Plant, Cell and Environment (1999) 22, 39–48
ORIGINAL ARTICLE
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Water stress induces different levels of photosynthesis and electron transport rate regulation in grapevines J. FLEXAS, J. M. ESCALONA & H. MEDRANO Instituto Mediterráneo de Estudios Avanzados -Universitat de les Illes Balears (UIB-CSIC), Departament de Biologia Ambiental, Carretera Valldemossa km. 7,5; 07071 Palma de Mallorca (Baleares), Spain
ABSTRACT Diurnal time courses of chlorophyll fluorescence and gasexchange rates were measured in young potted grapevines (Vitis vinifera L. cv. Tempranillo) subjected to different conditions of water supply under Mediterranean summer conditions. The irrigated plants exhibited typical diurnal patterns for all measured parameters, showing a correspondence between electron transport rate, net CO2 assimilation and stomatal conductance. Mild decreases in soil-water availability led to different degrees of down-regulation of photosynthesis and increased nonphotochemical quenching of chlorophyll fluorescence. A good correspondence between electron transport rate and CO2 assimilation was still maintained, suggesting a coregulation of both photosynthetic processes. In contrast, a severe water deficit induced a drastic down-regulation of photosynthesis and breakage of the above-mentioned link. Both midday net CO2 assimilation and electron transport rate significantly correlated with pre-dawn water potential (ΨPD) (r2 = 0·65 and r2 = 0·92, P < 0·001, respectively). However, when field data were analysed, the relationship between electron transport rate and ΨPD was not maintained, although net CO2 assimilation was similarly correlated with ΨPD. Interestingly, the steady-state chlorophyll fluorescence yield was a good indicator of plant water stress. Key-words: Vitis vinifera L.; Vitaceae; chlorophyll fluorescence; electron transport rate; grapevines; photosynthesis; water stress. Abbreviations: A, net CO2 assimilation rate; E, leaf transpiration; ETR, electron transport rate; Fs, fluorescence yield at steady state; Fm and Fm', maximal fluorescence levels when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively; Fo and Fo', initial fluorescence levels when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively; Fv/Fm, efficiency of excitation capture by open PSII in dark-adapted leaves; ∆F/Fm', actual photochemical efficiency of PSII; g, stomatal conductance; NPQ, non-photochemical quenching of chlorophyll fluorescence; PPFD, photosynthetic photon flux density; ΨPD and ΨMD, leaf water potential at pre-dawn and Correspondence: H. Medrano. Fax: 34/71/173184; e-mail:
[email protected] © 1999 Blackwell Science Ltd
midday, respectively; Rl, estimated photorespiration rate; I1 and I2, Irrigation treatments; R, Recovery treatment; D1 and D2, drought treatments; HD1 and HD2, hard drought treatments.
