Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 53, No. 375, pp. 1781-1791, August 1, 2002
© 2002 Oxford University Press

Effects of water deficit and its interaction with CO₂ supply on the biochemistry and physiology of photosynthesis in sunflower

Received 20 August 2001; Accepted 15 April 2002

W. Tezara^3,1, V. Mitchell², S. P. Driscoll² and D. W. Lawlor²

¹ Instituto de Biología Experimental, Universidad Central de Venezuela, Apartado 47114, Caracas 1041 A, Venezuela
² Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK

³ To whom correspondence should be addressed. Fax: +58 212 7535897. E-mail: wtezara{at}strix.ciens.ucv.ve

	Abstract

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Photosynthetic responses of sunflower plants grown for 52 d in ambient and elevated CO₂ (A=350 or E=700 µmol mol^–1, respectively) and subjected to no (control), mild or severe water deficits after 45 d were analysed to determine if E modifies responses to water deficiency. Relative water content, leaf water potential (_w) and osmotic potential decreased with water deficiency, but there were no effects of E. Growth in E decreased stomatal conductance (g_s) and thereby transpiration, but increased net CO₂ assimilation rate (P_n, short-term measurements); therefore, water-use efficiency increased by 230% (control plants) and 380% (severe stress). Growth in E did not affect the response of P_n to intercellular CO₂ concentration, despite a reduction of 25% in Rubisco content, because this was compensated by a 32% increase in Rubisco activity. Analysis of chlorophyll a fluorescence showed that changes in energy metabolism associated with E were small, despite the decreased Rubisco content. Water deficits decreased g_s and P_n: metabolic limitation was greater than stomatal at mild and severe deficit and was not overcome by elevated CO₂. The decrease in P_n with water deficiency was related to lower Rubisco activity rather than to ATP and RuBP contents. Thus, there were no important interactions between CO₂ during growth and water deficit with respect to photosynthetic metabolism. Elevated CO₂ will benefit sunflower growing under water deficit by marginally increasing P_n, and by slowing transpiration, which will decrease the rate and severity of water deficits, with limited effects on metabolism.

Key words: Key words: Elevated CO₂, fluorescence, metabolism, photosynthesis, water deficit.

	Introduction

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Atmospheric CO₂ concentration (C_a), which was about 280 µmol mol^–1 before the industrial revolution, is now 360 µmol mol^–1 and is increasing by 1.8 µmol mol^–1 year^–1 (Houghton et al., 2001). This will affect vegetation, as elevated CO₂ substantially increases photosynthetic CO₂ assimilation rate (P_n), and thereby growth and total biomass, particularly of plants with the C₃ photosynthetic metabolism (Lawlor and Keys, 1993; Bowes, 1996; Drake et al., 1997). Also, increased C_a has already resulted in warmer temperatures globally, a trend expected to continue. Changes in temperature are likely to alter precipitation worldwide, decreasing it in many areas (Houghton et al., 2001). This, together with the higher evapotranspiration resulting from warmer conditions, is expected to subject vegetation, both natural and agricultural, to greater risk of more severe and prolonged water deficiency (Sengupta and Sharma, 1993; Samarakoon and Gifford, 1995; Ellsworth, 1999). Decreasing water supply will greatly decrease growth. Such changes in CO₂ and water supply affect many key metabolic and physiological processes in plants, the mechanisms of which are still unclear. Particularly, there is limited quantitative understanding of the effects of interactions between CO₂ and water deficiency (Chaves and Pereira, 1992; Tschaplinski et al., 1995, 1996; Samarakoon and Gifford, 1995).

The effect of increased C_a results from the increased supply of CO₂ to the photosynthetic carbon reduction cycle enzyme ribulose bisphosphate carboxylase-oxygenase (Rubisco), which is not saturated with CO₂ in the current atmosphere (Bowes, 1996; von Caemmerer, 2000). Consequently, more carbohydrates are synthesized, stimulating faster and greater growth of biomass (Lawlor and Mitchell, 1991; Drake et al., 1997). Also, carbohydrates accumulate as a result of carbohydrate supply exceeding the demands for growth of the whole plant (Eamus and Jarvis, 1989). Establishment of new sinks, or stimulation of existing ones, is beneficial under elevated CO₂ to consume carbohydrates and avoid imbalance in metabolism (Field and Mooney, 1986; Stitt, 1991). The increased P_n occurs despite smaller g_s caused by elevated CO₂. In spite of the general stimulation in P_n, there is considerable variation in the response of photosynthetic capacity (i.e. the maximum rate of P_n attained under particular conditions of light and temperature at saturating CO₂ supply, etc.) when the plant is grown in elevated CO₂ (DeLucia and Thomas, 2000). The response ranges from greatly decreased capacity via no change to increased capacity (Drake et al., 1997; Morison and Lawlor, 1999). Changes in P_n may result (Lawlor and Keys, 1993; Moore et al., 1998) from altered regulation of the photosynthetic mechanism without modifications to amounts of components: increased P_n is often called ‘up-regulation’ and decreased P_n, ‘down-regulation’. Also longer-term changes in P_n due to altered photosynthetic capacity may occur: such ‘acclimation’ may include increased (Habash et al., 1995), or decreased capacity (Drake et al., 1997). Loss of capacity (using the common terminology it will be referred to as acclimation) is the more common response of plants grown in elevated CO₂. It is associated with altered tissue composition (‘machinery’) and, particularly, decreased amounts and activities of Rubisco (Lawlor and Keys, 1993; Moore et al., 1998). With elevated CO₂, the capacity for carboxylation may exceed the rate of ribulose bisphosphate (RuBP) regeneration, which initially results in decreased activity of Rubisco (down-regulation). Other mechanisms within the leaf may respond to this imbalance and the amount of Rubisco decreases (Stitt, 1991; Sage, 1994; Moore et al., 1998). In some cases there may be a shift towards increased electron transport leading to greater production of ATP and NADPH, and thus of RuBP by the PCR cycle. Physiological re-optimization of the photosynthetic machinery away from CO₂ fixation (especially Rubisco) towards increased RuBP synthesis, involving increased components for electron transport and ATP synthesis does not always occur.

