The Role of ABA and the Transpiration Stream in the Regulation of the Osmotic Water Permeability of Leaf Cells

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

Raphae¨ l Morillon and Maarten J. Chrispeels*

Division of Biology, University of California at San Diego, La Jolla, CA 92093-0116

Contributed by Maarten J. Chrispeels, September 5, 2001 The transpiration stream that passes through a plant may follow an pressed PIPs are in the PIP1 and PIP2 subfamilies. These apoplastic route, with low resistance to ﬂow, or a cell-to-cell route, subgroups also have a conserved phosphorylation site that may in which cellular membranes impede water ﬂow. However, pas- be a target for regulation of aquaporin activity and overall sage of water through membranes can be facilitated by aquaporins plasma membrane hydraulic conductivity (7, 8). thereby decreasing resistance. We investigated the relationship In the study presented here we questioned how the magnitude between transpiration, which can be down-regulated by abscisic of the transpiration stream affects the hydraulic conductivity of acid (ABA) or by high humidity, and the osmotic water permeability the membranes. The transpiration stream can be modulated by (Pos) of protoplasts. By using leaf protoplasts of wild-type (wt) changing the relative humidity (RH) of the atmosphere or by the Arabidopsis thaliana plants and of mutants that are low in ABA application of the hormone abscisic acid (ABA), which causes (aba1) or insensitive to ABA (abi1 and abi2), we found that stomatal closure. For plants growing at 20°C, the transpiration protoplasts from aba1 and abi mutants have very low Pos values stream will decrease more than 80% when the RH is raised from compared with those from wt plants when the plants are grown at 45% to 85%. Plants sense this change in humidity and open their 45% relative humidity. High values of Pos were found 3 h after the stomates further (9). Although not all of the plants react in the addition of ABA to the culture medium of aba1 plants; addition of same way, the most common response consists of sensing of the ABA to abi plants did not restore the Pos to wt levels. There was no stomatal transpiration rate through the guard cells (10). Other such increase in Pos when excised leaves of aba1 plants were mechanisms, such as cuticular transpiration (11, 12) or leaf water treated with ABA. When the transpiration stream was attenuated potential (12, 13), could also be involved in sensing RH. All of by growing the plants at 85% relative humidity, the Pos of proto- these mechanisms find experimental support in the literature. plasts from all plants (wt and mutants) was higher. We suggest However, ABA is not involved in this sensing mechanism (14). that attenuation of the transpiration stream in whole plants is This is in contrast to its role in bringing about stomatal closure required for the up-regulation of the P of the membranes, and os in response to a decrease in soil water potential. that this up-regulation, which does not require ABA, is mediated by The synthesis of ABA is enhanced in response to soil water the activation of aquaporins in the plasma membrane. deficit, causing the rapid closure of stomates and thereby decreasing the intensity of the transpiration stream. In addition, lant growth requires a tradeoff between the need to acquire ABA, which is involved in long-distance signaling between root PCO2 for photosynthesis and the need to minimize water loss and shoot (15), may directly affect the hydraulic conductivity of from the leaves. Water lost through the transpiration stream is both root and shoot tissues. A number of studies carried out with replaced by water that is taken up from the soil. The force that decapitated root systems show that ABA applied to the roots drives this water uptake is the tension created by the evaporation increases the exudation volume from the cut stump of the stem of water from the leaf cells. Water that moves through living by increasing the hydraulic conductivity (Lr) of the root system tissues can follow an apoplastic or cell-to-cell path. Careful about 2-fold. ABA thus functions to allow faster uptake of water measurements of the hydraulic properties of plant tissues and by the roots and better transport of water within the plant organs show that the relative contribution of each pathway to (16–18). This effect of ABA appears to be rapid (within 30 min) overall hydraulic conductivity may change substantially depend- and long lasting (17). ing on the intensity of water flow and other factors (1). When the ABA-deficient mutants are very useful for investigating the rate of water flow is high (open stomates, low relative humidity), role of this hormone in plant water relations. The cut stump of water flows along the apoplast and around the protoplasts a plant of the wilty tomato mutant flacca has an exudation rate because the apoplastic path has the lower hydraulic resistance. that is