Raphaël Morillon

Plasma Membrane Aquaporins Play a Significant Role during Recovery from Water Deficit

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.

A PIP1 Aquaporin Contributes to Hydrostatic Pressure-Induced Water Transport in Both the Root and Rosette of Arabidopsis

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (Kros) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased Kros by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of Kros by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11–18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of Kros and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells

The transpiration stream that passes through a plant may follow an apoplastic route, with low resistance to flow, or a cell-to-cell route, in which cellular membranes impede water flow. However, passage of water through membranes can be facilitated by aquaporins thereby decreasing resistance. We investigated the relationship between transpiration, which can be down-regulated by abscisic acid (ABA) or by high humidity, and the osmotic water permeability (Pos) of protoplasts. By using leaf protoplasts of wild-type (wt) Arabidopsis thaliana plants and of mutants that are low in ABA (aba1) or insensitive to ABA (abi1 and abi2), we found that protoplasts from aba1 and abi mutants have very low Pos values compared with those from wt plants when the plants are grown at 45% relative humidity. High values of Pos were found 3 h after the addition of ABA to the culture medium of aba1 plants; addition of ABA to abi plants did not restore the Pos to wt levels. There was no such increase in Pos when excised leaves of aba1 plants were treated with ABA. When the transpiration stream was attenuated by growing the plants at 85% relative humidity, the Pos of protoplasts from all plants (wt and mutants) was higher. We suggest that attenuation of the transpiration stream in whole plants is required for the up-regulation of the Pos of the membranes, and that this up-regulation, which does not require ABA, is mediated by the activation of aquaporins in the plasma membrane.

Plasma Membrane Aquaporins Are Involved in Winter Embolism Recovery in Walnut Tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.

Brassinolide may control aquaporin activities in Arabidopsis thaliana

Abstract.u2002It is usually assumed that aquaporins present in the cellular membranes could be an important route in the control of water flux in plants, but evidence for this hypothesis is scarce. In this paper, we report measurements of the osmotic permeability (Pos) of protoplasts isolated from hypocotyls of wild-type and mutant Arabidopsis thaliana (L.) Heynh. Mutants were affected in their growth and exhibited different sensitivities to the phytohormone, brassinolide. For the two mutants studied (cpd: constitutive photomorphogenesis and dwarfism; bri1: brassinosteroid insensitive), hypocotyl length was correlated to Pos for the protoplasts. Under experimental conditions where hypocotyl growth had ceased, restoration of root, hypocotyl and petiole growth by brassinolide was correlated with an increase in Pos of the hypocotyl protoplasts. We consider that the increase in Pos of the hypocotyl cells was needed because these cells were part of the transcellular water pathway of the plant. This is the first time, to our knowledge, that brassinolide has been shown to be involved in the modification of the water-transport properties of cell membranes. Our results also emphasize the importance of aquaporins and the transcellular pathway in water transport under normal growth conditions.

Osmotic water permeability of isolated vacuoles

Abstract. We measured the osmotic water permeability (Pos) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, Pos values were in the range 200–1000u2009μmu2009s−1. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar Pos values for red beet and onion were reduced by 1u2009mM HgCl2, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5u2009mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl2-treated onion vacuoles, the reduced Pos (56u2009μmu2009s−1) had a low activation energy (approx. 6u2009kJu2009mol−1), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low.

Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts

Abstract. The effect of low water potentials on root growth of flax (Linum usitatissimum L. cv. Ariane), rape (Brassica napus L. de Candolle, cv. Bristol), hard wheat (Triticum turgidum L. cv. Cham1) and soft wheat (Triticum aestivum L. cv. Ritmo) was studied by measuring the osmotic water permeability (Pos) of root protoplasts and the protein abundance of PIP1 and PIP2 aquaporins. These different species require more or less water, the most sensitive to water deficit being flax and rape. Ritmo, is a cultivar of wheat adapted to temperate zones, while the other cultivar Cham1 is adapted to low-rainfall areas. The seedlings were germinated and grown in water, salt or sugar solutions at different water potentials. The values of Pos for flax, rape and Cham1 wheat were normally distributed and could be characterized by mean ± SD. Root protoplasts from water-grown seedlings had Pos values of 485±159xa0µm s–1 (flax), 582±100xa0µm s–1 (rape), and 6.3±3.5xa0µm s–1 (Cham1). At the same age, the protoplasts from Ritmo exhibited a much wider range of values than the protoplasts of Cham1. When seedlings were grown under conditions of osmotic or salt stress, the mass of the roots was reduced for all species. With 0.25xa0mol kg–1 sorbitol or 0.125xa0M NaCl, the Pos for flax, rape and Cham1 remained constant or slightly increased, while for Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos. Only Cham1 and Ritmo were able to germinate at a lower potential (0.5xa0mol kg–1 sorbitol). For Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos when grown in this stress condition and both wheats exhibited low Pos values. The expression of the PIP1 and PIP2 aquaporins families was also studied by immunoblotting. We did not observe any difference in protein expression for any of the species, whatever the growing conditions. We suggest that the high Pos values for flax and rape could play a role in the sensitivity of these plants at low water potential. The low native Pos for Cham1 in spite of the expression of both families of aquaporins may reflect its adaptation to low-rainfall conditions by a functional regulation of the water channels. For a similar reason, the low-water-potential-induced Pos of Ritmo may also correspond to a down-regulation of the aquaporins, reflecting adaptation of this wheat to water-deficit conditions.

Modulation of Aquaporin Gene Expression in Arabidopsis Leads to Altered Membrane Water Permeability

A single plant contains at least thirty different MIP genes, most of which appear to be expressed under normal growth conditions. The expression of some may be linked to specific physical stresses such as water deficit. The proteins are found in the tonoplast (vacuolar membrane) and the plasma membrane. It is not known whether all these genes encode aquaporins (or aqua-glyceroporins) because the activities of many have not yet been tested. On a phylogenetic tree we can distinguish the tonoplast aquaporins (TIPs) from the plasma membrane aquaporins (PIPs) and among the latter there are different sub-families. The activity of putative aquaporins has been tested almost exclusively in heterologous systems, primarily in Xenopus oocytes. In planta function must be inferred from such tests. TIPs appear to always be very active when expressed in oocytes, and among the PIPs, the PIP2 family is active, but the PIP1 family is largely inactive. This inactivity is not caused by the failure of these proteins to be transported to the plasma membrane (see Chaumont et al., this volume) but could be due to the inability of oocytes to activate PIPs. Such findings point up the need to assay aquaporin activity in planta. Because plants have so many aquaporin genes, single knockouts are probably not a particularly effective way to understand function. However, using an antisense construct, it is possible to downregulate an entire family of sequence related genes.