Circadian regulation of leaf hydraulic conductance in sunflower (Helianthus annuus L. cv Margot)

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2005? Original Article Plant, Cell and Environment (2005) 28, Circadian regulation of leaf hydraulic conductance A. Nardini et al. Circadian regulation of leaf hydraulic conductance in sunflower (Helianthus annuus L. cv Margot) ANDREA NARDINI, SEBASTIANO SALLEO & SERGIO ANDRI Dipartimento di Biologia, Università di Trieste, Via L. Giorgieri 10, Trieste, Italy ABSTRACT The circadian regulation of leaf hydraulic conductance (K leaf ) was investigated in Helianthus annuus L. (sunflower). K leaf was measured with an high pressure flow meter during the light and dark period from plants growing at a photoperiod of 12 h. K leaf was 4.0 e-4 kg s -1 m -2 MPa -1 during the light period (LL) and 30 40% less during the dark period (DL). When photoperiod was inverted and leaves were measured for K leaf at their subjective light or dark periods, K leaf adjusted to the new conditions requiring 48 h for increasing from dark to light values and 4 d for the opposite transition. Plants put in continuous dark showed K leaf oscillating from light to dark values in phase with their subjective photoperiod indicating that K leaf changes were induced by the circadian clock. Several cuts through the minor veins reduced leaf hydraulic resistance (R leaf ) of both LL and DL to the same value (1.0 e + 3 MPa m 2 s kg -1 ) that equalled the vascular resistance (R v ). The contribution of the non-vascular leaf resistance (R nv ) to R leaf was of 71.9% in DL and of 58.4% in LL. The dominant R nv was shown to be reversibly modulated by mercurials, suggesting that aquaporins play a role in diurnal changes of K leaf. Key-words: aquaporins; circadian clock; hydraulic conductance; leaf; light; mercury chloride; sunflower. INTRODUCTION Correspondence: Andrea Nardini. Fax: ; nardini@univ.trieste.it Hydraulic architecture is an important determinant of the water balance of a plant (Zimmermann 1983; Tyree & Ewers 1991) in that maximum gas exchange rates that can be sustained without causing excessive evaporative desiccation are closely related to the efficiency of water transport through the different plant organs (e.g. Sperry 2000; Meinzer 2002). On the basis of the Ohm s law hydraulic analogue (Tyree & Zimmermann 2002), the water transport capacity of plants can be adequately described and measured in terms of conductance to water in the liquid phase (hydraulic conductance, K) or in the gaseous phase (in the case of stomatal conductance). During the 1980s, most attention was focused upon mechanisms modifying the hydraulic conductance of plants (K plant ) through the cavitation-induced loss of conductance of xylem conduits (Sperry, Donnelly & Tyree 1988; Tyree & Sperry 1989) and, more recently, the conductance recovery due to conduit refilling (Salleo et al. 1996; Tyree et al. 1999). Since the above studies, the hydraulic conductance of xylem has been recognized to change on a both diurnal and seasonal time scale (Zwieniecki & Holbrook 1998; Nardini & Pitt 1999) causing corresponding changes in leaf water status and stomatal behaviour (Salleo et al. 2000; Brodribb & Holbrook 2004). Over the last decade, scientists have become more aware of large short- and long-term changes in K plant as due to changes in the K of roots, stems and leaves in response to changes in environmental and endogenous factors. As an example, xylem hydraulic conductance has been found to change due to temperature-dependent changes in water viscosity (Cochard et al. 2000; Matzner & Comstock 2001) or in response to modifications of the ionic strength of xylem sap that would change the porosity of the pit membranes through which water flows from one conduit to another (Zimmermann 1978; Van Ieperen, Van Meeteren & Van Gelder 2000; Zwieniecki, Melcher & Holbrook 2001). Increasing attention has also been paid to changes in the conductance to water of the non-vascular compartment of roots and leaves. In particular, the hydraulic resistance (R = 1/K) of the non-vascular pathway in the root (i.e. of the radial water path from soil to the stele) has been estimated to represent up to 80% of the total root resistance (Frensch & Steudle 1989; Amodeo et al. 1999). Similarly, water leaving xylem elements in the leaf veins has to move through or around several cells before reaching the substomatal cavity where it evaporates. The extra-vascular hydraulic resistance of a leaf has been estimated to build up 50 90% of the whole leaf resistance (Tyree & Cheung 1977; Tyree, Nardini & Salleo 2001; Trifilò et al. 2003; Cochard, Nardini & Coll 2004). Although there is no general consensus on this partitioning of R in a leaf (Zwieniecki et al. 2002; Sack, Streeter & Holbrook 2004), the hypothesis has been advanced (but not verified up to now) that changes in leaf hydraulic conductance may be the result of active processes occurring in the mesophyll living cells and including new expression or activation of aquaporins that can greatly enhance the water permeability of cell membranes (Eckert et al. 1999; Otto & Kaldenhoff 2000; Morillon & Chrispeels 2001). Light is one of the signals inducing stomatal opening in Blackwell Publishing Ltd