The decline in P_n and acclimation in elevated CO₂ is correlated with suboptimal growth conditions, for example, small rooting volume, which affects nutrient supply (Sage, 1994; Drake et al., 1997; Morison and Lawlor, 1999). Indeed, such alterations in composition may occur predominantly with deficient nutrient, especially N, supply: in wheat, for example, this depends on the nitrogen supply (Mitchell et al., 2000). However, the effects of water deficiency are not well established (Tyree and Alexander, 1993; Samarakoon and Gifford, 1995; Tschaplinski et al., 1995, 1996).

Water deficits affect plants in very different ways when subjected to elevated CO₂. Long-term, slowly developing water deficits decrease growth, by slowing rates of cell division and expansion due to loss of turgor and increased synthesis of abscisic acid (Lawlor and Cornic, 2002). Also, g_s decreases, thus decreasing transpiration but also limiting P_n. With current C_a, C_i may decrease if photosynthetic capacity is not affected by water deficit, or is decreased less than g_s. With more severe deficits the photosynthetic capacity is decreased (Tezara et al., 1999), although there is still lack of agreement about the severity of the deficit at which capacity decreases, and the nature of the changes in photosynthetic mechanism responsible (Lawlor and Cornic, 2002; Lawlor, 2002). The argument may be simplified into stomatal limitation of CO₂ supply or metabolic limitation. If it is accepted that in leaves with mild water deficits, the restriction of water loss and of CO₂ uptake by small g_s predominates, then elevated C_a should ameliorate the development of water deficits and increase or maintain P_n. This would help maintain use of captured light energy for NADPH and ATP synthesis by providing more sink capacity and thus diminish accumulation of excitation energy in the photosynthetic pigments, which is a major cause of photo-oxidative damage (Scarascia-Mugnozza et al., 1996). Elevated atmospheric CO₂ should therefore be beneficial for plants in dry environments, offsetting some of the consequences of global environmental change. However, acclimatory changes in the photosynthetic machinery consequent upon growth in elevated CO₂ may reduce the benefits under water deficits. For example, smaller Rubisco carboxylation capacity and greater light capture with greater capacity for synthesis of NADPH and ATP (an apparent benefit in elevated CO₂ for regeneration of ATP) would predispose plants to photo-oxidative damage (Scarascia-Mugnozza et al., 1996).

There have been relatively few studies of the interaction between CO₂ and water stress (Vu et al., 1987; Tschaplinski et al., 1995, 1996) and understanding of the mechanisms is less well developed than that of CO₂ and temperature (Morison and Lawlor, 1999). Does growth in elevated CO₂ cause positive or negative acclimation of the photosynthetic apparatus? Are effects of CO₂ altered by water deficit? Does elevated CO₂ decrease the impact of water deficits on P_n and what processes in photosynthetic metabolism are affected? Is there interaction between elevated CO₂ and water deficits of varying severity? It is hypothesized that elevated, compared with ambient, CO₂ will increase P_n under water deficit and be beneficial for plants in dry environments, thus offsetting some of the consequences of global environmental change. To answer these questions and test the hypothesis, ATP, RuBP and Rubisco (both content and activity) were measured, and photosynthetic energy transduction and dissipation were assessed by measurement of chlorophyll a fluorescence. The focus of this study was on sunflower as its responses to water deficits and elevated CO_2, both as single environmental factors,have been analysed in detail (Kramer and Boyer, 1995; Tezara et al., 1999).

	Materials and methods

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Plant material and growth conditions
Sunflower plants (Helianthus annuus L. cv. Avante) were grown from seed in plastic pots filled with 3.0 l of sintered argilite clay (Terra-green, Silvaperl Products Ltd, Harrogate, UK), which has high water-holding capacity. Plants were watered daily and fertilized weekly with sufficient nutrient solution (Vitafeed 412: Vitax, Skelmersdale, UK) for maximum growth (based on previous experiments). Plants were grown in naturally lit growth chambers (Lawlor et al., 1993) with supplementary light giving an approximately 16 h light period and 8 h dark period. Supplementary light of photosynthetic photon flux (PPF: 400–700 nm wavelength) of 350 µmol m^–2 s^–1 was applied for 12 h giving an average PPF of approximately 500 µmol m^–2 s^–1 and a maximum of c. 1100 µmol m^–2 s^–1. Temperature was maintained at 25/18 °C (±2 °C day/night) and 70–85% relative humidity in the day and above 90% at night (day/night). Plants were grown in one chamber under ambient CO₂ concentration (A, 350±20 µmol mol^–1) and one under elevated CO₂ concentration (E, 700±20 µmol mol^–1). The CO₂ concentrations were continuously measured in each chamber by independent infrared gas analysers (WMA, PP Systems, Hitchin, UK) calibrated at least weekly with standard CO₂ mixtures calibrated against gas mixing pumps (Wösthoff, Bochum, Germany). When the CO₂ concentrations fell below the set-points, pure CO₂ was injected, under control of solenoid valves, into the air flow of the chambers to maintain concentration. Chamber conditions (light, temperature, humidity) were very similar, but to avoid bias due to small differences, which might have had cumulative effects on the plants over the relatively long duration of the experiment, the CO₂ treatments and plants were exchanged between chambers approximately every 4 d.

