Journal of Experimental Botany, Vol. 51, No. 352, pp. 1893-1902,
November 1, 2000
© 2000 Oxford University Press
Original Papers |
An overnight chill induces a delayed inhibition of photosynthesis at midday in mango (Mangifera indica L.)
1 Photosynthesis Research Unit of USDA/ARS and Department of Plant Biology, University of Illinois, Urbana, IL 61801-3838, USA
2 Institute of Horticulture, ARO The Volcani Center, PO Box 6, Bet-Dagan 50250, Israel
Received 4 May 2000; Accepted 13 June 2000
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Abstract |
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The effect of a cold night on photosynthesis in herbaceous chilling-sensitive crops, like tomato, has been extensively studied and is well characterized. This investigation examined the behaviour of the sub-tropical fruit tree, mango, to enable comparison with these well-studied systems. Unlike tomato, chilling between 5 °C and 7 °C overnight produced no significant inhibition of light-saturated CO2 assimilation (A) during the first hours following rewarming, measured either under controlled environment conditions or in the field. By midday, however, there was a substantial decline in A, which could not be attributed to photoinhibition of PSII, but rather was associated with an increase in stomatal limitation of A and lower Rubisco activity. Overnight chilling of tomato can cause severe disruption in the circadian regulation of key photosynthetic enzymes and is considered to be a major factor underlying the dysfunction of photosynthesis in chilling-sensitive herbaceous plants. Examination of the gas exchange of mango leaves maintained under constant conditions for 2 d, demonstrated that large depressions in A during the subjective night were primarily the result of stomatal closure. Chilling did not disrupt the ability of mango leaves to produce a circadian rhythm in stomatal conductance. Rather, the midday increase in stomatal limitation of A appeared to be the result of altered guard cell sensitivity to CO2 following the dark chill.
Key words: Chilling temperatures, chlorophyll fluorescence, circadian rhythm, leaf gas exchange, stomata.
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Introduction |
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Demand for sub-tropical fruits such as mango (Mangifera indica L.) has increased dramatically in the USA, Western Europe and Japan during the last decade. USA mango consumption increased from 32 to 202 thousand tons between 1988 and 1998, and less than 2% of this is from domestic production (USDA, 1999
). This rapidly emerging demand is encouraging production in cooler areas, such as Israel and parts of the American continent, even though moderately cool temperatures are known to limit the photosynthetic productivity of sub-tropical fruit trees (Schaffer and Anderson, 1994). The mechanistic basis for the chill-induced limitation of photosynthesis in these tropical woody species is unknown.
Chilling under even moderate illumination has been demonstrated to cause photoinhibition of photosynthesis in many chilling-sensitive species. Slower enzymatic reactions of the thylakoid and carbon metabolism at cool temperatures can lead to a down-regulation of the efficiency of photosystem II (PSII) electron transport as a result of increased quenching of excitation energy by the xanthophyll cycle and other processes in the antennae. Under most circumstances, the aggregate effect of these and other protective processes, coupled with a considerable capacity for repair, effectively prevents chronic photoinhibition by excess irradiance. However, photoinhibition is frequently exacerbated by low temperature in plant species evolutionarily adapted for growth in warm climates (Long et al., 1994). High light and low temperature can increase the net damage to the D1 component of PSII as well as slow down the repair processes responsible for recycling non-functional reaction centres (for reviews see Krause, 1994; Long et al., 1994). In addition, highly chilling-sensitive herbaceous species such as tomato (Lycopersicon esculentum) experience dysfunction that is not attributable to PSII damage. The persistence of an inhibition of net CO2 assimilation rate (A) following the chill in tomato arises from the inability of the chilled plants to light-activate fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase, two key enzymes of the photosynthetic carbon reduction cycle (Sassenrath et al., 1990; Hutchison et al., 2000).