2 Circadian regulation of leaf hydraulic conductance 751 well-watered C 3 and C 4 plants (Willmer & Fricker 1996). Stomatal movements are known to be sensitive to the circadian clock (Gorton, Williams & Assmann 1993; Webb 2003) through a circadian oscillator probably located in the guard cells and different from the oscillators regulating other typical circadian behaviours of plants (Wilkins 1992; Webb 2003). Tsuda & Tyree (2000) have hypothesized that leaf hydraulic conductance (K leaf ) increases during the day in response to the increased water demand due to stomatal opening. Diurnal changes in K leaf have been measured in only a few species up to now and the underlying mechanisms are largely unknown, but some excellent papers have appeared in the literature showing the influence of aquaporins in determining the diurnal changes of the hydraulic conductance of roots (Martre et al. 2002; Siefritz et al. 2002). Henzler et al. (1999) have shown that the root hydraulic conductance (K root ) of Lotus japonicus changed over a five-fold range with a circadian rhythm and that this was due to the corresponding circadian new expression of a PIP1 aquaporin located in the endodermal and stelar cells, during the light period. Because leaves are the organs of plants most exposed to light and they control the water flow out of the plant through stomata, it is not unreasonable to hypothesize that their hydraulic conductance may be finely regulated by metabolism-dependent changes in the water permeability of the living cells possibly regulated by the circadian clock. Light-dependent, long-term changes of K leaf have been documented by Sack et al. (2003) who have reported that sun leaves of several woody species had significantly higher K leaf than shade ones. In addition, shortterm modifications of K leaf induced by irradiance have been reported by Sack et al. (2002) and by Tyree et al. (2005). The present study reports measurements of diurnal changes of K leaf on sunflower (Helianthus annuus L). This species was preferred because it is well known to have several circadian behaviours (e.g. Buda et al. 2003) the best known of which is the typical rotation of the inflorescence. Our main objectives were to investigate: (1) the possible circadian regulation of K leaf ; (2) the partitioning of R leaf between the vascular and the non-vascular leaf compartment; and (3) the prevailing mechanism, if any, adopted by sunflower for regulating the efficiency of water transport within the leaf blade. MATERIALS AND METHODS Plant material All experiments were undertaken on 4- to 5-week-old plants of Helianthus annuus L. cv. Margot (seeds were provided by Maisadour Sementi Italia SpA, Verona, Italy). Seeds were planted in greenhouse trays and after cotyledons were fully expanded, seedlings were transferred to 1.5 L pots filled with a mixture (1 : 1) of peat and sand (one seedling per pot). After 10 d, each plant received 7.5 g of fertilizer (Nitrophoska Top, BASF Italia SpA, Milan, Italy; 15% N, 10% P 2 O 5, 15% K 2 O, 2% MgO, 12% SO 3, 0.02% B, 0.01% Zn). Plants were grown in a room where air temperature was adjusted to vary between 25 and 20 C (day/night), relative humidity was set at 65 ± 5% and light was provided by lamps (HQI-T 1000 W/D; Osram GmbH, München, Germany) with a photosynthetically active radiation (PAR) of 400 ± 50 mmol m -2 s -1 as measured at the leaf surface using a quantum sensor (LI-190S-1; LiCor Inc., Lincoln, NE, USA). The photoperiod was set at 12 h except for experiments under constant dark conditions (see below). Plants were maintained well irrigated and each plant received 200 ml of water per day. All measurements were performed on mature leaves sampled between node 8 (node 1 bearing the cotyledons) and node 11. These leaves were chosen because a previous study by some of the present authors (Lo Gullo et al. 2004) had shown that apical leaves of mature sunflower plants have maximum hydraulic efficiency. Measuring leaf hydraulic conductance Leaf hydraulic conductance (K) was measured using a high pressure flow meter (HPFM; Tyree et al. 1995). Leaves were cut under distilled water leaving about 30 mm petiole for connection to the HPFM. They were immediately transported to the laboratory where they were connected to the HPFM using compression fittings, within 5 min