Water deficit treatments
Water deficit was induced in 45-d-old plants, with the fourth pair of true leaves just fully expanded, by differential watering. Plants were weighed and water lost replaced early each morning. Well-watered (control) plants received sufficient water to maintain soil water content close to pot capacity (an average of c. 300 ml d^–1), or two-thirds (mild deficit) or one-third (severe deficit) of that. After 12 d of differential watering, plants were not watered the following morning and measurements were made during the day. To accommodate the measurement programme, the experiment was started on different days so that some replicates were measured on different occasions. Data presented are averaged over time. Thirty out of 90 grown at each CO₂ concentrations were harvested for growth analyses before the beginning of the water deficit treatments; the remaining 60 plants were subjected to three different water treatments (20 plants for each water treatment). Several experiments were done to measure the physiological parameters described below, and on each experiment not all the parameters were measured concomitantly, except for _w, RWC and .

Physiological measurements
Leaf water status: Leaf water potential (_w) was measured with a custom-made pressure chamber (IACR-Rothamsted, UK) using nitrogen gas on the fourth leaves from five individual plants, chosen at random from the 20 replicate pots per water deficit in each CO₂ treatment chamber. After the measurement of _w, relative water content was determined using five leaf discs from each leaf; RWC was determined as (RWC=(F_w–D_w)/(T_w–D_w), where F_w is fresh mass of the discs, T_w is turgid mass after 6 h floating on distilled water at room temperature, and D_w is dry mass after oven drying for 24 h at 80 °C). Osmotic potential () was determined with a vapour pressure osmometer (Wescor, Logan, USA) on five discs, which were taken from each leaf and frozen and thawed before measurement. Trials demonstrated that RWC and osmotic potential were unaffected by using leaves from the pressure chamber or by storage for . For these determinations, n=30.

Plant growth: Thirty plants were harvested in each CO₂ treatment when the water deficits were started. Roots were washed from the soil, and plants separated into roots, stems, leaves, and developing flowers. Parts were dried at 80 °C for 48 h.

Gas exchange: All measurements of P_n, g_s and C_i were done on the fourth, fully expanded intact leaf using a six-chamber computerized open gas-exchange system with infrared CO₂ measurements (WA-225-MK3 IRGA; Analytical Development Co., Hoddesdon, UK) as described by Jacob and Lawlor (1991). Ten cm² of the lamina (avoiding major veins) of leaves attached to the plant were sealed into each chamber, which was illuminated with PPF of 1400 µmol photons m^–2 s^–1; leaf temperature was 25 °C and the air contained 0.21 mol mol^–1 O₂ and 350 or 700 µmol mol^–1 CO₂. The vapour pressure deficit between the leaf and chamber air was maintained at 1.2±0.2 kPa. Measurements of gas exchange were taken during steady-state photosynthesis after a 1 h period of adjustment of the leaf to the chamber conditions. The calculations of P_n, g_s and C_i were made according to Farquhar and Sharkey (1982).

Determination of P_n/C_i response curves: Response of CO₂ assimilation (P_n) to intercellular CO₂ concentration (C_i), the P_n/C_i response curve, was measured in the open gas exchange system by increasing C_a from 0 to 1000 µmol mol^–1. Carboxylation efficiency was calculated from the initial slope (dP_n/dC_i) of the response curve and P_nmax (the CO₂ saturated rate of photosynthesis) from the asymptote of the fitted response function (von Caemmerer and Farquhar, 1981; Farquhar and Sharkey, 1982). Stomatal and metabolic limitations (L_s and L_m, respectively) were analysed as L_s=100x(P'_n–P_n)/P'_n, where P'_n is the photosynthetic rate to be expected when C_i=C_a. The relative mesophyll limitation, L_m, was calculated from Jacob and Lawlor (1991) and L_m=100x(P_n(c)–P_n(d))/P_n(c), where P_n(c) is the photosynthetic rate in control leaves at C_i of 500 µmol mol^–1, and P_n(d) is the rate in droughted leaves at the same C_i. L_m is thus a measure of the capacity of the mesophyll to fix CO₂ at 500 µmol mol^–1 C_i and is zero in control leaves.

Photosynthetic light response curves: Response of P_n to PPF (the light response curve) was measured at two different C_a (350 and 2500 µmol mol^–1) and a leaf temperature of 25 °C by illuminating the leaf at increasing PPF until P_n was constant. Incident PPF on the leaf was modified using neutral density filters (Lee Filters; AC Lighting, Bucks., UK). The apparent quantum yield of CO₂ fixation (_CO2) was calculated as the slope of the linear portion of the response curves between 0 and 150 µmol m^–2 s^–1 PPF.