only half that of a control plant (19). By using this same Conversely, when the rate of water flow is small, a larger mutant, Bradford (20) showed that spraying the shoot with ABA proportion of water follows the cell-to-cell path and needs to pass not only caused stomatal closure, but also raised the hydraulic through cellular membranes (and possibly through plasmodes- conductivity of the root system. mata). Water flow along this path has a much higher hydraulic Application of the cell pressure probe allows a direct approach resistance and can be modulated by aquaporins, proteins that to studying the effect of ABA on transmembrane water trans- facilitate transmembrane water transport (2–4). An important et al. unanswered question is whether the magnitude of flow in the port. With this technique, Hose (21) investigated the effect apoplastic path affects the resistance in the cell-to-cell path. of ABA on the hydraulic conductivity of maize roots and cortex In plants, aquaporins form a family of 35–37 sequence-related cells. They found that ABA facilitates the cell-to-cell component proteins (5, 6) that transport water and neutral solutes, such as of water transport across the root cylinder. The effects on glycerol, across membranes. Aquaporins have been found in the tonoplast (vacuolar membrane) and the plasma membrane, and Abbreviations: ABA, abscisic acid; PIP, plasma membrane intrinsic protein; Pos, osmotic many among them can increase the Pos of the Xenopus laevis water permeability; RH, relative humidity; wt, wild type. oocyte plasma membrane by 10- to 20-fold. Sequence compar- *To whom reprint requests should be addressed. E-mail: [email protected]. isons show that they fall into four different clades, and the The publication costs of this article were defrayed in part by page charge payment. This plasma membrane intrinsic protein (PIP) clade has about a article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. dozen members. The most abundantly and ubiquitously ex- §1734 solely to indicate this fact.

14138–14143 ͉ PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.231471998 Downloaded by guest on October 2, 2021 cellular hydraulic conductivity were large (7- to 27-fold) but mis stuck to the glass. Then, under the microscope, stomatal highly transient and postulated to be mediated by aquaporins. apertures were recorded with a video recorder for 10 min, To understand how the transpiration stream affects the hy- allowing the image capture of more than 100 stomata. Measure- draulic conductivity of cellular membranes, we used the tech- ments of the stomatal apertures were performed subsequently nique of Ramahaleo et al. (22) to measure the Pos of leaf on the filmed images. In each case more than 100 measure- protoplasts derived from wild-type (wt) Arabidopsis ecotype ments were performed. Landsberg erecta (Ler) plants and from mutants that are im- paired in ABA synthesis (aba1) or are insensitive to ABA (abi1 Preparation of Protoplasts and Pos Measurements. Leaf protoplast and abi2) (23, 24). In these mutants, the stomates are wide open. preparation and osmotic water permeability measurements on As a result, the apoplastic water flow, with its low hydraulic isolated protoplasts were performed as described (22). resistance, dominates the water transport path. What happens to To determine whether ABA affects the permeability of pro- the permeability of the membranes in these plants? Our results toplasts, Pos was measured on leaf protoplasts from aba1 plants. show that attenuating the transpiration stream by closing the The protoplasts were incubated for 10 min with 1 or 10 ␮MABA stomates with ABA or increasing the RH causes an increase in in the measuring solutions. the Pos of the protoplasts. Aba1 and abi mutant plants have very Ϯ low Pos values compared with wt. When ABA was added to the Statistical Analysis. The Pos values are given as the mean SD or growth medium of the aba1 plants, stomatal closure preceded a as frequency histograms. We used SIGMASTAT from SPSS (Chi- ͞ ͞ dramatic increase in Pos of the leaf protoplasts up to the levels cago; www.spss.com software science) to analyze the data. The found in wt plants. ABA had no effect on the Pos of protoplasts t test and ANOVA test were used to detect differences between isolated from excised leaves that were incubated in ABA solu- the lines and the growing conditions at the usual probability level ϭ tion. We conclude that leaves regulate the Pos of the plasma of P 0.05. membrane in response to the magnitude of the transpiration stream. Results aba and abi Mutants of Arabidopsis Have Plasma Membranes with a Materials and Methods Low Pos. To find out whether mutant plants that are defective in Plant Material and Growing Conditions. Throughout this study, ABA synthesis or ABA perception have an altered Pos of the Arabidopsis thaliana ecotype Ler was used as the wt. Plants were plasma membrane, we applied the method of Ramahaleo et al.