Although the simultaneous occurrence of low temperatures and moderate to high irradiance levels is an agriculturally relevant situation for numerous important row and field crops grown in temperate regions, in many emerging mango producing regions chilling in the light is much less common than cool nights followed by warm sunny days. Therefore, this study focuses on the effect of chilling in the dark on leaf photosynthesis during the following warm day. The dysfunction of photosynthesis on the day following an overnight (i.e. dark) chill has been well characterized in herbaceous crops of tropical and subtropical origin, such as tomato, cucumber (Cucumis sativus) and maize (Zea mays). After 16 h in the dark at 4 °C, tomato A is inhibited by 60% instantaneously upon rewarming (Martin et al., 1981). The dark chill causes a combination of chill-induced inhibition of photosynthetic metabolism and stomatal closure. There is no reduction in the maximum quantum efficiency of CO2 assimilation (Martin et al., 1981), and the electron transport capacity of isolated thylakoids is always in excess of that required to support light- and CO2-saturated photosynthesis (Kee et al., 1986) ruling out photoinhibition/photodamage to PSII as a significant cause of the inhibition to net photosynthesis. Furthermore, in tomato, dark chilling can disrupt circadian rhythms in enzyme activity (e.g. sucrose phosphate synthase (SPS), nitrate reductase (NR) [Jones et al., 1998]) and/or gene expression (e.g. chlorophyll a/b binding protein and Rubisco activase [Martino-Catt and Ort, 1992]) of proteins critical to photosynthesis and the partitioning of its products. It is argued that chilling tomato in the dark inhibits photosynthesis primarily through the disruption of circadian co-ordination among component reactions of photosynthesis, rather than the direct inhibition of the reactions themselves (Jones et al., 1998).
Chronic photoinhibition (reduced maximum quantum efficiency of PSII photochemistry, Fv/Fm) of mango during the Israeli winter correlates with the previous minimum night air temperature (2–15 °C) but not the midday temperature (24–30 °C) (Nir et al., 1997). Chilling entire potted mango plants in the dark reveals that the photoinhibition of PSII is accompanied by simultaneous inhibition of leaf A and reduced stomatal conductance (gs) during the following day in full sunlight (Nir et al., 1997). It is not known if the photoinhibition is the direct cause of impaired photosynthetic performance, or is secondary, initiated by a smaller sink for the products of electron transport as a result of a direct chill effect on other components of photosynthesis or on stomatal closure.
This study was conducted to investigate the response of photosynthesis to a night chill and a subsequent warm photoperiod in the subtropical fruit tree mango, to allow comparison with well-studied herbaceous chilling-sensitive species like tomato. Examination was made of the impact of chilling on the circadian rhythms in whole leaf photosynthesis measured under constant, free-running conditions. Particular attention was focused on the separation of the dark chill effects on PSII electron transport, carbon reduction cycle enzymes, stomatal conductance and end-product inhibition, in mango trees grown and measured under conditions conducive for maximal photosynthesis. The significance of results from experiments conducted on plants in controlled environments were validated by measurements on mango trees exposed to additional stresses experienced under natural conditions.
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Materials and methods |
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Growth conditions
Mango plants (Tommy Atkin scions on Turpentine rootstock) used for the controlled environment experiments were obtained from a commercial nursery in Florida (USA). The trees were maintained in a climate-controlled glasshouse in Urbana, Illinois with day/night air temperatures of 30/25 °C throughout the year, and supplemental sodium lamps used during the winter. The trees were grown in 11.0 l pots containing peat, composted pine bark, coarse sand, and perlite (5:3:1:1) and fertilized weekly with 15-5-15 NPK. Regular pruning was used to maintain 1 m tall plants with a single apical shoot. All measurements were made on the youngest photosynthetically mature flush of 3–8 replicate plants.
Outdoor experiments were conducted with Tommy Atkin scions on 13/1 rootstock, of a similar age and size to the Urbana plants, that were obtained from a commercial Israeli nursery. These plants were grown in 15 l pots of peat, sandy loam and foam pellets (1:1:1, by vol.) under ambient conditions in Bet-Dagan (near Tel Aviv), Israel except that solar irradiance was reduced 50% by black neutral density shade nets suspended above the plants. The mean daily maximum and minimum air temperatures were 29.4 °C and 12.5 °C during the 3 weeks prior to measurements, with a mean maximum daily vapour pressure deficit (VPD) of 2.7 kPa and no significant precipitation. All outdoor measurements were made on the youngest photosynthetically mature flush in April/May 1999.