Chlorophyll a fluorescence measurements: Fluorescence from PSII chlorophyll a of the leaves was measured simultaneously with gas exchange, through the glass window of the chambers at leaf temperature of 25 °C as described by Habash et al. (1995), using a modulated fluorometer (MSMF; Hansatech, Kings Lynn, UK). A modulated beam of less than 0.5 µmol m^–2 s^–1 PPF, set at 580 nm using a narrow-band pass filter (585 DF 44; Omega Optical Inc., Brattleboro, VT, USA) was used and the resulting fluorescence selectively measured at 695 nm using a band pass filter (695 DF 30; Omega Optical). Actinic illumination from metal-halide lamps, was used for both gas-exchange and fluorescence measurements. To reduce Q_A fully, a 2 s saturating flash of 7000 µmol m^–2 s^–1 PPF of 400–635 nm was applied from a pulse light source (PLS2; Hansatech, Kings Lynn, UK). Far-red light of 15 W m^–2 from the PLS2 through a far-red filter (RG 715; Schott, Mainz, Germany) allowed the determination of minimum fluorescence (F'_o) immediately after steady-state photosynthesis ceased. The protocol for fluorescence measurement was similar to that described by Genty et al. (1989), except that the measurements were performed on attached leaves. The relative quantum yield of PSII (_PSII) at steady-state is defined as (F'_m–F_s)/F'_m where F_s and F'_m are steady-state fluorescence and maximum fluorescence in the light, respectively. The coefficients of photochemical (q_P) and non-photochemical (q_NP) quenching of chlorophyll a fluorescence were calculated from measurements of fluorescence. Whole chain electron transport rate in leaves (J) was estimated by the method of Krall and Edwards (1992) from the equation: J=_PSIIxPPFxaxf where a is the fraction of incident PPF absorbed by the leaf, and f the absorption of PSII divided by the absorption of (PSI+PSII). It is assumed that the two photosystems are equally involved in linear electron transport, so f=0.5.

Biochemical determinations
Total soluble protein content (TSP), Rubisco content and activity, and RuBP, ATP, and chlorophyll contents were determined on samples immediately after gas exchange measurements at C_a=350 µmol mol^–1 and 1400 µmol m^–2 s^–1 PPF. When stable P_n was reached, the section of lamina in the chamber was frozen to –20 °C within 0.1 s by freeze clamping (Lawlor et al., 1989) and stored in liquid N₂. The TSP was determined in an aliquot of the crude extract by Coomasie blue binding (Bradford, 1976) with BSA as standard. Rubisco was extracted from a 5 cm² area of the freeze-clamped leaf, which had been kept in liquid nitrogen. Frozen samples were ground in liquid nitrogen, and then at 0–4 °C, in 1 ml buffer (100 mol m^–3 Bicine, pH 8.0; 20 mol m^–3 MgCl₂, 50 mol m^–3 mercaptoethanol, 10 ml 40 mol m^–3 phenylmethylsulphonyl fluoride, and 10 mg acid-washed PVP). Initial Rubisco activity was assayed as described by Parry et al. (1993). Rubisco content was measured by separation of proteins in the extract on 15% SDS-PAGE gels: Rubisco was identified and quantified by comparison with standard Rubisco protein (Lawlor et al., 1989). RuBP was extracted in 5% (v/v) perchloric acid on independent freeze-clamped leaf samples and measured by ¹⁴C incorporation into acid-stable products using purified Rubisco and NaH¹⁴CO₃ (Giménez et al., 1992). ATP content was determined enzymatically (Stitt et al., 1989).

Statistical analysis
Plants used for measurements were randomly selected from the 20 replicate plants per water stress treatment, grown in each CO₂ chamber. They constitute pseudoreplicates as only a single chamber per CO₂ treatment was used (because of space and costs). Frequent movement of CO₂ treatments and associated plants between rooms, with carefully standardized conditions, is an accepted method of ensuring that the CO₂ treatment can be separated from the effects of other factors (Lawlor et al., 1993). The statistical analyses were carried out using Genstat (IACR-Rothamsted), and curve fitting by Sigmaplot. All linear regressions, correlations and variance analyses (single- and two-factor) were tested for significance at P 0.05. Results are presented as means (5n10)±1SE.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Application of three different watering regimes decreased RWC, _w and substantially for plants grown under both ambient and elevated CO₂, with no significant effect of elevated CO₂ concentration on them (Table 1). Gas exchange measurements (Fig. 1) showed that decreasing _w progressively and significantly decreased P_n (Fig. 1A, B) and g_s (Fig. 1C, D) for plants grown and measured in both ambient and elevated CO₂. There were no differences between P_n or g_s related to CO₂ concentration during growth and no interaction between water deficits and CO₂ concentration. P_n of leaves of plants not subjected to water deficit was significantly (P <0.01) larger when measured in C_a=700 µmol mol^–1 (Fig. 1B) than in C_a=350 µmol mol^–1 for both ambient and elevated CO₂-grown plants (c. 40% and 55%, respectively; Fig. 1A compared to Fig. 1B). P_n of severely stressed leaves of plants grown in elevated CO₂ was slightly but not significantly smaller than that of ambient grown-plants when measured with C_a=700 µmol mol^–1 (Fig. 1B), but not when measured in C_a=350 µmol mol^–1. P_n of the water-deficient leaves measured in elevated CO₂ was higher than those grown in ambient CO₂ (Fig. 1B compared to Fig. 1A).