grown either on a soil–peat mixture (J.M. McConkey and Co., (22) to leaf protoplasts obtained from wt, aba1, abi1, and abi2 PLANT BIOLOGY Sumner, WA) during 4–5 weeks in a growth room (20°C Ϯ 1°C) plants. aba1 mutants have very low levels of ABA, and the wilty with a continuous light and a RH of 45 Ϯ 5% or in hydroponic phenotype of the mutant can be restored by ABA treatment (23, system, as described (25). The Magenta boxes were covered with 24), whereas abi mutants have defects in ABA perception and a vented lid allowing gas exchange and were autoclaved before stomatal closure (24, 28). We generally determined the Pos of 40 use. The plants were grown in the same room as the plants grown protoplasts obtained from 4–5 different protoplast preparations, in soil. The effect of RH was studied as follows. Ten days after with each preparation being made from the fully expanded the seeds germinated, the lids of the boxes were removed. During leaves of 3–4 plants. Fig. 1A shows the change in volume the next 4 weeks, the plants grown without lids at the RH of the (shrinkage) for three different leaf protoplasts transferred from growth room (45%), and some water was added every week to a solution of 0.4 mol⅐kgϪ1 to 0.6 mol⅐kgϪ1 sorbitol. Each prep- maintain a constant level. To study plant growth at high RH, the aration usually has protoplasts with a range of Pos values, and this lids were replaced on the boxes (after 4 weeks) to maintain the range of values is reproducible from one experiment to the next. RH at 85%. The RH was measured with a recording thermo– The range of Pos values obtained for protoplasts derived from wt hygro pen (Fisher Scientific) placed inside the box. The leaf plants is shown in Fig. 1B Upper. The histogram shows two temperature and box temperature were recorded with a ther- subpopulations with half the protoplasts in the 1.25–10 ␮m⅐sϪ1 mometer with two thermocouples (Doric Scientific, San Diego). range and the other half in the 20–540 ␮m⅐sϪ1 range. Experi- The temperature was recorded several times during a week. ments in which the Arrhenius energy of activation for cell swelling was measured (22, 29, 30) indicate that values around ABA Treatment of Whole Plants and Excised Leaves. The control and 5–15 ␮m⅐sϪ1 for plant membranes correspond to an energy of ABA treatments were performed by transferring the pots for 3 activation of 50–70 kJ. Such a high value for the energy of or 24 h to a basin containing water or 1 ␮M ABA (Sigma) activation of water transport corresponds to a situation where solution buffered with 10 mM Mes͞Tris at pH 5.5 (21). there are no water channels, or the water channels are not in Excised leaves were incubated under vacuum in 1 ␮MABA operation. ͞ solution, 10 mM Mes Tris, pH 5.5, for 1 or3hinaPetri dish In the aba1 and abi mutants, the distribution of Pos values was before measurement of the size of the stomatal pore or the much narrower with essentially all of the plants in the 1.25–40 ␮ ⅐ Ϫ1 preparation of protoplasts. m s range. The mean Pos value of protoplasts of the aba1 plants (6 ␮m⅐sϪ1) is only one-tenth of that of the protoplasts from Isolation of Protein Fractions and Immunodetection. For each gel the wt plants (69 ␮m⅐sϪ1), whereas the abi mutant protoplasts lane, the same amount of protein (about 30 ␮g) was loaded. The have a mean value of about one-fifth (15 ␮m⅐sϪ1)ofwt. proteins were denatured with 1% SDS and 100 mM ethanedi- Statistical analysis using Dunn’s test for ANOVA shows that the thiol at 40°C. The immunodetection of PIP1 and PIP2 proteins Pos values of the protoplasts from the mutant plants are signif- was performed as described in Daniels et al. (26) by using, icantly different (P ϭ 0.05) from the wt values (Table 1). The low respectively, a serum against the amino acid sequence (KSLGS- Pos values in the mutants indicate that plasma membrane aqua- FRSAANV) found in PIP2 aquaporins or a serum against the N porins may be inactive in most protoplasts. terminus of PIP1a aquaporins (27). ABA Treatment of Intact aba1 Plants Increases the Pos of Protoplasts. Measurements of Stomatal Aperture. Two leaves of each line were To understand how increasing the level of ABA affects the Pos cut and the lower surface was stuck to a microscope slide with values, we placed the pots for 24 h in a basin containing a solution a medical adhesive (Hollister, Libertyville, IL). Three repeats of 1 ␮M ABA. First, we determined the effect of ABA on from three different plants were done in each case. After 1 min, stomatal aperture (Fig. 1C). Under well-watered conditions, the leaf was peeled away under water, leaving the lower epider- stomatal pore size was about 3 ␮m for wt Ler plants. The