from cutting. Leaves were kept immersed in a water bath during measurements. This procedure was aimed at stopping transpiration (Sack et al. 2002) while buffering eventual leaf temperature changes upon illumination (see below). Degassed water filtered at 0.1 mm was forced into the petiole at a pressure (P) of 0.15 MPa and K was measured (as the flow to pressure ratio) at 16 s intervals until values became stable (i.e. the coefficient of variation of the last 20 readings was less than 3%), which usually took about 20 min. At the end of each experiment, leaf surface area (A L, one side only) was measured using a leaf area meter (LI-3000 A, LiCor Inc.) and K was scaled by A L, thus obtaining the leaf specific hydraulic conductance (K leaf, kg s -1 m -2 MPa -1 ). Preliminary experiments were performed to check the effects of light conditions during HPFM measurements of K leaf (Sack et al. 2002) for leaves collected in the middle of their light period (Light leaves, LL) or in the middle of the dark period (Dark leaves, DL). K leaf was measured under normal laboratory irradiance (PAR < 10 mmol m -2 s -1, dark conditions) for 30 min. Then, leaves were illuminated using a fibre-optic light source (FL-460 Lighting Unit; Heinz Walz GmbH, Effeltrich, Germany) providing PAR = 400 mmol m -2 s -1 (i.e. the same PAR level at which plants were growing) as measured at the leaf surface using a quantum sensor (see above). K leaf was measured for 30 min under this irradiance. The light was then switched off and K leaf was recorded for 20 min more, under dark conditions. During measurements, the temperature of the water bath (T w ) was measured at 60 s intervals using a thermocouple thermometer (Digi-Sense, mod ; Cole Parmer Instrument Co., Vernon Hills, IL, USA) and all K leaf readings were corrected for temperature changes. T w did not change more than 2 C during measurements.

3 752 A. Nardini et al. As preliminary experiments had revealed that LL and DL had distinctly different K leaf when measured under a PAR < 10 mmol m -2 s -1, it was decided to perform all subsequent measurements under this irradiance level (corresponding to the usual laboratory irradiance). Dependence of K leaf on photoperiod In order to check whether the differences observed between LL and DL in terms of K leaf were dependent upon the actual light conditions at the time of sampling or they were due to a circadian regulation of leaf hydraulic properties, eventual rhythmic changes of K leaf were measured in plants subjected to the inversion of the photoperiod. The hydraulic conductance of leaves during their dark period (DL) was first measured for four consecutive days (four measurements per day, one leaf per plant). On the fourth day, the photoperiod was inverted and K leaf was measured for 5 d on samples collected in the middle of their actual light period (corresponding to their subjective dark period). Then, the photoperiod was inverted again and K leaf was measured for five more days in leaves collected in the middle of their actual dark period. Because circadian rhythms are typically maintained for some time even under constant environmental conditions (Webb 2003), a second experiment was designed in which sunflower plants habituated to a photoperiod of 12 h were put in continuous dark for 2 d. K leaf was first measured at 12 h intervals corresponding to the middle of their light and dark periods and then measured again after plants were put in continuous dark at the same time intervals (these times corresponding to the middle of the putative subjective photoperiod perceived by plants). Leaf conductance to water vapour (g L ) was also measured at 12 h intervals using a steady-state porometer (LI-1600; LiCor Inc.). K leaf and g L were measured on at least five leaves per time interval. Partitioning of leaf hydraulic resistance Experiments were designed to evaluate the partitioning of hydraulic resistance within the leaf blade of both LL and DL. To this purpose, five LL and five DL were measured before and after cutting increasing numbers of minor veins in order to by-pass the extra-vascular water pathway. The procedure was basically similar to that recently described by Sack et al. (2004). Minor veins were cut at random locations throughout the lamina by making mm incisions with a scalpel. In our case, however, a higher number of cuts was made, namely up to 500 versus as reported in the study by Sack and coworkers. In the case of sunflower, this resulted in about 3 4 cuts cm -2 leaf. Only veins of the fourth order or higher were cut open. We assumed that R leaf (= 1/K leaf ) measured after 500 cuts equals the hydraulic resistance of the leaf vascular system (R v ) after the non-vascular compartment (or the majority of it) has been by-passed. The hydraulic resistance