View this table: [in this window] [in a new window]   Table 1. Leaf water status of sunflower plants grown under ambient (A) and elevated (E) CO2 concentrations, and subjected to no (C), mild (M), and severe (S) water deficit Measurements under the conditions of growth. Values are means (n=30); * indicates statistically significant difference at P 0.05 (ANOVA) and NS not significant.

 

View larger version (30K): [in this window] [in a new window]   Fig. 1. Net photosynthetic CO2 assimilation, Pn (A, B), stomatal conductance, gs (C, D), and instantaneous water use efficiency, WUE (E, F) of leaves of sunflower plants grown at elevated (E, 700 µmol mol–1 CO2) and ambient (A, 350 µmol mol–1 CO2) CO2 concentrations with three different water deficits: none (control, C, white bars) mild (M, hatched bars), and severe (S, black bars). Measurements were made on leaves attached to the plants at 350 (left panels) or 700 µmol mol–1 (right panels) CO2.

 
Stomatal conductance was affected by water deficits and CO₂ during growth. For plants not subjected to water deficit, g_s of those grown in elevated CO₂ was 42% lower (significant at P <0.01; Fig. 1C, D) than those grown in ambient CO₂, irrespective of the CO₂ concentration during measurement. With mild and severe water deficit, g_s was much smaller than in the well-watered plants (c. 0.05 compared to 0.6 mol m^–2 s^–1), and similar in plants grown and measured in both CO₂ concentrations.

The WUE was not significantly altered by the mild water deficit, but was decreased by the severe deficit for plants grown in both ambient and elevated CO₂ when measured at 350 µmol mol^–1 CO₂. However, WUE decreased progressively with water deficit when measured at elevated CO₂ (Fig. 1E, F). Elevated CO₂ concentration during growth did not affect WUE when measured in ambient or elevated CO₂. However, WUE measured in elevated CO₂ was substantially higher than that in ambient CO₂ (by 230% and 380% in control and severely stressed plants, respectively: Fig. 1E, F).

The response of P_n to C_i (Fig. 2), shows that neither P_nmax nor carboxylation efficiency, were affected by growth of the leaves in elevated CO₂ at any water deficits. However, both P_nmax and carboxylation efficiency declined substantially and significantly with decreasing _w (Fig. 2; Table 2). Neither stomatal nor mesophyll limitation of P_n (Table 2) was significantly affected by elevated CO₂, although stomatal limitation was somewhat greater in plants grown in elevated CO₂, where g_s was smaller, than in those grown in ambient CO₂. However, L_s with water deficit in elevated CO₂ was smaller than in ambient CO₂ (Table 2). The L_m increased substantially with water deficit, and was slightly (but not significantly) less in leaves grown in elevated than ambient CO₂. The operating C_i was greater with C_a of 700 µmol mol^–1 than ambient (545 cf. 265 µmol mol^–1) (Table 2) but was not affected by the CO₂ during growth when measured in ambient CO₂.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Response of net photosynthetic CO2 assimilation (Pn) to intercellular CO2 concentration (Ci) in leaves of sunflower plants grown at ambient (open symbols) and elevated (closed symbols) CO2 concentrations and subjected to no (circles), mild (squares) or severe (triangles) water deficits. Values are means (n=6) and standard errors are shown when greater than the size of the symbol.

 

View this table: [in this window] [in a new window]   Table 2. Maximum Pn, carboxylation efficiency (dPn/dCi) relative stomatal (Ls) and mesophyll limitations (Lm) to photosynthesis and operating Ci of sunflower plants grown in ambient (A) and elevated CO2 concentrations (E) and subjected to no (C), mild (M), or severe (S) water deficit Values are means (n=6); leaves in ambient or elevated CO2; * indicates statistically significant difference at P 0.05 (ANOVA) and NS not significant.

 
Growth under elevated CO₂ did not significantly alter the parameters of the light response curves (Fig. 3) at any _w when measurement C_a was 350 µmol mol^–1. However, when measurement C_a was 2500 µmol mol^–1, P_nmax was significantly lower in plants grown under water deficits with elevated CO₂ compared to those grown in ambient CO₂, due to a 43% decrease in g_s (values not shown) of plants grown with mild deficit and 33% with severe deficit. The _CO2 (Fig. 3) was not affected by growth under elevated CO₂. However, it was higher when measured at C_a=2500 µmol mol^–1 at all _w. At large PPF, electron transport rate, J, was greater (Table 3), in leaves grown with ample water, in elevated than at ambient CO₂ (210 cf. 164 µmol e^– m^–2 s^–1) when measured at C_a=350 µmol mol^–1. The difference disappeared when _w decreased and J fell by 28% and 48% in the mildly and severely deficient plants. Elevated CO₂ during measurement increased J in all the treatments, particularly in the severely stressed leaves (71% cf. to 45% in the control). However, over all treatments, there was no significant effect of CO₂ during growth, but a significant effect of water deficit.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Responses of photosynthetic rate (Pn) to photosynthetic photon flux (PPF) for sunflower plants grown at ambient (open symbols) and elevated (closed symbols) CO2 concentrations, and measured at ambient CO2 of 350 and 2500 µmol mol–1 CO2, and subjected to no (circles), mild (squares) or severe (triangles) water deficits. Values are means ±SE (n=6).