Morillon and Chrispeels PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 14139 Downloaded by guest on October 2, 2021 Fig. 2. The effect of3hofABAtreatment on stomatal aperture and Pos values of leaf protoplasts from wt and mutant Arabidopsis plants. (A) Mean stomatal aperture of well-watered plants and plants treated for 3 h with 1 ␮M Fig. 1. The effect of 24-h ABA treatment on stomatal aperture and Pos values Ϯ of leaf protoplasts from Arabidopsis wt and mutant plants. (A) Time courses ABA. The aperture is given as the mean SD. (B) Mean Pos values of leaf protoplasts of Arabidopsis wt and mutants. In each case, the P value of 20 showing rates of shrinkage and calculated Pos values for three different os Arabidopsis leaf protoplasts. The initial diameters of the protoplasts were protoplasts was measured from three preparations. Bars with the same sign Ϫ1 Ϫ1 are not significantly different (Dunn’s test) and the results are presented as the about 30 ␮m in each case (OP1 ϭ 0.4 mol⅐kg ;OP2 ϭ 0.6 mol⅐kg ). (B) mean Ϯ SD. (C) Immunoblot analysis of microsomal extracts from Arabidopsis Histograms of Pos values. Measurements were performed on leaf protoplasts from the wt and the mutants, aba1, abi1, and abi2. Leaves of 4-week-old leaves of the wt and mutants using anti-PIP1 serum (Upper) and anti-PIP2 plants were harvested and 4–5 different preparations of protoplasts were serum (Lower). The minus signs correspond to a water treatment and the plus sign to a 3-h ABA treatment. Equal amounts of leaf microsomal protein (40 ␮g) used for each condition. The Pos of 40 protoplasts was measured for each line and in each condition. (C) Mean stomatal aperture of well-watered plants and were loaded in each lane. The arrow indicates the position of the protein. The plants treated during 24 h with 1 ␮M ABA. Bars represent the mean Ϯ SD. (D) numbers on the right indicate the molecular mass standards. Pos values of protoplasts from plants treated with 1 ␮M ABA for 24 h. (Fig. 1D), with more protoplasts in the 20 to 640 ␮m⅐sϪ1 range. ␮ ⅐ Ϫ1 stomates of the mutants were more widely open (4–5 ␮m). This The mean value of the wt plants went from 69 m s to 126 ␮ ⅐ Ϫ1 conforms to expectations for plants that have low levels of ABA m s as a result of the addition of ABA, and the value for aba1 ␮ ⅐ Ϫ1 ␮ ⅐ Ϫ1 (aba1) or are deficient in ABA sensing (abi). Addition of ABA plants went from 6 m s to 67 m s . A statistical analysis of the effect of ABA on wt or aba1 plants in a pairwise comparison caused closure of the stomates in the wt and aba1 plants, but the Ͻ abi plants were, as expected, much less sensitive to ABA and the shows that the effect of ABA is significant (t test; P 0.05). In stomates remained open (28). the abi1 and abi2 plants there was no shift in the histogram The 24-h treatment of the wt and aba1 mutant plants with (compare Fig. 1 B and D) and differences in the mean values were not significant (t test; P Ͼ 0.05). We interpret these findings ABA pushed the histogram of protoplast Pos values to the right to mean that ABA presence or perception is necessary to maintain protoplasts in a high Pos state, and that this effect is ؊1 Table 1. Statistical analysis of Pos values (␮m⅐s ) of leaf most likely mediated by aquaporins. protoplasts from the wt and aba mutants A 3-h ABA Treatment Is Sufficient to Raise the Pos. ABA could exert ABA treatment Mutant lines Mean SD Median Dunn’s test its effect on plasma membrane Pos at the level of aquaporin Ϫ Wt 69 116 9.5 A activity, aquaporin abundance, and͞or gene expression. We Ϫ aba1–1 6 4 4.6 B repeated the entire experiment3haftertheaddition of ABA to Ϫ abi1 14 32 5.0 B the growth medium and the results were almost identical. The Ϫ abi2 14 39 4.9 B effect of ABA on stomatal opening is shown in Fig. 2A. Once ϩ Wt 126 132 96.0 C again, ABA induced rapid stomatal closure in wt and aba1 ϩ aba1–1 67 129 11.0 C plants, but not in abi plants. Fig. 2B shows mean Pos values rather ϩ abi1 12 23 6.0 D than histograms as in Fig. 