of the extra-vascular compartment (R nv ) was calculated as R nv = R leaf - R v assuming vascular and extra-vascular compartments as hydraulic resistances arranged in series which can be considered as approximately true at least as a first modelling approach to leaf hydraulics (Cochard et al. 2004). Effects of mercury chloride (HgCl 2 ) and mercaptoethanol on K leaf As the experiments aimed at discriminating the partitioning of hydraulic resistances within the leaf blade revealed that the different hydraulic efficiency of LL and DL was mainly due to the contribution of the extra-vascular water pathway, experiments were designed to check the eventual mercury-sensitive nature of water transport within sunflower leaves, which might suggest a possible role for aquaporins in determining K leaf and its diurnal changes. HgCl 2 is known to inhibit aquaporins (Maurel & Chrispeels 2001). LL were cut off under distilled water and immediately transported to the laboratory. Here, they were rehydrated for 1 h with solutions of HgCl 2. To the best of our knowledge, this was the first attempt at feeding whole leaves with mercurials. Therefore, a dose response curve was assessed by rehydrating leaves with solutions at increasing [HgCl 2 ], i.e. 50, 100 and 200 mm. After rehydration, leaves were measured for K leaf. Five to seven leaves were measured for each concentration of HgCl 2 tested. As HgCl 2 is highly toxic to cells, especially at the highest concentration used in our study (Zhang & Tyerman 1999), the reversibility of the effects of HgCl 2 was tested by rehydrating LL and DL with a 200 mm solution of HgCl 2 for 1 h and then with a 30 mm solution of mercaptoethanol (ME) for 1 further hour. ME is a well known scavenger of Hg 2+ and its application to mercury-treated cells and roots has been shown to reverse the inhibitory effects of mercurials on aquaporin functionality (Martre, North & Nobel 2001; Kamaluddin & Zwiazek 2002). Control experiments were performed with LL and DL rehydrated for 1 and 2 h with distilled water. Rehydration of leaves was performed under forced air flow for security reasons and for speeding up the solution uptake. Statistics Data were analysed with the SIGMASTAT 2.0 (SPSS, Chicago, IL, USA) statistics package. One-way-ANOVA was used to test differences between experimental groups. We used Tukey s test to make post-hoc comparisons between all means. RESULTS Preliminary experiments addressed to ascertain the possible influence of light during HPFM measurements of K leaf are reported in Fig. 1. Here, leaves collected while in their light period (light-leaves, LL, open circles) and leaves collected while in the dark (dark-leaves, DL, solid circles) were measured under alternate 30 min time intervals of light and dark. In the dark LL reached stable K leaf levels of about 4.0 versus 2.7 e-4 kg s -1 m -2 MPa -1 recorded for DL,

4 Circadian regulation of leaf hydraulic conductance 753 K leaf (kg s 1 m 2 MPa 1 ) Time (min) Figure 1. Hydraulic conductance (K leaf ) of leaves of sunflower measured during 30 min-time intervals under alternate dark and light conditions (PAR = 400 mmol m -2 s -1 ). Vertical bars denote SD of the mean (n = 5). namely K leaf of DL was about 32% less than that of LL. When the lamp was switched on, LL maintained their K leaf unchanged (and it was kept at nearly similar levels during the subsequent dark time interval). K leaf of DL, on the contrary, tended to increase slightly when light was turned on (from 2.7 to 3.2 e-4 kg s -1 m -2 MPa -1, i.e. by about 18%) but this difference was not statistically significant, after 30 min. During the second dark time interval, K leaf of DL continued to increase until reaching values similar to those recorded for LL under similar conditions. Plants subjected to a photoperiod of 12 h and measured for K leaf in the middle of the dark period (black segments in the abscissa of Fig. 2) showed K leaf values of about 2.5 e- 4 kg s -1 m -2 MPa -1 (consistent with those of DL of Fig. 1) that were maintained approximately stable for 4 d. When the photoperiod was inverted (it was opposite in phase with respect to the previous one, first arrow in Fig. 2), leaves were measured for K leaf in the middle of the light period (white segments in the abscissa of Fig. 2). Leaves responded to the change in the photoperiod s phase by P < K leaf (e-4 kg s 1 m 2 MPa 1 ) Time (days) Figure 2. Hydraulic conductance (K leaf ) of leaves measured in the middle of their light or dark periods. The light regime is represented by the white (light on) and black (light off) boxes. The vertical arrows indicate the time when the photoperiod was inverted. Vertical bars denote SD of the mean (n = 4). Different letters indicate significant differences (P < 0.05) for Tukey pairwise comparisons.