 

View this table: [in this window] [in a new window]   Table 3. Calculated rates of electron transport, J (µmol e– m–2 s–1), for sunflower plants grown at ambient (A) and elevated (E) CO2 concentrations (350 and 700 µmol mol–1, respectively), and measured at 350 and 2500 µmol mol–1 CO2 under 800 µmol m–2 s–1, and subjected to no (C), mild (M), or severe (S) water deficit Values are means ±SE (n=6); * indicates statistically significant difference at P 0.05 (ANOVA) and NS not significant.

 
The _PSII of leaves grown at both CO₂ concentrations, decreased progressively as the PPF increased and as _w became smaller when measurements were done at 350 µmol mol^–1 CO₂ (Fig. 4A), whilst _PSII of plants measured at 2500 µmol mol^–1 was less affected by _w (Fig. 4B). In plants grown without water deficit and measured at 350 µmol mol^–1, _PSII was slightly higher in plants grown in elevated CO₂ than in those grown in ambient CO₂, and this effect disappeared when _w decreased. However, when measurements were done at 2500 µmol mol^{– 1} the effect of declining _w on _PSII was far smaller and there was no difference between plants grown at different CO₂ concentrations.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Quantum yield of electron transport through photosystem II, as a function of photosynthetic photon flux (PPF) for sunflower plants grown at ambient (open symbols) and elevated (closed symbols) CO2 concentrations, measured at 350 and 2500 µmol mol–1 of CO2, and subjected to no (circles), mild (squares) and severe (triangles) water deficits. Values are means ±SE (n=6).

 
The q_P and q_NP were significantly affected (Table 4) by water deficiency when plants were grown in both CO₂ concentrations, and measured at 350 and 2500 µmol mol^–1, with the exception of q_NP measured at low PPF (150 µmol m^–2 s^–1) at both CO₂ concentrations. However, growth under elevated CO₂ caused no significant differences in q_P or q_NP irrespective of measurement at C_a=350 or 2500 µmol mol^–1 CO₂ or measurement at a PPF of 150 or 800 µmol m^–2 s^–1 (Table 4). The q_NP measured at PPF of 800 µmol m^–2 s^–1 was smaller when measured with a C_a of 2500 µmol mol^–1 than with C_a of 350 µmol mol^–1, irrespective of the CO₂ concentration during growth or water deficit. The F_v/F_m ratio was not affected by either the CO₂ or water treatments, averaging 0.84±0.03.

View this table: [in this window] [in a new window]   Table 4. The coefficient of photochemical fluorescence quenching (qP) and the coefficient of non-photochemical fluorescence quenching (qNP) of sunflower leaves grown at ambient (A) or elevated (E) CO2 concentration and subjected to no (C), mild (M), or severe (S) water deficit Measurements were done at PPF=150 or 800 µmol m–2 s–1 and at 350 µmol mol–1 or 2500 µmol mol–1 of CO2. Values are means (n=6); * indicates statistically significant difference at P 0.05 (ANOVA) and NS not significant.

 
Total soluble protein and Rubisco contents of leaves of well-watered plants were significantly reduced by growth in elevated compared to ambient CO₂ (17% and 25%, respectively); however, the initial activity of Rubisco was 32% higher in well-watered plants grown in elevated than in ambient CO₂ (Table 5). Under both growth CO₂ concentrations, the initial activity of Rubisco decreased with _w. The RuBP content in well-watered plants decreased slightly with growth in elevated versus ambient CO_2, but increased with water deficit. Severe water deficit decreased RuBP content more in plants grown in ambient CO₂ than in elevated CO₂. The ATP content of well-watered plants was the same, irrespective of the CO₂ concentrations during growth, but decreased by 50% with decreasing _w in both CO₂ concentrations.

View this table: [in this window] [in a new window]   Table 5. Total soluble protein (TSP], Rubisco content, Rubisco initial activity, and RuBP and ATP content in leaves of sunflower grown under ambient (A) and elevated (E) CO2 concentration, and subjected to no (C), mild (M), and severe (S) water deficit measurements made at ambient CO2 Values are means (n=6); * indicates statistically significant difference at P 0.05 (ANOVA) and NS not significant.

 
Plant growth was markedly increased by growth in elevated CO₂; after 45 d at the start of the water deficit treatment the total biomass per plant was 27.5±0.92 g in ambient and 35.7±0.7 g in elevated CO₂, a 30% increase. The largest effect was on the roots (53% increase), then stem (40% increase) with little effect on leaves (11%). However, with elevated CO₂, leaf area per plant decreased by 6%, and leaf weight ratio, specific leaf area and leaf area ratio also declined.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
This study tested the hypothesis that growth of sunflower plants in elevated CO₂ would stimulate the rate of photosynthesis and allow adjustment of cellular water balance, so decreasing the impact of water deficits on photosynthetic mechanisms (Samarakoon and Gifford, 1995). Elevated CO₂ would also provide a greater sink for electrons and decrease the potential for photoinhibitory damage (Scarascia-Mugnozza et al., 1996). As a result, photosynthesis would show a strong interaction between water deficit and CO₂ during growth. Acclimation of photosynthetic mechanisms, frequently caused by growth in elevated CO₂ (Wullschleger, 1993; Bowes, 1996), and the impact on the photosynthetic responses to water deficits were also assessed.