1. It is clear that ABA addition 3 h ϩ abi2 26 56 6.6 D before protoplast preparation was sufficient to raise the Pos of The data are extracted from the histograms in Fig. 1. For each condition, a the aba1 protoplasts to wt levels. Fig. 2C shows the effect of ABA Dunn’s test ANOVA was carried out separately and used to detect significant on the abundance of two major classes of plasma membrane variation: the same letter indicates no significant difference (P ϭ 0.05). Forty aquaporins (PIP1 and PIP2) by using an immunoblot and two protoplasts were measured for each line in each condition. different and specific antisera. Equal amounts (40 ␮g) of mi-

14140 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.231471998 Morillon and Chrispeels Downloaded by guest on October 2, 2021 Fig. 4. Histograms of Pos values from leaf protoplasts of plants grown in hydroponics respectively at 45% and 85% of RH. Leaves of 5-week-old plants were harvested, and 2–3 different preparations of protoplasts were used. The Pos of 20 protoplasts was measured for each line and in each condition. Bars with the same sign are not signiﬁcantly different (Dunn’s test), and the results are presented as the mean Ϯ SD.

The Role of RH in Changing the Pos of the Protoplasts. To find out whether a change in the transpiration stream might affect the Fig. 3. ABA and membrane permeability. (A) The effect of treating aba1 permeability of the cells, we switched to a hydroponic system in plants for 1, 2, and 3 h with ABA on stomatal aperture and Pos values of leaf protoplasts. Details are as in Fig. 2. (B) The effect of 1 or3hofABAtreatment which single plants were floated on rafts in Magenta boxes either of excised leaves from the wt and aba1 mutant on stomatal aperture and Pos without a lid (45% humidity) or with a loose lid (85% humidity). values of leaf protoplasts. The aperture of the stomates of the plants grown at the higher humidity was significantly greater for the Landsberg ecotype (usually around 4.5 ␮m) when compared with the aperture of the crosomal protein were loaded in each lane. The results show that plants grown at 45% RH (usually around 3.5 ␮m), indicating that PLANT BIOLOGY the mutant plants may have somewhat lower levels of the the plants responded to this change in humidity. No significant aquaporins (the bands appear less intense), but that3hofABA difference in stomatal aperture was observed when the aba1 or treatment did not alter those levels. These results strongly abi mutants or the wt plants were grown at different RH values suggest that the effect of ABA must be at the level of aquaporin (data not shown). Measurements of leaf temperature with a activity. thermocouple of plants growing in a box without a lid or with a lid showed the temperatures to be the same: 20°C Ϯ 1°C. The Effect of ABA Is Seen Only in Whole Plants, Not in Excised Leaves. Determination of the Pos values revealed striking differences in An experiment was also conducted to determine the time course the Pos of the protoplasts, depending on the RH. The data are of the effect of ABA on stomatal closure and protoplast Pos. The presented in Fig. 4. The histograms clearly show that, in all plants apperture of the stomatal pores was examined 1, 2, and3hafter examined, raising the RH to 85% increased the proportion of ABA treatment of the plants. At these times we also prepared protoplasts with a high Pos value significantly for the aba and abi protoplasts from the leaves and determined their Pos. The results mutants. Thus, attenuation of the transpiration stream by raising in Fig. 3A show that after2hofABAtreatment the stomates the RH increased the Pos of the protoplasts of the mutant plants. began to close, but the change in Pos had not yet taken place. Thus, attenuating the transpiration stream comes before the Pos Discussion change. Nevertheless, the effect of ABA may be direct and According to the composite model of water transport, developed require more time. by Steudle and his collaborators over the last 10 years (1), water To find out whether ABA can have a direct effect on leaf cells, transport through the plant can follow either an apoplastic path as it does on cells of excised root systems, we tested its effect on or a cell-tocell path. How much water travels along each pathway excised leaves. Leaves from control and aba1 