5 754 A. Nardini et al. increasing K leaf to the typical light values, namely to about 4.0 e-4 kg s -1 m -2 MPa -1 (LL, Fig. 1) but only about 48 h after the photoperiod had been inverted. In other words, leaves tended to retain their K according to their subjective light period instead of immediately adjusting K to the actual light conditions. Interestingly enough, leaves responded to a new inversion of the photoperiod s phase (with leaves measured for K in the middle of the dark period), with K leaf decreasing again to dark values but this response was completed only 4 d after the light/dark rhythm had been inverted. In other words, changes in K leaf occurred much more rapidly in response to the transition from dark to light conditions than in response to the opposite transition. A typical experiment for assessing the eventual circadian origin of light-dependent processes is to adapt plants to a given circadian rhythm and then put them under continuous light or dark conditions in order to investigate the eventual persistence of oscillations of the physiological variable under study. In our case (Fig. 3), when plants were put in continuous dark their K leaf continued to oscillate between values of LL and values of DL according to their subjective photoperiod. Stomatal conductance to water vapour (g L ) showed some circadian response (Fig. 3) but this disappeared on the second day after the light period had been suppressed. In Fig. 4, the partitioning of R leaf between the vascular and the non-vascular leaf compartment is reported as estimated by cutting open increasing numbers of minor veins and measuring the corresponding R leaf in LL and DL (Fig. 4). The initial difference in R leaf (R leaf = 1/K leaf ) between LL and DL was of the same order of magnitude of that measured during experiments reported above (Figs 1 & 2). At increasing numbers of minor veins cut open, however, these differences tended to become smaller and smaller until the R leaf of LL and that of DL became the same. Under these conditions, the hydraulic resistance of the non-vascular compartment (R nv ) as computed by difference of R leaf minus the new R obtained after 500 minor veins had been cut open (see above), was estimated to represent 58.4% of the total leaf resistance for LL and 71.9% for DL; that is, leaves during the dark period showed a consistently higher R nv (or lower K nv ) than did leaves during the light period. The response of K leaf to increasing HgCl 2 concentrations is reported in Fig. 5. K leaf of LL infiltrated with 200 mm HgCl 2 for 1 h during forced leaf transpiration, equalled that recorded in DL (horizontal dashed range in Fig. 5) and an intermediate effect on K leaf was recorded in LL exposed to 100 mm HgCl 2, already. When leaves were rehydrated with HgCl 2 for 1 h, the K leaf of LL was reduced to dark levels whereas the K leaf of DL remained unchanged. Leaves rehydrated for 1 or 2 h with distilled water showed K leaf values not statistically different from controls. Light leaves infiltrated with HgCl 2 for 1 h and with ME for one more hour showed to have recovered their normal K leaf, namely about 4.0 e-4 kg s -1 m -2 MPa -1. g L (mmol m 2 s 1 ) K leaf (e-4 kg s 1 m 2 MPa 1 ) DISCUSSION Time (days) Figure 3. Leaf hydraulic conductance (K leaf ) and leaf conductance to water vapour (g L ) measured in the middle of plant actual (a, upper horizontal box) and subjective (b, lower horizontal box) photoperiod. Vertical bars denote SD of