of PIPs for water transport in plants has been demonstrated by experi- ments where an antisense PIP has been expressed constitutively in transgenic Arabidop- sis plants. This resulted in a strong decrease of osmotic swelling for the protoplasts pre- pared from these plants (Kaldenhoff et aI., 1998). In addition, the morphology and de- velopment of the antisense lines were normal, except that, due to a compensation mechanism, the root system was 5-fold more developed than in wild type plants.
Although TIPs and PIPs both belong to the MIP family and possess some homology, they form two distinct phylogenetic groups and their membrane localization can be in- ferred from the sequence. PIPs (30-31 kD) are somewhat bigger than TIPs (25-28 kD).
The isoelectric point of PIPs (around pH 9.0) is more basic than that of TIPs (around pH 6.0), due to the presence of several basic amino acids in the C-terminal part of the PIP sequences. The PIPs family is divided into two subfamilies on the basis of sequence analysis: PIPl and PIP2. PIP] possess longer N-terminal cytoplasmic tails and shorter C terminal tails than PIP2. In addition, PIP] does not possess a Ser 274 residue, which is conserved and phosphorylated in the terminal tail of PIP2 (Schaffner, 1998). Although PIPs possess a common topological structure, they may respond differentially to pertur- bations of the membrane environment. Thus, PMIP3l and PMIP27, two topologically related PIPs expressed in the storage tissue of sugar beet, undergo different conforma- tional changes upon inclusion of sulfhydryl reagents in the medium used for cell frac- tionation and preparation of plasma membrane vesicles (Barone et at., 1998).
Although all TIPs studied so far exhibit the classical sensitivity to heavy metals when they are expressed in oocytes, this is not the case for all PIPs. One PIP from Arabidopsis thaliana (RD28, Daniels et al., 1994) and one from tobacco (NtAQPl, Biela et aI., 1999) were insensitive to mercury chloride.
Both NOD 26, expressed in the peribacteroid membrane of legume nodules and
WATER TRANSPORT AND AQUAPORINS IN GRAPE 253 PM28A, a PIP2 expressed in spinach leaf, are phosphorylated in planta in the C-terminal domain. In the Xenopus oocyte expression system, phosphorylation of PM28A is associ- ated with an increased water transport activity (Johansson et al., 1998). These conclu- sions were supported by the fact that okadaic acid, a protein phosphatase inhibitor, in- creases water permeability, while K252a, a protein kinase inhibitor has the opposite ef- fect. In vitro phosphorylation of PM28a is mediated by a calcium dependent protein ki- nase associated with the plasma membrane (Johansson et aI., 1996). In addition, it was shown that PM28a is phosphorylated/dephosphorylated along with changes in the apoplast water potential, and that Ser274 was the amino acid involved. Addition of ABA in the grinding medium used to prepare the membranes did not affect the phosphoryla- tion ofPM28A (Johansson et aI., 1998).
With the exception of Arabidopsis PIP1 proteins, most PIP1 identified in various plants have no or very low activity in oocytes. In the case of maize PIP1 proteins, it has been shown that this lack of activity is not due to their failure to reach the plasma membrane of the oocytes (Chaumont et aI., 2000). One of these PIP (ZmPIPlb) was also unable to transport small soluted such as glycerol, choline, ethanol, urea, and amino acids. NOD26 transports water only with a low conductivity, but it allows the movement of glycerol and formamide but not urea (Rivers et al., 1997). NtAQP1, a member of PIP 1 family expressed in Nicotiana tabacum can mediate glycerol transport in addition to water flow, but is un- able to stimulate Na+, K+ and cr uptake when expressed in oocytes (Biela et aI., 1999).
Expression of RD 28, an Arabidopsis PIP2, is induced by dessication (Yamaguchi- Shinozaki et aI., 1992). TRAMP, a MIP homolog of tomato, is also more strongly ex- pressed in wilted leaves (Fray et al., 1994). Likewise, emip, a MIP gene homologous to the RD28a PIP, is induced by wilting, salt stress, and heat shock in epidermal strips of barley (Hollenbach and Dietz, 1995).
An interesting study by Clarkson and co-workers (2000) has demonstrated a marked diurnal cycle in the abundance of PIP 1 and PIP2 transcripts in roots of Lotusjaponicus.
The maxima and minima of PIP transcripts occur 2 to 4 h before the corresponding diur- nal fluctuations in root hydraulic conductivity. Furthermore, nitrogen deprivation also lowered the root hydraulic conductivity, possibly by a decrease of the activity and/or density of PIPs, which underline the interactions existing between ion and water move- ment. In Arabidopsis mesophyll, PIPI aquaporins seem to be concentrated in specific invaginating domains of the plasma membrane, called plasmalemmasomes. These struc- tures, which protrude deeply into the vacuoles, have been suggested to allow a rapid ex- change of water with the apopJast (Robinson et aI., 1996).
5.3. Grapevine aquaporins
In spite of the potential role of aquaporins for long distance and intercellular transport of water, resistance to water stress, and berry yield, searches in sequence databases indicate that this class of membrane proteins has not yet been investigated in detail in grapevine.