Elevated CO₂ was applied night and day for 45 d and continued for the next 12 d as the water deficits developed. Sunflower increased in biomass, particularly in root and stem, although leaf area decreased a little. Such stimulation of growth has been described for many annual, herbaceous (Lawlor and Mitchell, 1991; Lawlor et al., 1993; Drake et al., 1997; Morison and Lawlor, 1999) and perennial, woody species (Gunderson and Wullschlegger, 1994). The experimental CO₂ treatment and its duration was sufficient to allow acclimation of photosynthetic processes as shown by the decrease in Rubisco.

The water treatments were not designed to maintain constant water contents or potentials in soil or plants; indeed this is not possible for transpiring plants in soil (Kramer and Boyer, 1995). Because of the strongly non-linear relationships between water content, hydraulic conductivity and water potential in soils, _w in the plant is not linearly related to soil water content and potential (Kramer and Boyer, 1995). In the plant, RWC and _w may also not be simply related, but in these studies on sunflower they are (Tezara et al., 1999) and are used interchangeably for assessing water status. As water deficits decrease g_s and affect metabolism (either directly or via synthesis of abscisic acid), leading to a reduction in growth, the effect of a particular treatment depends on the integral of _w over the period (Kramer and Boyer, 1995). As the differential watering caused relatively large differences in _w over 12 d, it was assumed that changes in tissue composition and metabolism would have occurred, particularly as measurements were made when _w were minimal.

Twelve days of differential watering resulted in RWC, _w and which were very different for the three water treatments, but were very similar in ambient and elevated CO₂. Lower values of _w than with water deficits may reflect development of negative wall potential (Kramer and Boyer, 1995; Hopkins, 1995), although not observed in this variety of sunflower in similar experiments (Tezara et al., 1999). Similarity in RWC, _w, and between the water treatments in the two CO₂ concentrations may have been fortuitous, given the smaller g_s and transpiration rates. Suboptimal watering of plants grown in elevated CO₂ may result in a smaller deficit, i.e. a higher _w, compared to ambient CO₂ (Tyree and Alexander, 1993), but this is not always so (Ferris and Taylor, 1995; Sgherri et al., 1998; Ellsworth, 1999), including the present study. Difference between species in the interaction of CO₂ with water supply has been well demonstrated by Samarakoon and Gifford (1995). Even when _w is larger in elevated CO₂ (Tyree and Alexander, 1993; Tschaplinski et al., 1995; Allen et al., 1998; Huxman et al., 1998b) the increase may not be as large as predicted from the reduction in g_s and transpiration rate measured in leaf chambers. This may be due to decreased evaporative cooling raising leaf temperatures (Lawlor and Mitchell, 1991). The absence of significant differences between components of the leaf water balance resulting from growth in elevated CO₂ may thus be expected.

The relative importance of stomatal and metabolic limitations was assessed from P_n/C_i curves (Fig. 2), which eliminates the effect of decreased g_s resulting from the response of g_s to elevated CO₂ or induced by drought. The validity of calculated C_i, particularly with respect to water deficits (Lawlor and Cornic, 2002) has been questioned but it is considered valid by the authors. Elevated CO₂ during growth did not significantly affect the shape of the P_n/C_i response of well-watered plants, measured in either ambient or elevated CO₂, so that _CO2 and light-saturated P_n were not affected, suggesting that there were no changes in photosynthetic capacity (Fig. 3). Yucca brevifolia responded similarly (Huxman et al., 1998a). However, different photosynthetic parameters vary in response to elevated CO₂. Light-saturated P_n was more stimulated than _CO2 (i.e. CO₂ increased photosynthetic capacity but not efficiency) in four tree species (DeLucia and Thomas, 2000). The P_n/C_i curves in six C₃ tropical species showed substantial quantitative differences (Bunce and Ziska, 1999). Elevated CO₂ increased capacity in wheat in one experiment (Habash et al., 1995), but not in others (Mitchell et al., 2000). Photosynthetic rate decreased in Larrea tridentata (Huxman et al., 1998b), Picea abies (Urban and Marek, 1999), Yucca whipplei (Huxman et al., 1998a), sorghum (Watling et al., 2000), and Picea sitchensis (Centritto and Jarvis, 1999).

In this study, stomata limited (L_s) photosynthesis more in elevated than in ambient CO_2, as g_s was smaller. By definition, L_m is zero in watered plants under either CO₂ concentrations. The absence of an effect on P_n in plants grown in elevated CO₂ suggests no acclimation. However, this conflicts with the c. 25% decrease in Rubisco content (Table 4), which shows acclimation of tissue composition. Decreased Rubisco content frequently results from growth in elevated compared to ambient CO₂ (Sage, 1994; Drake et al., 1997), for example, in Picea sitchensis (Rubisco content was 36% lower; Centritto and Jarvis, 1999). Wheat responds variably, but this is probably caused by altered rates of development and leaf senescence, and attendant changes in N-content, than by CO₂ per se (Garcia et al., 1998; Mitchell et al., 2000). The factors determining Rubisco content in leaves grown under elevated, compared to ambient CO₂ have been extensively discussed (Stitt, 1991; Lawlor and Keys, 1993; Bowes, 1996; Moore et al., 1998). Causes of variation in acclimation were discussed in the introduction: the conclusion that variability may reflect nutrition, not CO₂ per se (Morison and Lawlor, 1999) is unlikely to apply in this study’s experiment, as nutrition was ample. The inhibition of Rubisco synthesis is possible (Moore et al., 1998). Decreased Rubisco content in sunflower was compensated by increased activity (32%), indicating much larger carboxylation activity per unit of Rubisco protein, which maintained P_n. This contrasts with the decreased initial activity described by Jacob et al. (1995) in Scirpus olneyi, which is often seen (Drake et al., 1997). The small decrease in RuBP in sunflower leaves grown in elevated CO₂, but measured in ambient CO₂, does not accord with the slightly decreased rate of P_n. Rather it is expected that increased P_n (e.g. under elevated CO₂) would increase demand for RuBP synthesis which is limited by the light reactions (von Caemmerer and Edmondson, 1986; von Caemmerer, 2000).