the mean (n = 5). Different letters indicate significant differences (P < 0.05) for Tukey pairwise comparisons. Our data show that leaves of sunflower tuned their K leaf during day/night cycles. Leaves collected while plants were in their light period had K leaf values higher than those recorded for leaves collected from plants during the dark period. Quantitatively, these differences were of the order of about 30 40%. In the present study, all K leaf measurements were made with the HPFM, namely under conditions substantially different from those experienced by leaves in vivo. In fact, during HPFM measurements leaves are infiltrated with water under positive pressure and air spaces filled with

6 Circadian regulation of leaf hydraulic conductance 755 R leaf (MPa m 2 s kg 1 ) Light: R nv = 58.4% Dark: R nv = 71.9% Figure 4. Leaf hydraulic resistance (R leaf ) as a function of the number of minor veins cut open to by-pass the leaf non-vascular compartment and measure R of the leaf vasculature (R v ). Note that at more than 250 cuts R of leaves during their light period (open circles) equalled R of leaves during the dark period (solid circles). R of the leaf non-vascular compartment (R nv ) as computed by difference (R nv = R leaf - R v ) is reported for leaves in the light and in the dark as a percentage of R leaf. Vertical bars denote SD of the mean (n = 5). water. Nonetheless, the K leaf values recorded were very similar to those obtained by Trifilò et al. (2003) for sunflower using a different technique (the vacuum chamber). Moreover, it has to be pointed out that the HPFM technique has been reported to yield correct and consistent K leaf values when compared with independent methods, including evaporative flux, in several recent studies (e.g. Tsuda & Tyree 2000; Nardini, Tyree & Salleo 2001; Sack et al. 2002). Therefore, K leaf variations reported in the present study are unlikely to be the result of experimental artifacts arising from the HPFM method per se. In particular, the HPFM measurements of K leaf have been recently reported to be unaffected by the degree of stomatal aperture (Tyree et al. 2005) in five different species. K leaf of sunflower LL was not affected by light conditions during measurements (Fig. 1). In fact, K leaf of LL measured in the dark was revealed to be significantly higher than that measured in DL and remained stable when the light was turned on and off. Sack et al. (2002) measured a 4.6 to 8.8 times increase of K leaf in leaves of Quercus rubra when measured using the HPFM under a PAR > 1200 mmol m -2 s -1 with respect to that measured at low PAR (<6 mmol m -2 s -1 ). On the basis of similar observations in leaves of several woody species, Tyree et al. (2005) inferred that sun leaves should be measured for K at high irradiance if typical light values are to be obtained. This light effect was not observable in sunflower where light leaves (LL) yielded the same K leaf values irrespectively of light conditions during K leaf (e-4 kg s 1 m 2 MPa 1 ) [HgCl 2 ] (mm) Figure 5. Hydraulic conductance (K leaf ) measured of leaves collected during their light period and rehydrated for 1 h with water or with mercury chloride (HgCl 2 ) solutions at increasing concentrations. The horizontal dashed range represents the K leaf measured for leaves collected during their dark period. Vertical bars denote SD of the mean (n = 5). Different letters indicate significant differences (P < 0.05) for Tukey pairwise comparisons.