INTRODUCTION Drought is considered to be a predominant factor both for determining the global geographic distribution of vegetation and for restricting crop yields in agriculture (Schulze 1986). Furthermore, water stress is a limiting factor for a wide range of physiological processes in plants (Cornic 1994; McDonald & Davies 1996) There is much evidence that water stress per se does not cause reductions in primary events of photosynthesis, i.e. PSII efficiency (Genty, Briantais & Vieira da Silva 1987; Cornic et al. 1989). However, water stress is often accompanied (particularly under Mediterranean conditions) by other limiting factors such as high temperature, leaf-to-air vapour pressure deficit, nutrient depletion and irradiance. It has been demonstrated that the combination of these factors favours photoinhibition, which limits the photosynthetic capacity of plants (Björkman & Powles 1984; Valladares & Pearcy 1997). In the species we have studied (Vitis vinifera L.), the photochemical apparatus maintains a high stability under different environmental conditions (Gamon & Pearcy 1990; Chaumont, Morot-Gaudry & Foyer 1995). A direct effect of high light intensity at the thylakoid level causing an afternoon depression of photosynthesis has been suggested by Correia, Chaves & Pereira (1990). Iacono & Sommer (1996) also reported that photoinhibition occurred under field conditions even in well-watered grapevines. However, lack of photoinhibition during the mid-morning depression of photosynthesis has been reported recently by Chaumont, Morot-Gaudry & Foyer (1997). Recent results of field experiments showed that moderate irrigation prevents photochemical down-regulation in grapevines (Flexas, Escalona & Medrano 1998). The combined measurements of chlorophyll fluorescence and gas-exchange rates proved to be a useful approach for distinguishing stomatal versus nonstomatal effects, as well as for estimating the importance of various types of energy use, such as thermal dissipation and photorespiration (Krall & Edwards 1992; Valentini et al. 1995). Previous studies on the effects of soil-water deficiency on photosynthesis and related processes through the growing 39
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season in field-grown grapevines showed a marked decrease in stomatal conductance and photosynthesis (Delgado et al. 1995). Nonstomatal effects were also observed in waterstressed plants, as shown by field-measured CO2 response curves (Escalona, Delgado & Medrano 1996). Chlorophyll fluorescence studies showed that a down-regulation of PSII efficiency took place in drought-stressed plants only on certain occasions. Under some extreme drought conditions, a certain degree of photoinhibition (measured by pre-dawn Fv/Fm) also occurred. Moderate irrigation, however, helped maintain a high PSII efficiency and prevented photoinhibition (Flexas et al. 1998). The aim of the present study was to assess the water stress-related effects on photosynthetic primary reactions and on their relationships with net carbon assimilation in grapevine (Vitis vinifera L. cv. Tempranillo), as well as to determine whether some chlorophyll fluorescence parameters could serve as indicators of plant water stress. MATERIALS AND METHODS Plant material and water treatments Main experiment Four 1-year-old plants of Vitis vinifera cv. Tempranillo were grown outdoors. They were planted in large pots (60 dm3) in a mixture of organic substrate (20%) and sandyloam soil (80%) maintained at field capacity and fertilized with nutrient solution (50% Hoagland; Hoagland & Arnon 1950) during the 3 months prior to beginning the measurements. The soil was covered with a 2 cm layer of perlite to diminish direct soil drying by evaporation. Before the start of the experiment, the pots were placed in a greenhouse at the UIB Plant Physiology Laboratory, where illumination and temperature were close to outdoor conditions. When measurements were started (July 1996), the plant shoots were about 1·5 m high, and their total leaf area averaged 0·49 ± 0·05 m2. After growth under favourable conditions, successive cycles of soil-water depletion were applied by withholding irrigation. Measurements were performed 20 d after withholding watering on two consecutive days (4 and 5 July, drought treatment, D1). Thereafter, the pots were immediately irrigated to field capacity with 50% Hoagland’s solution (6 dm3 pot–1) in the evening. Measurements were taken on the following day (8 July) (recovery treatment, R). Irrigation was then maintained at approximately 2·5 dm3 pot–1 d–1. New measurements were taken under well-watered conditions after 10 d, on 16 and 18 July (irrigation treatment I1). Watering was withheld again, and a second cycle of soilwater deficit was induced until 2 August. Measurements corresponding to the ‘hard drought’ (HD1) treatment were made. We call this treatment hard drought because the plant appearance indicated stronger effects of the water deficit than after the first soil-water depletion cycle, even though the soilwater content was similar to that of the first drought cycle. However, the plant leaf area was much larger (0·94 ± 0·05 m2), which caused a higher water demand.