Chilling treatment
Glasshouse-grown plants were watered and then placed in a growth chamber overnight (11 h) with an air temperature of 7 °C (25 °C for controls) and a VPD <0.3 kPa in March 1999. To ensure that only the aerial parts of the plant experienced the chill, as would occur in the field, the soil was maintained at approximately 25 °C by placing the pots in buckets submerged in a warmed water bath, and insulating the soil surface with polystyrene foam. One hour prior to the end of the night, all plants were re-warmed to 30 °C in the dark. Plants were rewarmed in the dark to prevent illumination while parts of the tree were still at low temperatures, as the aim of this study is to characterize only dark chill effects. This dark rewarming also allowed identification of any lasting effects of night temperature on leaf gas exchange and chlorophyll fluorescence in the dark, when control and chilled plants were measured at the same temperature. The air temperature was thereafter maintained at 30 °C with a VPD <1 kPa with 650–800 µmol m-2 s-1 photosynthetically active photon flux density (PPFD) incident at the leaf surface. These conditions of warm temperature, high humidity and saturating light intensity were selected to facilitate maximal photosynthetic rates, and were not dissimilar from growth conditions in the glasshouse.
Outdoor grown plants were watered and then placed overnight (11 h) in a room at 5 °C (with 23 °C controls). Soil temperature in chilled plants was maintained close to control values by circulating heated water through latex tubing wrapped around the outside of the pots and insulating with polystyrene foam. Chilled plants were warmed to 23 °C for 1 h in the dark before moving plants to either the 50% shade or full sunlight conditions for the photoperiod.
A/ci and fluorescence analysis
To examine the time-course of effects on photosynthesis of the previous night temperature under controlled conditions, leaf gas exchange (Li-6400, Li-Cor Inc., Lincoln, Nebraska, USA) was used to measure the relationship between light-saturated A and the intercellular CO2 concentration (ci) in conjunction with chlorophyll fluorescence (PAM-2000, Walz, Effeltrich, Germany). The A/ci relationship was used to estimate the maximum in vivo carboxylation velocity of Rubisco (Vc,max), the maximum rate of electron transport contributing to RuBP regeneration (Jmax) (McMurtrie and Wang, 1993), stomatal limitation of A(l) (Farquhar and Sharkey, 1982), and chloroplastic inorganic phosphate (Pi) limitation of A (Sharkey, 1984) calculated as described earlier (Allen et al., 1997; Baker et al., 1997). Fluorescence signals were analysed as described previously (Genty et al., 1989) to provide an estimate of the relative quantum efficiency of PSII photochemistry, 
PSII (given by [
, and also known as 
), and were measured simultaneously with light-saturated A at an ambient CO2 concentration of 360 µmol mol-1. Fv/Fm was determined after 20 min of dark-adaptation. Leaves of glasshouse-grown plants were irradiated at 700 µmol m-2 s-1 PPFD from fluorescent lamps with the leaf temperature maintained at 30 °C and a VPD <1 kPa.
For outdoor-grown mango, leaf gas exchange and chlorophyll fluorescence analyses were undertaken as described above, except an incident PPFD of 1000 µmol m-2 s-1 was produced by a red and blue LED light source (6400-02B, Li-Cor Inc., Lincoln, Nebraska, USA), at ambient humidity and using a 1 h dark-adaptation period before Fv/Fm determinations. Fluorescence from five leaves or gas exchange from two leaves was measured and averaged on each of the three replicate plants per treatment. The means and standard errors presented were calculated from the plant average (n=3). The ambient air temperature and humidity were recorded (Hobo H8, Onset Computer Corp., Pocasset, Massachusetts, USA) throughout the photoperiod in addition to a quantum sensor measuring PPFD incident at leaf level (9901-013, Li-Cor Inc., Lincoln, Nebraska, USA).
Leaf water potential measurements
Leaf water potential (
L) measurements were conducted on intact excised leaves using a pressure chamber (PMS Instrument Co., Corvallis, Oregon, USA). The means and standard errors presented are from five replicate glasshouse-grown plants.