plants were depends on the magnitude of the total water flow and especially incubated with ABA for 1 or3haftervacuum infiltration to on the nature of the driving forces. When the stomatal pores are facilitate entrance of the hormone into the leaf, before proto- open and there are strong pressure gradients because of evap- plast preparation. An examination of the stomatal apertures orative demand, water flows largely around the protoplasts showed that1hofABAtreatment was sufficient to close the because this is the path of least hydraulic resistance. In the stomates. However, there was no increase in the Pos of the absence of transpiration, water moves cell-to-cell by osmotic protoplasts prepared from the leaves either after 1 or3hofABA forces through semipermeable membranes; membranes provide treatment (t test, P Ͼ 0.05). These results are not consistent with a variable resistance to water flow depending on the abundance a direct effect of ABA on the cells, though we cannot rule out and activity of aquaporins or water channel proteins. Steudle (1) that the ABA had a direct effect on the guard cells but not on postulated that the two pathways interact with each other and the mesophyll, which yields most of the protoplasts. We suggest that such interactions are responsible for regulating the circu- that attenuation of the transpiration stream is an important lation of water in the plant. The results presented here support component of the up-regulation of Pos by ABA. We also tested such an interaction: when the stomates were wide open, as in the the effect of ABA directly on protoplasts of aba1 mutant plants. aba1 and abi plants, the Pos of the protoplasts was found to be Incubation for 10 min with ABA did not increase the Pos of the very low, much lower than in control plants that had partially protoplasts (data not shown). Although 10 min is a brief opened stomates; when the stomates were closed because ABA exposure, it should be sufficient for the hormone to enter the had been added to the growth medium, the Pos increased more cells and trigger an activation pathway. For example, a 5-min than 10-fold. The relationship between apoplastic flow, cell-to- exposure of guard cells to ABA is sufficient to dramatically cell flow, stomatal aperture, and ABA is illustrated in Fig. 5A. enhance potassium-channel currents (31). The effect of ABA is to dramatically reduce the apoplastic flow

Morillon and Chrispeels PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 14141 Downloaded by guest on October 2, 2021 of chilling sensitive lines. A direct effect of ABA on aquaporins in these maize plants was postulated on the basis of experiments in which HgCl2 was used to shut down plasma membrane aquaporins (36). An elegant recent study by Hose et al. (21) that used cell and root pressure probes on maize roots led to the conclusion that ABA added to excised root systems increased the root hydraulic conductivity by 3- to 4-fold and the cell conductivity by 7- to 27-fold. The effect on cellular hydraulic conductivity was transient, lasting only an hour. The authors concluded that ABA acts at the plasma membrane presumably by activating aquaporins. This would facilitate the uptake of water by the roots under nontranspiring conditions when the apoplastic pathway of water transport is minimal and the cell-to-cell pathway needs to be maximized. In all of these experiments, ABA was applied to excised root systems, eliminating the possibility that the effect is mediated by stomatal closure and a reduction in the transpiration stream. Surprisingly, mutants of maize, or Arabidopsis that contain very low levels of ABA or are insensitive to ABA, have not yet been used for hydraulic conductivity measurements on excised root systems. We were able to dissociate the effect of ABA on Pos of leaf cells from its effect on stomatal closure, by examining these parameters 1, 2, and3haftertheaddition of ABA to the medium. We found that after 2 h the stomates began to close, but the Pos of the membranes had not yet changed. This means that closing of the stomates may be a necessary condition, though it may not be sufficient to change the Pos; ABA may also have a Fig. 5. A model of water flux in a leaf depending of the intensity of the function in Pos regulation. By applying ABA to excised immersed transpiration stream. (A) In