Second experiment In the main experiment described above withholding watering also involved the interruption in the supply of nitrogen and other nutrients. The importance of a nitrogen deficit during water stress on plant photosynthetic performance has been recently reported (McDonald & Davies 1996; Heckathorn, De Lucia & Zielinski 1997). In order to discriminate the effects of nutrient versus water deficiency, a second complementary experiment was made on the same plants in the summer of 1997. In this experiment six plants were grown under field conditions during the spring and regularly watered with 50% Hoagland’s solution. However, 15 d before the onset of the experiment all the plants were irrigated daily with water only. By early June, these plants had a similar leaf area to that in mid-July 1996. In contrast to the plants of the main experiment, the pots remained in the experimental field and were therefore subjected to higher wind and leaf-to-air vapour pressure deficit oscillations. This was reflected in the higher watering demand, measured with a sap flow meter (Escalona, Flexas & Medrano 1998; and unpublished results). The treatments consisted of irrigating plants 1 and 2 (controls) at a rate of three dm3 of water per day, whereas plants 3–6 were irrigated at half this rate. The irrigation was stopped 6 days before measurements in plants 3 and 4, and 3 days later in plants 5 and 6. Therefore, by 13 June three different plant water-stress levels were present: irrigated (I2), drought (D2) and severe drought (HD2). In this experiment, measurements corresponding to the different treatments were performed on the same day. Field data Some results from field-grown grapevines are shown for comparison with data obtained in pot-grown plants. Experimental conditions were as described by Flexas et al. (1998). The data are from the summers of 1994, 1995 and 1996. Environmental conditions and plant water status Leaf temperature and PPFD incident on the leaf surface were, respectively, measured with a thermocouple and a quantum sensor, both incorporated in the portable PAM2000 fluorometer (Walz, Effeltrich, Germany). The PPFD incident over a horizontal surface was measured with the external quantum sensor of a Li-6400 infrared gasexchange analyser (IRGA) (Li-Cor Inc., Lincoln, Nebraska, USA). The accuracy and reliability of both sensors were tested previously. The leaf-to-air vapour pressure deficit was measured with the Li-6400. The leaf water potential was measured with a Scholander chamber (Soil Moisture Equipment Corp., Santa Barbara, USA) at pre-dawn (0600 h) and midday (1400 h, local time) in leaves of the same age and position as those used for fluorescence and gas-exchange measurements. The soil-water content was measured with time domain reflectometry © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
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(TDR TRIME-system; IMKO, Ettlingen, Germany), previously calibrated for the soil used in this experiment.
RESULTS
Chlorophyll fluorescence and gas-exchange measurements
Irradiance, air temperature and humidity were typical of Mediterranean summer conditions for all the experiments, with small differences between months (June–July–August), i.e. the conditions were comparable at all sampling times. The diurnal time courses of PPFD were also similar for all the days of the experiment except in treatment R, when midday irradiance was about 20% higher than average. Figure 1(a) shows the diurnal time course of PPFD incident on a horizontal surface and over the leaf surface, averaged over the 6 days of measurements. The unusual shape of the curve showing an increase of morning light was due to the
Chlorophyll fluorescence was measured in diurnal time courses using a portable saturation-pulse fluorometer PAM-2000 (Walz, Effeltrich, Germany). All measurements were carried out on the south-facing side of the vines, on four just fully expanded leaves (one for each shoot). The four measured leaves had a very similar appearance, and exactly the same orientation, and showed similar gasexchange rates before starting the experiment. Saturation pulses of about 8000 µmol photons m–2 s–1 were applied to achieve a complete saturation of the PSII reaction centres (i.e. measurement of Fm and Fm'). Three to five single measurements on each leaf were made at each sampling. Leaf gas-exchange parameters (CO2 assimilation rate, transpiration rate, leaf temperature and related parameters) were continuously recorded with a Li-6400 IRGA, at a rate of one measure each 15 min. The IRGA chamber was attached to one of the four leaves used for chlorophyll fluorescence measurements while trying to maintain the leaf in its natural position. No movements during the day were observed in the other three leaves. In both the second experiment and in field experiments the gas-exchange parameters were measured as single measurements several times per day, using the Li-6400.