Measurement of circadian rhythms
Gas exchange and chlorophyll fluorescence were followed for 2 d on a leaf maintained under constant conditions of PPFD, VPD, leaf temperature, and CO2 concentration (ca) in the gas exchange cuvette. Fluorescence measurements were repeated every 30 min, gas exchange data were measured every 1.5 s and averaged each hour. The remainder of the glasshouse-grown plant was maintained at constant room temperature and in near darkness (<10 µmol m-2 s-1 PPFD) in the laboratory. The aerial parts of the plant were exposed to a 10 °C or 25 °C night (11 h) prior to the 48 h of light (as described above), or chilled at 7 °C with 160 µmol m-2 s-1 incident PPFD for 6 h early during the continuous light conditions.
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Dark chilling causes delayed inhibition of A in mango
Chilling mango plants overnight at 7 °C (Fig. 1a
) does not cause any detectable inhibition of light-saturated A during the first 2 h of a 650–800 µmol m-2 s-1 PPFD photoperiod at 30 °C. However, by the middle of the photoperiod there was a 50% reduction in light-saturated A (Fig. 1a). This decline is followed by recovery of CO2 assimilation back to control values by the end of the photoperiod.
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) or 7 °C (
). The net rate of CO2 assimilation (A) at an ambient CO2 concentration of 360 µmol mol-1 (a), stomatal limitation of A(l) (b), maximum velocity of Rubisco carboxylation (Vc,max) (c), and maximum potential rate of electron transport contributing to RuBP regeneration (Jmax) (d) were calculated from the A/ci response measured at a saturating PPFD of 700 µmol m-2 s-1 and a leaf temperature of 30 °C. The light-adapted quantum yield of PSII electron transport (The dark chill-induced decline in midday photosynthesis was accompanied by declines in gs (data not shown). To investigate whether closure of stomata was causally related to the decline in A, stomatal limitation of light-saturated A(l) was calculated. This computation compares the measured light-saturated A at an ambient ca of 360 µmol mol-1, with A at a ci of 360 µmol mol-1 (i.e. as would be expected if stomatal conductance were infinite). An increase in stomatal limitation at midday (Fig. 1b
) coincided with the decline in A suggesting that chill-induced stomatal closure may be responsible for at least a portion of the decline in net photosynthesis. Under growth chamber conditions chilling had no effect on L (Table 1) eliminating leaf water status as a direct contributor to the inhibition of A or to the partial closure of stomata.
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Following the 7 °C dark chill, up to a 40% decline in the midday Vc,max (Fig. 1c
) occurred simultaneously with the inhibition of photosynthesis, yet the decline observed in Jmax (Fig. 1d) was much smaller (<20%). There was no evidence for an increase in Pi-limitation causing the chill-induced declines in photosynthesis, as A was not correlated with reductions in the proportionate increase in light-saturated A in response to a switch from 21% to 2% O2 (i.e. non-photorespiratory conditions) (data not shown).
To determine whether this dark chill-induced inhibition of midday photosynthesis was apparent under conditions relevant to mango plants in the field, a similar experiment was undertaken in Bet-Dagan. After a night at either 5 °C or 23 °C, potted plants growing outdoors under 50% shade netting were returned to the shade or placed in full sun, with ambient conditions of temperature and humidity. Control photosynthetic rates (Fig. 2a, h) were lower than in the plants grown and measured under more conducive controlled conditions of temperature and humidity (Fig. 1). However, maximum A of control leaves grown and measured outdoors under 50% shade netting (which is typical for commercial nursery mango plants in Israel) was not dissimilar to the only literature values for mango, the authors found, for similar conditions during growth (i.e. means of 3.8 [Schaffer and Gaye, 1989] and 4.2 [Nir et al., 1997] µmol m-2 s-1). As was observed in controlled environments, there was no inhibition of photosynthesis during the first 2 h under ambient sunlit conditions after the dark chill. However, by midday A was reduced by 80% with gross CO2 fixation rates only slightly larger than respiratory losses (Fig. 2a). By mid-afternoon there was a substantial decline in the photosynthesis of control plants, following PPFD peaking around 1800 µmol m-2 s-1, and concurrent with the air temperature exceeding 30 °C and VPD approaching 3 kPa (Fig. 2a, f, g). The chilled plants however, showed some recovery in net photosynthesis later in the afternoon (Fig. 2a). Very similar results were observed when plants were placed under shade netting following the dark chill, where air temperature and VPD were similar, but with a c. 50% reduction in PPFD throughout the day (Fig. 2h, m, n). Under both light regimes, coinciding with the midday decline in A, was a similar reduction in Vc,max and Jmax and an increase in stomatal limitation of A. As observed in the controlled environment study, midday inhibition of A was not associated with a reduction in the proportionate increase in A in response to a switch to non-photorespiratory conditions, under full-sun or shade (data not shown).