7 756 A. Nardini et al. the measurements. It has to be noted, however, that the PAR under which K leaf was measured in the present study was of 400 mmol m -2 s -1, namely the PAR under which plants were growing and not over 1200 mmol m -2 s -1 as in the study by Sack et al. (2002). Transpiration-induced changes of leaf hydraulic conductance of sunflower were reported by Koide (1985). More recently, Tsuda & Tyree (2000) reported diurnal changes of both root and shoot hydraulic conductance in sunflower and hypothesized that plant hydraulic conductance would directly respond to the transpiration rate. Although our data confirm the existence of diurnal rhythms of the hydraulic efficiency of sunflower plants, we feel that the above hypothesis is not confirmed by our experimental observations. In fact, when the photoperiod was interrupted and plants were put in continuous dark (Fig. 3) stomata showed very limited circadian oscillations for 24 h and no further stomatal opening occurred after 48 h. In spite of the more or less cuticular g L levels measured 48 h after plants had been put in the dark, however, K leaf continued to oscillate from typical light to dark values according to the plants subjective photoperiod. Therefore, we conclude that light-dependent changes in K leaf were not due to corresponding changes in transpiration consequent to stomatal behaviour. In other words, the circadian clock was clearly regulating K leaf of sunflower, independently of possible oscillators located in the stomatal guard cells (Webb 2003). Leaves from plants whose photoperiod had been inverted, adjusted K from dark to light values (Fig. 2) in a matter of 48 h from the phase inversion. Opposite K adjustment occurred following the new inversion of the photoperiod (i.e. from light to dark) but K leaf required about 4 d for returning to typical dark values. This suggests that the possible metabolic control over the perception of the dark-tolight transition was more efficient than that operating in the opposite direction. This would help explain why DL (Fig. 1) when measured for K in the light, tended to increase their K leaf and maintained the same tendency even when returned to dark conditions. In agreement with a previous paper by some of the present authors (Trifilò et al. 2003), leaves of sunflower showed the majority of their hydraulic resistance (R) to be located in the non-vascular compartment. In the abovecited study, sunflower leaves were either pre-immersed in 70% ethanol for 1 h to kill mesophyll cells or the leaf blade was cut along the intervenous areas, thus allowing the water flow to by-pass the extra-vascular compartment. In both cases, experiments revealed that only about 20% of R leaf resided in the vascular compartment. Analogous data have been measured by some of the present authors in other species such as Prunus laurocerasus and Juglans regia (Salleo et al. 2003). Sack et al. (2004) have properly argued that cutting the leaf blade between the major veins might cause water flow to by-pass the minor veins where the majority of the vascular resistance would be located. Accordingly, Sack and coworkers proposed a different technique consisting of making up to 150 small cuts through the leaf blade at the minor vein level to effectively by-pass the nonvascular compartment while maintaining intact the water pathway through minor veins. Under these conditions, they reported 64 74% of R leaf to reside in the leaf xylem in the case of red oak and sugar maple. Although we agree with their methodological suggestion, our conclusions for sunflower are opposite to theirs in that even using the technique suggested by Sack et al. (2004), R nv was calculated to represent up to about 70% of R leaf. When up to 500 cuttings across minor veins were made (Fig. 4), R leaf of light and dark leaves tended to become the same. This suggests that this new R leaf represented R v of sunflower leaves which was the same regardless if measured of leaves in the light or in the dark period. A novel aspect of the contribution of R nv to total R leaf appeared in this study, however, consisting of different R nv /R leaf ratios in LL with respect to DL (Fig. 4). In fact, R nv turned out to represent a fraction of R leaf as high as 71.9% in leaves during the dark period versus only 58.4% during the light period. A possible explanation is that leaves might modulate their hydraulics through active modifications of membrane water permeability in phase with the circadian clock. On the basis of measurements of the temperature response of water flow through intact leaves of red oak and sugar maple, Sack et al. (2004) have shown that the resistance to water flow through the nonvascular compartment arises, at least partly, from cell membranes. These authors concluded that the most likely location for a transcellular component of water flow across leaves is the bundle sheath. The best known source of variable cell membrane permeability to water is represented by activation and/or expression of aquaporins. In the present study, HgCl 2 reduced K leaf of light leaves to that typical of dark leaves (Fig. 6), thus suggesting that mercury-sensitive water transport proteins had been inhibited and that, consequently, putative water channels may contribute to modulating K leaf. We are fully aware that heavy metals are toxic to cells and that 200 mm HgCl 2 can be a toxic mercury concentration (Zhang & Tyerman 1999). To the best of our knowledge, however, there