Environmental conditions and plant water status
Calculations The ETR was estimated after Krall & Edwards (1992), by multiplying ∆F/Fm' × incident PPFD × 0·5 (two photons are used for exciting one electron, as we have assumed an equal distribution of excitation between photosystems II and I), and × 0·84, which is considered the most common leaf absorbance coefficient for C3 plants (Björkman & Demmig 1987) as well as for Vitis vinifera leaves under a wide range of environmental conditions and leaf ages (Schultz 1996). The Stern–Volmer NPQ was calculated using the expression NPQ = (Fm – Fm')/Fm'. From combined measurements of fluorescence and gasexchange we estimated the photorespiration rate, Rl, according to Valentini et al. (1995). This must be considered as a gross estimation because this method assumes that all photochemical energy is used only in photosynthesis and glycolate photorespiration. Thus, the estimated rates of photorespiration should be the maximum values expected. Statistical analysis Analysis of variance (ANOVA) was applied to the treatment effects on the light response curves of ETR, using light intensity as a covariant (Statgraphics Plus for Windows; Manugistics Inc., Rockville, MD, USA). The location of significant effects was identified with the LSD test. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
Figure 1. Diurnal time courses of: photosynthetic photon flux density (PPFD) incident over a horizontal surface (■) and over the leaf blade (▲). Data are average for the 6 days of measurements (a); leaf-to-air vapour pressure deficit (VPD), (b); and leaf temperature (c). The treatments are: irrigation 1 (▲), recovery (●), drought 1 (▲ ▲) and hard drought 1 (▼ ▼). Values are means ± SE except in (b), which are single values.
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position and structure of the greenhouse. As observed in the field for the same cultivar (Flexas et al. 1998), quite different values of irradiance were obtained on actual leaf position in relation to a horizontal surface. The difference was particularly pronounced at midday, when the PPFD was reduced by about 500 µmol photons m–2 s–1 due to leaf orientation. The irradiance in the second experiment was slightly lower. Leaf-to-air vapour pressure deficit (Fig. 1b) reached high midday values of 3 KPa in the HD1 treatment. The highest leaf-to-air temperature increase (4 °C) was found in HD1 (Fig. 1c). These peak leaf temperatures were at the limit of those directly affecting chlorophyll fluorescence yields (Pastenes & Horton 1996). Similar values for leaf temperatures were found in the second experiment (Fig. 2b) and under field conditions (data not shown). In response to soil-water depletion, ΨPD showed a clear gradient from high values in I1 to low values in HD1 (Table 1). However, ΨMD showed a much narrower range of variation regardless of the treatment. This is in agree-
ment with field observations on the same cultivar. The leaves showed a great capacity of Ψ recovery because, on the following morning, 8 h after irrigation at 2200 h, ΨPD reached almost its highest values. Pre-dawn water potentials and soil-water contents of the second experiment were quite similar to those of the main experiment (Table 1). The range of pre-dawn water potentials found under field conditions was similar to that of the pot experiments. Chlorophyll fluorescence and gas-exchange parameters As expected, in all treatments the diurnal patterns of A followed that of PPFD during the morning (Fig. 2a). After midday, A began to decline in all treatments. The slope of this decline was similar in I1, R and D1, although this decline started from higher values in the irrigated plants. In HD1 the depression started earlier and was more pronounced. Maximum rates in HD1 were higher than in D1,
Figure 2. Diurnal time courses of (a) net CO2 assimilation (A); (b) electron transport rate (ETR); (c) estimated photorespiration rate (Rl); (d) leaf transpiration (E); (e) stomatal conductance (g); and (f) NPQ in the main experiment. For treatment symbols see Figure 1. Values are means ± SE of 12 values, except in (a, c, d and e), which are single values. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
Water stress and photosynthesis Table 1. Mean soil-water content (SWC) and leaf water potential (Ψ) at pre-dawn(ΨPD) and midday(ΨMD) during the four treatments Treatment
SWC (%)
ΨPD (MPa)
ΨMD (MPa)
Hard drought
(10·9 ± 0·2 (6·9 ± 0·2)
(– 0·58 (– 0·50)
– 1·17
(9·7 ± 1·0 (8·5 ± 0·5)
(– 0·41 (– 0·3)
– 1·14
Drought Recovery
(18·8 ± 0·2
(– 0·19
– 1·11
Irrigation
(21·2 ± 0·4 (18·5 ± 0·8)
(– 0·13 (– 0·03)
– 1·25
The SWC are mean values ± SE of four to six replicates; Ψ are mean values from two equivalent leaves. Values in brackets are for the second experiment.