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) or 5 °C (•). The net rate of CO2 assimilation (A) at an ambient CO2 concentration of 360 µmol mol-1 (a, h), stomatal limitation of A(l) (b, i), maximum velocity of Rubisco carboxylation (Vc,max) (c, j), and maximum potential rate of electron transport contributing to RuBP regeneration (Jmax) (d, k) were calculated from the A/ci response measured at a saturating PPFD of 1000 µmol m-2 s-1 and a leaf temperature of 30 °C. The maximum quantum efficiency of PSII photochemistry (Fv/Fm) (e, l) was measured after 1 h dark-adaptation. Dark respiration rate (negative A in a and h) and pre-dawn Fv/Fm were determined after rewarming plants to 23 °C in the dark for 1 h before 08.00 h. Ambient air temperature, VPD (g, n) and PPFD at leaf level (f, m) profiles during the photoperiod are shown. PPFD profile under 50% shade shows spikes due to small gaps in the netting. The black/white boxes at the top of the figure indicate the dark/light conditions. Statistical analysis of this data, in conjunction with that in Fig. 1Table 2
summarizes a two-way analysis of variance examining the effects on leaf gas exchange of night temperature (chill and control) and experiment (growth chamber, full-sun and 50% shade) at five times (I–V) during the warm day. Selection of pairs of chill and control data from the growth chamber study (Fig. 1) was based on proximity to the time into the photoperiod of the near-field measurements (Fig. 2), as identified in the figures. This analysis demonstrates that there was a significant chill-induced inhibition of A only around midday (Time III; c. 4 h into the photoperiod) in these three experiments. This analysis further confirms that the inhibition of A coincided with a significant night chill-induced increase in stomatal limitation and decrease in Vc,max, but without a significant impact on Jmax (Table 1). This analysis also demonstrates that A, Vc,max and Jmax were significantly lower in the plants growing and measured outdoors than the glasshouse-grown plants measured under controlled conditions of light, humidity and temperature.
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Chill-induced depression of photosynthesis in mango is not caused by photoinhibition of PSII
The delayed depression of A by previous night chilling was not a result of either damaging (chronic) or protective (dynamic) photoinhibition of PSII electron transport, as there were no declines in dark-adapted Fv/Fm (Fig. 1e) or light-adapted PSII (Fig. 1f) sufficient to account for the extent of the decline in photosynthesis under controlled conditions.
Control plants under full-sun had a small degree of non-rapidly reversible photoinhibition by early afternoon, revealed by a decline in Fv/Fm, but this was not observed in the shaded plants (Fig. 2e, l). Dark-chilled plants had a large photoinhibitory reduction in Fv/Fm under full-sun conditions, but a substantially smaller midday decline in Fv/Fm under shade, despite A still declining by 80% relative to the warm night controls.