the well-watered condition without ABA, the leaves, we found that ABA caused stomatal closure, as expected, water flux is large in the wt and even larger in the mutant plants; the Pos of the but did not result in a change in the Pos of the membranes. We protoplasts from the mesophyll of the mutant plants is low. ABA treatment of presume thata3hofABAtreatment would be sufficient, as wt and the aba1 mutant causes closure of the stomates, which leads to a ABA brings about stomatal closure in 1 h. ABA, in the absence decrease of the transpiration stream and an activation of the aquaporins in of a transpiration stream, does not appear to be sufficient to the plasma membranes of the mesophyll cells. (B) In hydroponics when the RH promote the increase in Pos of leaf cells. This surprising result is low (45%), the water flux is large in the wt and even larger in the mutant contrasts with the already mentioned direct increase of the plants; the Pos of the protoplasts from the mesophyll of the mutant plants is hydraulic conductivity induced by ABA on the roots of decap- low (same as above). An increase of the RH to 85% leads to a decrease of the transpiration stream and strong increase of the water permeability of the itated plants, independently of the transpiration stream. We mesophyll cells for all of the lines. suggest that both a change in the transpiration stream and ABA are needed to alter the Pos of the membranes of the leaf cells. High humidity also attenuates the transpiration stream by and to increase the cell-to-cell flow. Does ABA play a direct role decreasing the vapor pressure deficit. We performed Pos mea- in this modification of the Pos or is its effect indirect and surement on protoplasts from plants grown in hydroponics at mediated by stomatal closure and the attenuation of the tran- different humidities. Leaf protoplasts from wt plants grown at scription stream? 85% RH had similar Pos values compared with those of plants grown at 45% RH. However, in the aba1 and abi mutants, raising Is the Effect of ABA Direct or Indirect? Most of the literature of the the humidity to 85% also raised the Pos to well above control role of ABA in the water economy of plants deals with the role values. Because the relative humidity directly depends on the of ABA in stomatal closure. When roots experience water temperature, we measured the leaf temperature and the temperature in the box with thermocouples. In both conditions, the deficit, but the leaves are still fully turgid, ABA moving up the Ϯ xylem causes the stomates to close through a collapse of the temperature was 20 1°C. Because the temperature was constant during the experiment, we may assume that the saturation osmotic pressure of the guard cells. This collapse is caused by the value of water vapor was also the same in both conditions. We efflux of potassium ions and anions as well as the conversion of can therefore assume that the transpiration water flux is deter- malate into starch (32) and by the efflux of water from the cells. mined mainly by the degree of stomatal aperture and by the Whether ABA directly regulates the Pos of guard cell plasma difference in vapor pressure between the stomatal chamber and membranes is not known. the air outside the leaf. If we assume that the RH at 20°Cinthe Both indirect and direct evidence indicates that ABA can have stomatal chamber is close to 100% (37) at both outside RH an effect on the aqueous permeability of the membranes of root values, the increase of RH corresponds to a 4-fold decrease in the cells. Thirty years ago, Tal and Imber (19) and Glinka and transpiration stream. Thus, there are two conditions that in- Reinhold (33) reported that the application of ABA to the root crease the Pos of the cells: decreasing the transpiration stream by medium of decapitated plants increases the exudation rate from closing the stomates with ABA or decreasing the transpiration the cut stump. Since that time, a number of studies have been stream by growing mutant plants that have low Pos at high RH. published on this subject (16, 17, 20, 34). Stress conditions such