but on a diurnal basis (i.e. the integral of the curve) the assimilatory rates were much higher in D1 (183·5 mmol m–2 d–1 in D1 and 134·5 mmol m–2 d–1 in HD1). For the I1 and R treatments, both the E and g diurnal time courses (Figs 2d & e) showed a pattern similar to that in A. In contrast, a drastic reduction in both parameters was imposed by both drought treatments (Fig. 2b). This reduction was much higher than in the A rate, and therefore water use efficiency increased. The values of A/E in the water-stressed plants were three times higher than in irrigated plants. The diurnal time courses of ETR also showed marked differences between treatments. In HD1, ETR followed a pattern that coincided well with that shown for A, with a peak at mid-morning followed by a depression at midday, which did not recover in the afternoon. In contrast, the I1 pattern did not show significant midday depressions. The R and D1 patterns were between these two extremes, showing a midday depression followed by an afternoon recovery. Contrary to results reported by Krause, Virgo & Winter (1995), the afternoon recovery of ETR in the R and D1 treatments was not accompanied by a recovery of A rates, suggesting an increase in alternative photochemical rates, such as photorespiration (Fig. 2c). Although in I1 the Rl of the plants followed a pattern similar to that of ETR, there was a clear afternoon increase of Rl in R and D1, with a peak at 1700 h, coinciding with the afternoon recovery peak of ETR. In contrast, the maximum Rl values for HD1 plants were reached at 1300 h and the rates were lower than those recorded for the other three treatments. Diurnal time courses of NPQ (Fig. 2f) showed a pattern that followed the PPFD, with a very clear gradient between treatments. Interestingly, the diurnal time courses of Fs were clearly dependent on the water status of the plants. For the two wellwatered treatments, the diurnal courses showed a sustained increase during the morning, reaching maximum values at midday (Fig. 3). Under drought, there was a strong quenching of steady-state chlorophyll fluorescence at midday, with a depression to values that were below the pre-dawn Fo (indicating a strong quenching of Fo'). © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
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In spite of the hard water stress and high irradiance levels, no treatment effect was observed on pre-dawn Fv/Fm, with all of the values being between 0·80 and 0·82, which suggests no permanent photoinhibition. The results of the second experiment (Fig. 4) were quite similar to those described above. The apparent differences in the patterns were due to differences in the PPFD pattern. The maximum rates of all measured gas-exchange and chlorophyll fluorescence parameters were almost identical in the controls of the two experiments (I1 and I2). In D2, as in D1, the ETR showed a midday depression and an afternoon recovery (Fig. 4e). This afternoon recovery was not matched by a recovery in A rates (Fig. 4c). The midday depression in HD2, as in HD1, was not recovered in the afternoon. Development of NPQ in response to water deficit (Fig. 4h) was also quite similar between the two experiments. Interestingly, we again observed a clear response of Fs to water stress (Fig. 4g). Interactions between photosynthetic parameters, light and water status The incident light dependency of ETR is shown by plotting ETR values against PPFD (Fig. 5). The curve is similar to that known for the light dependence of CO2 assimilation (Harbinson & Foyer 1991). Similar values of ETR were observed for the four different conditions at low light intensities of up to 200 µmol photons m–2 s–1, but the maximum ETR at high light intensities decreased with water stress. At saturating light (more than 600 µmol photons m–2 s–1 PPFD), the average ETR was maintained at saturation and almost constant, except for those in HD1, in which the maximum ETR was achieved at less than 600 µmol photons m–2 s–1. An ANOVA (light intensity as covariant) revealed a significant treatment effect. The sub-
Figure 3. Diurnal time courses of steady-state chlorophyll fluorescence (Fs) in the main experiment. For treatment symbols see Figure 1. Values are means ± SE of 12 values.