Circadian regulation of photosynthesis persists following a low temperature night
Mango plants exhibited very strong rhythms in A (Fig. 3a) with an interval of c. 24 h when maintained under constant conditions of PPFD, leaf temperature, VPD, and ca. These 
35% circadian rhythms in photosynthesis are synchronized with larger (65%) rhythms in gs (Fig. 3a). The net result of these two processes, which regulate CO2 diffusion into and CO2 utilization by the leaf, produced a circadian rhythm in ci that peaked simultaneously with A and gs during the subjective day (Fig. 3b). An overnight dark chill at 10 °C immediately prior to the free-running period of continuous light, while inhibiting A and gs, did not upset the periodicity during the subjective day and night (Fig. 3d–f). Similarly, the circadian pattern was preserved after a 6 h chill at 7 °C and 160 µmol m-2 s-1 PPFD applied near the start of the continuous light period (Fig. 3g). No circadian rhythm in PSII was apparent under any treatment (Fig. 3b, e, h).
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Discussion |
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The depressions in photosynthesis observed during the day following a dark chill, were not due to an immediate direct inhibition of components of photosynthesis, as chilled plants achieved similar rates to the control plants during the first 2 h of the photoperiod (Figs 1
, 2; Table 2). Therefore mechanisms previously shown to be involved in the instantaneous inhibition of photosynthesis at low temperatures in other plants, including effects on carbon reduction cycle enzymes and electron transport (see reviews by Baker, 1994; Leegood and Edwards, 1996) were not responsible for the inhibition observed in mango. That the delayed response to a dark chill was observed at different levels of irradiance on the subsequent day and under controlled as well as natural conditions (Figs 1, 2), demonstrates that this is a robust observation in mango. Coffee (Coffea arabica), another subtropical species, also exhibits a delayed inhibition of photosynthesis following a shoot chill in the dark, with a 60% inhibition developing during the first 2 h of light (Bauer et al., 1985). Similarly, chilling maize for 12 h in the dark at 5 °C results in only a 15% inhibition of A 100 min into the following warm photoperiod, but a 50% reduction compared to controls develops by the end of the day (Long et al., 1983). In contrast, tomato shows a large inhibition of A immediately following the dark chill that persists throughout the subsequent day (Martin et al., 1981).
In mango, photoinhibition (photodamage or photoprotection) of PSII electron transport was not the primary cause of the midday inhibition, as it was observed independent of changes in Fv/Fm and PSII (Figs 1, 2). This behaviour is very similar to the response recently reported for grape leaves chilled at night (Flexas et al., 1999). However, the effects of the dark chill are very different to chilling concurrent with high light where reduced quantum yield of PSII, as a result of photoprotection and photodamage, are commonly observed (see reviews by Baker, 1994; Krause, 1994; Long et al., 1994). Dark chilling did exacerbate subsequent high light photoinhibition (Fig. 2e), but this was likely the result of removal of the main sink for the products of electron transport, carbon assimilation, rather than the cause of it. The mechanisms behind the dark chill-induced inhibition of photosynthesis need to account for the delay of a few hours after the chill, when control rates of photosynthesis were observed (Figs 1, 2; Table 2). This depression was associated with a rise in stomatal limitation of A and a decrease in Rubisco activity (Figs 1, 2; Table 2).
Reports of increases in stomatal limitation of A following a chill are commonplace across numerous species susceptible to chill-induced inhibition of photosynthesis including tomato (Martin et al., 1981), olive (Bongi and Long, 1987), coffee (Bauer et al., 1985), grapevine (Flexas et al., 1999), and peanut (Bell et al., 1994). Chilling of roots is known to reduce hydraulic conductivity (McWilliam et al., 1982; Bagnall et al., 1983; Radin, 1990) and thereby lower photosynthesis through increases in stomatal limitation (Day et al., 1991; Bassirirad et al., 1993). However, to mimic natural conditions more closely, only the aerial parts of the mango trees were chilled since soil temperature does not usually drop substantially in response to a single cold night in the field. All plants were watered prior to chilling.