Special interest should be given to aquaporins expressed in the berries, which may have
254 S. DELROT et af.
a direct impact on the sugar concentration at harvest, and to root aquaporins which may be involved in the resistance to water stress often undergone by the grape plant during the summer season. In order to characterize clones encoding the sugar transporters and the aquaporins that might be specifically expressed during the ripening of the berry, we have developed a strategy based on the screening of a cDNA library and of a genomic library with probes obtained by RT-PCR. The permeability of the tonoplast is much higher than that of the plasma membrane (Maurel et af., 1997; Niemietz and Tyerman, 1997), which may indicate that cells regulate the influx of water at the plasma membrane (Chaumont et af., 1998). For this reason, we have focused our attention on PIPs. How- ever, given the large volume and swelling of the vacuole during grape ripening, TIPs are also worthy of investigation for a fuller understanding of water fluxes in the berries.
The cDNA library was prepared from RNA extracted from berries of Vitis vinifera cv Pinot noir collected at the veraison stage. Due to the high concentration of sugars and polyphenol, a specific RNA extraction procedure had to be designed (Fillion et af., 1999). The mRNA was purified using oligodT columns, and used for the preparation of the library with the ZAP-cDNA synthesis kit (Stratagene). Degenerate primers for PCR were designed from known PIP sequences (Arabidopsis thaliana, maize, sugar beet, to- bacco, and pea). PCR was used to amplifY a 268 bp PIP fragment which was cloned and used as a probe to screen the cDNA library. Out of approximately 600,000 clones, 28 clones were analyzed in further detail. They were sorted by restriction analysis, and se- quenced. Two full-length and several partial-length PIP clones were obtained, which were sorted in two families based on sequence analysis: Vilis vinifera PIPI A and VvPIPIB. Both families belong to the PIPI group, which, in contrast to the PIP2 group, does not possess a phosphorylation site in the C terminal part ofthe sequence.
PIPIA (Fig. 10.3) and PIPIB exhibit 76 % identity at the nucleotide level, and 88%
at the protein level. The molecular mass is 30 690 Da; the pI is 8. I The amino acids which differ to those in PIPlb (Mw 30745 Da) are shown in pink. There are signature sequences for MIP, and for aquaporins. Computer analysis permits a prediction of sev- eral types of potential phosphorylation sites.
A phylogenetic analysis conducted with the DNAstar program suggests that both VvPIPIA and B are closely related to aquaporins cloned from the ice plant Mesernbryan- thernurn crystallin urn (Picaud et af., in preparation). The ice plant is able to respond to salt stress and drought by a switch from C3 to CAM metabolism, and by the induction of various genes including MIPs that are expressed in the root tips (Yamada et af., 1995).
As grapevine is also resistant to water stress, this phylogenetic relationship may pose the question of whether these aquaporins are specifically involved in water-stress resistance.
To functionally characterise the clones obtained in this study, heterologous expression in Xenopus oocyte was perfonned. Both clones were transferred in the pGEM4ZT7Ts vector (Abrami et al., 1994) allowing expression in Xenopus. After in vitro transcription, capping with methyl guanosine, and addition of a polyA tail, the resulting cRNA were injected into the oocyte. Control oocytes were injected with an equivalent amount of water. Three days later, the oocytes were placed into an hypoosmotic medium, and their swelling was monito-
WATER TRANSPORT AND AQUAPORlNS IN GRAPE
A ... '"111.~~
o C_i'KC...,.~ . .
o C_~L.&wIlICC. ~bt . . "..,
~PKA~ ... k
C _ _ n~t~'lte
255
Figure 10.3. Deduced amino acid sequence and membrane topoplogy of VvPIP1A, a plasma membrane aquaporin cloned from grape berries. Remarkable sites deduced from computer analysis (http://www.expasy.ch/cgi-bin) are reported.
red. VvPIP 1 A injection resulted in a faster swelling than the control, due to the presence of water channels encoded by this clone. The water penneability coefficient was about 2- 3 fold higher than that of the control, which is not very high. It has been reported that aquaporins from the PIPI family increase the membrane penneability of oocytes in a much less efficient way than PIP2 members (up to 20-fold for PIP2, only 2-3 fold for PIP1) (Daniels et aI., 1994; Yamada et at., 1995; Weig et ai., 1997; Biela et al., 1999;
Chaumont et aI., 2000). This suggests that this clone encodes a water channel, but other substrates might be transported and should be tested. Different PIPIB clones have been obtained which differ only by the length and sequence of the 3' non coding regions and the length of the polyA tail. This is relatively common for plant transcripts and may af- fect the stability ofmRNA.
The expression of PIPIA and lB has been studied by northern analysis in different or- gans of the vine, and in the berries at different stages of ripening. Both VvPIPs are most strongly expressed in the berries, and somewhat in the roots. In the berries, VvPIPIA was strongly and transiently induced at the time of veraison and a smaller peak appeared at mid maturation. In contrast, VvPIPlB expression showed little variation during the course of berry ripening (Picaud et al., in preparation). The pattern of expression of the two PIPs was compared with that of several sugar transporters cloned from the same material (Fillion et aI., 1999; Ageorges et al., 2000). PIPIA was induced at the same time as the sucrose trans- porters SUT I, SUT2 and to a lesser extent Vvht J, a hexose transporter.