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Figure 4. Diurnal time courses of (a) photosynthetic photon flux density (PPFD); (b) leaf temperature; (c) net CO2 assimilation (A); (d) leaf transpiration (E); (e) electron transport rate (ETR); (f) stomatal conductance (g); (g) Fs; and (h) NPQ in the second experiment. The treatments are: irrigation 2 (▲),drought 2 (● ●) and hard drought 2 (▼ ▼). Values are means ± SE of six values.
sequent LSD test on ETR showed that the response of the I1 plants was significantly different from the responses in D1 and HD1 plants, and that the R plants were intermediate between I1 and D1 (P < 0·01). When statistical analysis was only applied to points with PPFD values above 500 µmol photons m–2 s–1, treatment effects were significantly different from each other (P < 0·01).
The ETR and A were compared by the ratio ETR/A (Fig. 6). In the middle of the day and during the afternoon, i.e. under high irradiances, the ratio in I1 plants was between 8 and 13 throughout the day. In R and D1, similar ratios were observed during the morning and midday, but a clear increase to values of 20–30 coincided with the estimated peak of photorespiration at 1700 h. In HD1, how© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
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Figure 5. Response of apparent electron transport rate (ETR) to incident photosynthetic photon flux density (PPFD) in the main experiment. Values are single measurements from four equivalent leaves, taken during the full daily cycle. ANOVA (light intensity as covariant) revealed a significant treatment effect. The subsequent LSD test discriminated two significantly different groups (a and b, P < 0·01) as follows: I1 = a; R1 = ab; D1 and HD1 = b. When tested only for values above 500 µmol photons m–2 s–1 each treatment was significantly different from the others.
ever, the ratio increased immediately as a response to high levels of light, with values as high as 70 at midday. The ETR values of the two experiments performed with pot-grown plants correlated highly significantly with ΨPD (P < 0·001, r2 = 0·92; Fig. 7c). In contrast, the ETR values under field conditions (Fig. 7b) were far from significantly correlated. However, the correlation between A and ΨPD using pooled data from the three experiments was highly significant (P < 0·001, r2 = 0·65; Fig. 7a). We found that the slopes of the lines of best fit between A and ΨPD for each experiment were similar in the three cases (data not shown).
DISCUSSION Relationships between photosynthetic primary reactions and net carbon assimilation
Figure 6. Diurnal time courses of the ratio ETR/A during the main experiment. For treatment symbols see Figure 1. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
The treatment responses in A and ETR show that water stress strongly affects both CO2 assimilation and PSII efficiency in grapevine leaves. Decreases in A and ETR are generally proportional, suggesting a close link between the photosynthetic processes (Foyer et al. 1990).
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Figure 7. (a), correlation between light-saturated CO2 assimilation (A) and pre-dawn leaf water potential (ΨPD). Data are from main, second and field experiments. (b), correlation between maximum recorded electron transport rate (ETRmax) and pre-dawn leaf water potential (ΨPD) for field data. (c), correlation between maximum recorded electron transport rate (ETRmax) and pre-dawn leaf water potential (ΨPD) for the main and second experiments. Values are means ± SE. (●), field experiment; (▲) main experiment; and (■), second experiment. Filled symbols correspond to irrigated treatments and unfilled symbols are nonirrigated treatments.