Low shoot temperature can increase cavitation of xylem vessels reducing hydraulic conductivity of the stems, however, freezing is usually required for significant embolism to occur (Sperry et al., 1988). Indeed, the L of control and chilled mango leaves in the controlled environment study were indistinguishable (Table 1). Throughout the photoperiod L remained high (-0.1 to -0.2 MPa), as measured with a pressure chamber (Table 1). This is presumably due to the well-watered status of the plants and the low VPD in the growth chamber. In all previous reports of which the authors are aware, L in well-watered mango measured with a pressure chamber range from -0.2 to -0.3 MPa, both in the early morning (Núñez-Elisea and Davenport, 1994; Pongsomboon et al., 1997) and at midday (Larson et al., 1991). The only other report of mango leaf L known to this study, gave much more negative values of -0.6 to -1.1 for pre-dawn, and midday minimums of -1.4 to -1.7 MPa in 2-year-old potted plants, measured with a psychrometer (Pongsomboon et al., 1992). Equivalent measurements to those given in Table 1, using isopiestic thermocouple psychrometry (Ehret and Boyer, 1979) gave values of -1.0 to -1.5 MPa, with no significant chill effect (data not shown). The discrepancy between psychrometry and pressure chamber measurements of L in this species may reflect errors in the former technique due to the presence of waxes and other materials on the leaf surface which take a long time to adsorb water vapour in the psychrometer (JS Boyer, personal communication). Overall, these data show that there was not a significant chill-induced limitation in water supply to leaves in the controlled environment study. Therefore, the observed increase in stomatal limitation at midday following a cold night arises due to low temperature impacts at the leaf level, independent of water availability.
In chill-sensitive herbaceous crops, such as cucumber, bean, cotton, soybean, and mung bean, low temperatures can result in the inability of stomata to close, despite a severe water stress, resulting in leaf wilting (Bagnall et al., 1983; Eamus, 1987; Guye and Wilson, 1987). Chilling mango did not appear to desensitize stomata to water stress as seen in herbaceous plants, as stomatal limitation was unchanged or increased following the chill under field conditions of high VPD (Fig. 2). Moreover, there was also no obvious loss of turgor in any of these field experiments. Chilling has been reported to induce stomatal opening at night (Honour et al., 1995), which potentially could result in lower pre-dawn L and stomatal closure later in the day. However, in these experiments the plants were chilled at close to saturation vapour pressure so that there would be no reduction of pre-dawn L in response to chilling thereby avoiding this potential complication. Temperature has also been demonstrated to reverse the response of stomata to abscisic acid (ABA), but since chilling results in stomatal opening in response to ABA (Rodriguez and Davies, 1982; Eamus and Wilson, 1983; Cornic and Ghashghaie, 1991; Honour et al., 1995) this cannot be the direct cause of the increase in stomatal limitation observed here. It has further been reported that chilling can make stomata more sensitive to CO2 concentrations (Drake and Raschke, 1974; Raschke, 1975). It is possible that a chill-induced inhibition of A, through lower Rubisco activity, after 2 h of the photoperiod, produced a transient increase in ci. If chilling had sensitized guard cell responses to CO2, then stomatal closure in response to this rise could, in turn, bring about the observed increase in stomatal limitation in the middle of the day.
Rubisco activity is regulated at numerous levels (Portis, 1995; Servaites and Geiger, 1995; Zhang and Portis, 1999) and it is not possible from these in vivo measurements to determine which may be involved in deactivating the enzyme in response to dark chilling. However, the time scale of the observed changes, and its ready reversibility later in the afternoon, indicate an effect on Rubisco activation state rather than on protein abundance or irreversible damage to the enzyme. Furthermore, the absence of balancing declines in PSII electron transport suggests that the reduction in A is not due to an energetic limitation related to a reduction in 
pH formation capacity. Low temperatures have been demonstrated to inhibit leaf starch mobilization at night (for review see Leegood and Edwards, 1996), and this could lead to end-product inhibition of photosynthesis, by reduced Pi return to the chloroplast (Sharkey, 1984; Stitt, 1991). However, no evidence for this being the cause of the midday depression in photosynthesis in this study could be detected, as the proportionate increase in A in response to a switch to non-photorespiratory conditions, was not reduced concurrently with A, following the dark chill. Chilling disrupts the circadian rhythms in the steady-state mRNA level of key photosynthetic proteins, including Rubisco activase, in tomato (Martino-Catt and Ort, 1992). The long lifetime of Rubisco activase suggests that this is unlikely to be the direct cause of the observed decline in Vc,max (Figs 1, 2). However, low temperature disruption of circadian rhythms in the activities of SPS and NR, enzymes influencing the partitioning of the products of photosynthesis, suggested that this possibility needed to be examined, especially as there were no other obvious candidates to explain the observed midday depression in photosynthesis. In particular, the possibility that the midday stomatal closure was a result of chilling disrupting the circadian regulation of stomatal opening, was examined.