as flooding, chilling, and salt cause a decrease in the hydraulic Why Should There Be an Increase in Pos of the Mesophyll Cells When conductivity of the root system that may be ameliorated by the Transpiration Stream Is Weak? Stomatal closure, whether it preadaptation or ABA treatment. For example, Perez de Juan et occurs at night or during the day as a result of water deficit, is al. (35) found that drought hardening of maize, which induces accompanied by the continued uptake of water from the soil and ABA synthesis, or ABA application, improved the water status the redistribution of water within the plant. As a result of these

14142 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.231471998 Morillon and Chrispeels Downloaded by guest on October 2, 2021 processes, the entire plant is fully rehydrated if sufficient water one involving ABA and one involving the sensing of the tran- is available in the soil. Furthermore, even when photosysnthesis spiration stream—may share common elements. The signal- is diminished because the stomates are closed, water needs to transduction pathway for stomatal closure has been analyzed in circulate between the phloem and the xylem to transport solutes. considerable detail (40, 41). Phosphorylation events are impor- We postulate that this cell-to-cell movement of water will be tant mediators of signaling in all cells and play a major role in accelerated by an increase in Pos when there is no transpiration ABA signaling in guard cells. Genes encoding protein phospha- stream, as illustrated in Fig. 5A. The lower osmotic pressure tases that regulate stomatal movement were identified several differences, which could be the result of a lowered rate of years ago (42). Guard cells contain an ABA-activated protein photosynthesis, would require higher Pos values to move water kinase (43). An ABA-activated serine–threonine kinase of guard cell-to-cell and support the high rate of xylem to phloem cells that is essential for stomatal closure was recently identified recycling that occurs in plants (38). Rehydratating a partially by Li et al. (44). Activation of anion channels by phosporylation dehydrated plant may be speeded up if the Pos of the cell mediated by this kinase is thought to result in stomatal closure. membranes is higher. Interestingly, phosphorylation of PIPs is regulated by apoplastic When the vapor pressure difference is high and the stomates water potential (7), and phosphorylation at a conserved site are open, the transpiration flux is maximum. In this condition, increased the activity of PM28A, a PIP2 aquaporin (8). This the Pos values of the leaf protoplasts are low, suggesting, as conserved phosphorylation site is in the cytoplasmic domain proposed by Steudle and Peterson (39) for the root, that the between the second and third transmembrane ␣-helices and is water flux in the plant is mainly apoplastic and does not involve situated close to the aqueous pore. Our results show that ABA a substantial symplastic or trans-cellular water flux. When the did not alter the abundance of PIP1 and PIP2 aquaporins—we vapor pressure difference is low, the transpiration flux is dra- define PIP2 aquaporins more narrowly than they are defined in matically decreased and the transcellular water flux becomes the most recent article of Johanson et al. (6)—but cannot, of much more significant (39). The significant increase of the Pos course, exclude that ABA causes an increase in PIPs not values at high RH we measured is in agreement with this recognized by our antibodies (like PIP 2.6, PIP 2.7, and PIP 2.8 hypothesis (Fig. 5B). In the case of the leaf, the increase of the in ref. 6). Genomics and proteomics approaches need to be water permeability of the cells would not allow to speed up the applied to these and other mutants to determine how gene rate of transpiration, but it could facilitate the recycling between expression and protein abundance change in this important xylem and phloem and other internal processes that require protein family and how this relates to the hydraulic permeability transcellular water transport. of cells and tissues. PLANT BIOLOGY

How Could ABA and Relative Humidity Exert Their Effects? The lack We thank Dr. Tony Schaeffner who generously provided the PIP1- of effect of ABA on the Pos of the protoplasts from the abi specific antiserum. This work was supported in part by the U.S. Depart- mutants indicates that the two signal-transduction pathways— ment of Agriculture Research Initiative Competitive Grants Program.

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