A gradual decline in A from mid-morning to evening has been described previously for a large number of species, including Vitis vinifera (Correia et al. 1990; Chaumont et al. 1994). In irrigated plants, this decrease corresponds to a decline in stomatal conductance. Demmig-Adams et al. (1989) and Correia et al. (1990) reported that the midday depression of photosynthesis could be due to photoinhibition and down-regulation of PSII in Arbutus and Vitis vinifera, respectively. Our results show no evidence of photoinhibition in I1 plants. The value of A started to decrease without any change in ETR, even though a downregulation of PSII was shown by increased NPQ (‘dynamic photoinhibition’ according to Osmond 1994). A close correlation was observed between water loss, CO2 assimilation rate and light use efficiency in irrigated plants, with relatively constant ratios of A/E and ETR/A during the course of the day. In R plants, g continued to increase even after A had started to decrease at midday. However, a midday depression occurred in ETR, suggesting that R plants were more subjected to nonstomatal down-regulation of photosynthesis in response to excess light intensity. A midday depression of ETR has been reported by Krause et al. (1995) in young leaves of tropical forest trees, which are considered to be very susceptible to photoinhibition. The present results in grapevines show that ETR depression could be caused by an increased NPQ in R compared to I1 plants. The ratio ETR/A showed a slow but sustained increase during most of the day in D1 plants. This increase is consistent with reported increases caused by rising temperature and photorespiratory rates (Leegood 1995), suggesting that at least part of the electrons are not used directly in photosynthetic CO2 fixation. In HD1 plants the ETR/A ratio showed strong changes, reaching values
of up to 70 at midday. This indicates that at such an extremely low A, a smaller proportion of ETR is used for CO2 assimilation. The decline of Fs below Fo values in D1 and HD1 indicates an increase in the activity of photoprotective processes such as thermal dissipation at the antenna level (Osmond 1994). This is also reflected in the large increase in NPQ, probably involving the xanthophyll cycle. According to Foyer et al. (1990), the activation of this cycle leads to a full down-regulation of the photosynthetic processes. The main function of this regulation must be the protection of PSII from photodamage. Consistent with reports by Leegood (1995) and Valentini et al. (1995), the ratio of photorespiration to net photosynthesis was about 0·4 at maximum Rl rates (1700 h) for both I1 and R. However, our results in D1 and HD1 show much larger increases. This could be related to the increase in leaf temperature in drought treatments (Leegood 1995), but it could also reflect some overestimation of Rl rates under drought in this model. In HD1 leaves, the high ETR/A ratio reached possibly invalidates the use of the relationships proposed by Valentini et al. (1995). There is increasing evidence that photorespiration (and also a direct reduction of oxygen in the Mehler reaction) protects the photosynthetic apparatus from photoinhibition (Kozaki & Takeba 1996; Park et al. 1996). Although the ratio of photorespiration to CO2 assimilation increases, the absolute values of Rl decrease under water stress, as shown by the estimations of the present experiment. Thus, the total amount of absorbed light energy dissipated by photorespiration should be reduced, particularly in respect to that which can be dissipated by the thermal sink (Cornic 1994; Osmond et al. 1997). The maximum estimated percentage of energy dissipation associated with photorespiration was © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
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about 18% (D1 leaves in the afternoon). However, the maximum percentage of nonphotochemical light energy dissipation under those conditions reached about 70%. Interactions with nutrient deficiency We performed the second experiment in order to explore whether the observed effects were due to water deficit only or also to deficiencies in nitrogen or other nutrients. The rapid development of water stress in this experiment should have decreased the extent of nitrogen retranslocation and drought-related growth dilution, which are two of the main causes of reduced nitrogen availability under drought (Heckathorn et al. 1997). The main effects of nitrogen deficiency on leaf photosynthesis are expected to be decreases in water use efficiency and a down-regulation of Rubisco (Ghashghaie & Saugier 1989; Heckathorn et al. 1997) and, in some species, an activation of thermal dissipation via the xanthophyll cycle (Verhoeven, Demmig-Adams & Adams 1997). No significant differences in treatment responses were observed between the