are no data in the literature about the [Hg 2+ ] concentration that can be supplied to whole leaves to inhibit aquaporins without causing permanent damage to membranes. The dose response curve had shown (Fig. 5) that a clear response of K leaf to mercury was observable in leaves supplied with 200 mm HgCl 2. If mercury per se had caused membrane disruption, K leaf could be expected to increase and not to decrease as observed (Fig. 6). Moreover, ME was able to reverse the effect of HgCl 2 in that K leaf of light leaves that was lowered to dark values as a consequence of the mercurial solution supplied, returned to typical light levels when leaves were put in contact with ME. On the basis of the above, we conclude that: (1) leaves during their light period have higher K than during the dark period; (2) this is due to parallel changes in the R of the non-vascular leaf compartment which is dominant over R of the vasculature; (3) the higher K leaf in the light period might be due to light-dependent new expression/activation of aquaporins; and (4) K leaf changes with a circadian rhythm. This last point is, in our opinion, further reinforced by the fact that the circadian clock has

8 Circadian regulation of leaf hydraulic conductance 757 K leaf (e-4 kg s 1 m 2 MPa 1 ) Figure 6. Hydraulic conductance (K leaf ) of leaves either measured immediately after excision (controls) or after 1 and 2 h rehydration with distilled water or after 1 h rehydration with a 200 mm HgCl 2 solution or after 1 h rehydration with a 200 mm HgCl 2 solution followed by 1 h rehydration with 30 mm mercaptoethanol (ME). Vertical bars denote SD of the mean (n = 5). Different letters indicate significant differences (P < 0.05) for Tukey pairwise comparisons. been reported to regulate transport mechanisms in the root (Henzler et al. 1999; Clarkson et al. 2000). Aquaporin transcription in the roots of plants of Lotus japonicus increased in phase with the plant s subjective photoperiod and caused root hydraulic conductance to peak (Henzler et al. 1999). If roots are sensitive to light signals, we may expect that leaves are the first organ responding to light by actively increasing their K and sending signals to roots that would adjust K root to the new water demand. Assuming that our interpretation of experimental data is correct, it can be concluded that expression and/or activation of mercurysensitive aquaporins increases K leaf by about 30 40%. This finding is in agreement with recent reports showing that aquaporins can account for up to 60% of root hydraulic conductance. In a recent paper, Martre et al. (2002) measured the hydraulic conductance of plants of Arabidopsis and compared wild-type (WT) to double antisense (das) plants with reduced amounts of PIP1 and PIP2 aquaporins. They found no difference in terms of K leaf between the two genotypes, although the water permeability of leaf protoplasts of das was strongly reduced with respect to WT. They attributed this result to the essentially apoplastic nature of the transpiration stream in Arabidopsis. Our findings are not in agreement with their conclusions. The discrepancy between the above data and ours might arise from the different species studied or from the different methods used to measure K leaf. Martre et al. (2002) measured plant hydraulic conductance using the evaporative flux method (EM). Several studies have shown that both EM and HPFM give correct estimates of K leaf (Tsuda & Tyree 2000; Nardini et al. 2001; Sack et al. 2002). Therefore, a first possible conclusion is that the prevailing water pathway within the leaf is different among species. A second point to be considered is that the relative contributions of vascular and extra-vascular compartments to leaf hydraulic resistance can be variable among species. Aquaporins can be expected to significantly improve leaf hydraulic conductance only if the extra-vascular water pathway represents a major fraction of the total leaf hydraulic resistance, as in the case of sunflower. Recently, the question was posed of the possible benefits of the circadian regulation of some plant processes. In terms of plant water balance, the hypothesis has been advanced that the circadian control of stomatal movements anticipates changing light conditions at dawn and dusk (Webb 2003). As an example, stomata might anticipate dawn light conditions and open, allowing plants to maximize gas exchange when temperatures are still low, thus limiting water loss and improving the water use efficiency of the plant. In the case of sunflower, the evident circadian response of K leaf to light might induce stomatal aperture and not vice-versa as proposed by Tsuda & Tyree (2000). Moreover, increasing K leaf before transpiration rate increases might allow plants to effectively buffer leaf water potential above the critical threshold inducing xylem cavitation while maximizing gas exchange and productivity (Salleo et al. 2000). In conclusion, our present data combined with data by Henzler et al. (1999), strongly suggest that water balance of plants results from a fine metabolically controlled tuning of the hydraulic conductance of all plant organs interacting with each other in response to environmental stimuli (Maurel & Chrispeels 2001). REFERENCES Amodeo G., Dorr R., Vallejo A., Sutka M. & Parisi M. (1999) Radial and axial water transport in the sugar beet storage root. Journal of Experimental Botany 50,

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