Under constant conditions the synchronization between ci, A and gs suggest that most of the circadian rhythm in CO2 assimilation was due to reduced ci during the subjective night (i.e. the times that would have been dark under diurnal growth conditions) as a result of stomatal closure (Fig. 3), as previously seen in bean (Hennessy and Field, 1991). It is not possible to determine, from the data presented here, whether there was an additional circadian rhythm in mesophyll photosynthesis, independent of the stomatal rhythm. Stomatal rhythmicity has been investigated over many years with Maskell reporting as early as 1928 (Maskell, 1928) the loss of the photosynthetic rhythm when the leaf surface was cut, allowing CO2 uptake to occur independently of the stomatal rhythm in Prunus laurocerasus. In an experiment conducted with considerably greater environmental control and sensitivity, a rhythm in A was observed even when ci was maintained constant (Hennessey and Field, 1991) indicating that a rhythm in photosynthesis exists, independent of the stomata, in bean. With no circadian rhythm in PSII detectable (Fig. 3b, e, h) it is likely that the relative rate of photorespiration to photosynthesis increased during the subjective night, when the ratio of CO2 to O2 inside the leaf fell, thereby maintaining a constant sink for electrons and reducing power.
A dark chill did not disrupt the large rhythm observed in A (Fig. 3d), showing that the stomatal rhythm was unaffected. To ensure that the dark-to-light transition between the chill and the free-running conditions was not resetting a chill-disrupted rhythm, data from a low light chill in which there was no light transition following the chill and observation of rhythms were also obtained (Fig. 3g). The maintenance of a circadian pattern after light and dark chills indicates that a low temperature disruption of the circadian rhythm in stomatal opening, was not behind the observed midday rise in stomatal limitation and depression of photosynthesis (Figs 1, 2). However, this does not exclude the possibility that a stoma independent photosynthetic rhythm could have been impacted.
In summary, the causes of a decline in photosynthesis following a dark chill in the subtropical fruit tree mango, contrast with the behaviour of the chill-sensitive herbaceous species, like tomato. Unlike tomato, but in common with another subtropical woody species, coffee (Bauer et al., 1985), mango had no inhibition of photosynthesis immediately following the dark chill, but exhibited a large reduction of A, after 2 h of the subsequent photoperiod. This midday inhibition was not primarily a result of PSII photoinhibition, but coincided with an increase in stomatal limitation of A and a decrease in Rubisco activity. Chill-induced stomatal closure was not caused by disruption of circadian regulation of stomatal conductance, but may have been the result of altered guard cell sensitivity to CO2 following the chill.
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Acknowledgments |
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This work was supported in part by a grant from the USA–Israel Binational Agricultural Research and Development Fund (BARD; IS-2710-96). We are grateful to Evan DeLucia for the use of a gas exchange system for the work in Israel and to John Boyer for valuable advice on water potential measurements.
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Notes |
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3 To whom correspondence should be addressed. Fax: +1 217 244 0656. E-mail: d\|[hyphen]\|ort{at}uiuc.edu
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PSII, relative quantum efficiency of PSII electron transport; L, leaf water potential; A, net CO2 assimilation rate; ca, CO2 concentration leaving the leaf gas exchange cuvette; ci, intercellular CO2 concentration; Fv/Fm, maximum quantum efficiency of PSII photochemistry; gs, stomatal conductance; Jmax, maximum potential rate of electron transport contributing to RuBP regeneration; l, stomatal limitation to A; PPFD, photosynthetically active photon flux density; RuBP, ribulose-1, 5-bisphosphate; Rubisco, RuBP carboxylase/oxygenase; Vc,max, maximum carboxylation velocity of Rubisco; VPD, vapour pressure deficit. |
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