main and second experiment. Both the maximum rates and the shapes of diurnal patterns of all measured parameters were almost identical between equivalent treatments of the two experiments (Figs 2 and 4). The value of A/E increased under water stress, showing that the drought effect was prevalent over the nutrient deprivation effects, and NPQ showed an identical response in both experiments. Therefore, we assume that most of the effects described previously are a direct consequence of water stress. The effects of nitrogen availability on photosynthesis have most frequently been studied in herbs and grasses, but those on grapevines can be less significant. It is likely that the effects are greater in grasses because they usually have higher photosynthetic and growth rates, shorter vegetative cycles and a relatively low nutrient storage capacity. Water stress sensitivity of fluorescence parameters The different soil-water availability levels were better reflected by midday ETR than by any of the gas-exchange parameters. The relationship between ETRmax and ΨPD gave high correlation coefficients, as observed by Björkman & Powles (1984) for Nerium oleander, and statistical analyses of the response of ETR to light intensity gave four significantly different groups, one for each treatment. Even though evidence in the literature shows that water stress per se does not produce decreases in the primary events of photosynthesis (Genty et al. 1987; Cornic et al. 1989), our results show that ETR is significantly dependent on ΨPD under the conditions detailed above. These results, however, contrast with the ETR response found in the field-grown Vitis plants, in which no correlation between ETR max and ΨPD was found (Fig. 7). However, the response of A to ΨPD was identical for both © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 39–48
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pot-grown and field-grown plants. This is probably due to differences in the way and rate with which water stress is imposed. It is likely that the slow development of water stress in the field enables the activation of different acclimation mechanisms, which lead to the maintenance of higher ETR values even when A decreases. Differences in accumulative photon exposure could also have occurred, because the lowest water potentials under field conditions occur in August, when days are shorter than in June and July. Similar discrepancies were found by Downton (1983) between grapevines in which water stress developed rapidly over several hours and others in which drought developed over several days. Whatever the causes of these differences, further research is needed to clarify the relationship between ETR and plant water status as well as to confirm the applicability of ETR measurements as an indirect assessment of plant water stress. The Fs patterns were clearly dependent on the water status of the plants in both experiments. From our results, it appears that Fs reflects the photosynthetic response to drought better than other commonly used parameters. Furthermore, Fs has the advantage of being a simpler and less intrusive parameter. A clear and early response to drought, though not gradual, has been observed for Fs under a wide range of conditions, even in the field, where response of ETR is not clear (unpublished). The effects of drought on the diurnal time course of Fs have been reported for different plants by Cerovic et al. (1996). In spite of the great interest of these first results, it would be convenient to strengthen the evidence before discussing the causes of the higher dependency of Fs on leaf water status. ACKNOWLEDGEMENTS We gratefully acknowledge the help of Dr J. Cifre, who gave advice on the statistical treatment of data. Drs S Jonasson, M. Chaves and E. Descals are acknowledged for critical comments and language corrections. Research scholarships from the Universitat de les Illes Balears for J. Flexas, and from the Spanish Ministry of Education and Science for J. Escalona are also acknowledged. This work is part of the AGF97–1180 Project, financially supported by the Spanish CICYT-Spanish Government. REFERENCES Björkman O. & Demmig B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489–504. Björkman O. & Powles S.B. (1984) Inhibition of photosynthetic reactions under water stress: interaction with light level. Planta 161, 490–504. Cerovic Z.G., Goulas Y., Goburnov M., Briantais J.-M., Camenen L. & Moya I. (1996) Fluorosensing of water stress in plants: diurnal changes of the mean lifetime and yield of chlorophyll fluorescence, measured simultaneously and at distance with τLIDAR and modified PAM fluorometer in maize, sugar beet and kalanchoe. Remote Sensing of the Environment 58, 311–321. Chaumont M., Morot-Gaudry J.-F. & Foyer C. (1994) Seasonal and
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