Elevated CO₂ during growth did not affect the energy dissipation of well-watered leaves, judging from the changes in q_P and q_NP (Table 4). Nor did it affect whole chain electron transport rate (J) in sunflower, averaged over the water deficits and measurement CO₂ treatments. But J was 34% higher when measured in elevated CO₂ than in ambient. In Cercis canadensis, Liquidambar styraciflua, Acer rubrum, and Carya glabra, J was, on average, 10% higher under elevated CO₂ than at ambient CO₂ and P_n was increased by 59% in A. rubrum but by 159–190% in the other species (DeLucia and Thomas, 2000). The J, and light-saturated P_n, were also increased in Pinus taeda (Hymus et al., 1999), and wheat (Habash et al., 1995). The _PSII of sunflower at low PPF was the same for leaves grown and measured in elevated and ambient CO₂, but at large PPF,_PSII was lower for plants grown and measured in ambient CO₂ than when measured in elevated CO₂. The decrease in _PSII due to low _w measured in ambient CO₂ disappeared in elevated CO₂. Similar responses occurred in C. canadensis, L. styraciflua and C. glabra (DeLucia and Thomas, 2000) and Habash et al. (1995). Electron transport and RuBP synthesis were modified; the maximum rate of J was 9% lower in ambient compared to elevated CO₂ and short-term exposure to elevated CO₂ decreased the maximum carboxylation rate by 46% and increased RuBP regeneration by 29% in Picea abies (Urban and Marek, 1999). The increase of J of sunflower plants growing at elevated CO₂ corresponded to an increase in _PSII, in contrast to Picea sitchensis where J decreased at elevated CO₂ (Centritto and Jarvis, 1999).

For sunflower, the slightly higher _PSII and possibly lower energy loss related to antenna-based quenching mechanism (q_NP), accorded with higher rates of J at large PPF when measurements were done at 2500 µmol mol^–1, indicating a larger electron sink in carboxylation at elevated than ambient CO₂ concentration. However, the response of q_P was unchanged by elevated CO₂ in this study, in contrast to the response of C. canadensis, L. styraciflua and C. glabra (DeLucia and Thomas, 2000) and wheat (Habash et al., 1995). This study’s results show that plants exposed to elevated CO₂ have the same proportion of open PSII centres at high PPF. At sub-saturating PPF (50–200 µmol m^–2 s^–1), P_n of sunflower was strongly limited by electron transport and RuBP regeneration rate, and by photorespiration in ambient CO₂ with the apparent quantum yield smaller than in elevated CO₂. These results suggest that the capacity for electron transport and the requirement for electrons in photosynthesis slightly increase with elevated CO₂. They do not support the hypothesis that elevated CO₂ during growth, under relatively low PPF, causes a substantial shift of capacity from carboxylation to electron transport. However, the decrease in TSP and Rubisco protein in well-watered sunflower plants grown at elevated CO₂ (Table 5), shows acclimation involving loss of Rubisco, relative to the capacity for RuBP synthesis, which was retained. Increased activation-state of Rubisco maintained P_n, thus ensuring the sink for electrons and agreeing with the absence of effect of elevated CO₂ on q_P and q_NP (Table 4) and the relatively small increase in electron flux. It is concluded that reductions in g_s and Rubisco content, caused by growth in elevated CO₂, are compensated and regulated by changes in electron transport, RuBP synthesis, and Rubisco activity and do not substantially affect photosynthetic energy dissipation.

Considering the effects of water deficiency, P_n decreased at mild and severe deficit but not because of small g_s as C_i increased, so photosynthetic capacity (P_nmax) must have decreased (Fig. 2; Table 2). There was no significant response of capacity to substantially elevated CO₂ (note particularly Fig. 3B) at mild and severe water deficits. However, when measured at elevated, compared to ambient CO₂, (Fig. 1A, B), P_n increased as expected from Fig. 2. Because transpiration was decreased in proportion to P_n in the mild deficit, the WUE was similar to controls when measured in ambient CO₂ but as P_n fell more with severe deficit, WUE diminished substantially.

The changes in P_nmax support the earlier conclusion (Tezara et al., 1999) that factors associated with decreased RWC and _w progressively inhibited photosynthetic capacity in sunflower. The mechanism was considered to be decreased ATP synthesis, shown by low ATP content and the consequent reduction in RuBP synthesis and content; limitation by inadequate inorganic phosphate supply or triose phosphate transport is not considered to be the cause of the decreased P_nmax (Lawlor and Cornic, 2002; Lawlor, 2002). However, the decreases in Rubisco content (62%) and initial activity (88%) were larger than those in RuBP (73%) and ATP (50%) comparing severely water deficient with well-watered plants grown under ambient CO₂. A larger proportion of Rubisco protein was lost in this study than previously observed (Tezara et al., 1999), where the fall in P_n with water deficits was clearly related to RuBP supply and ATP content; Lawlor, 2002). Therefore the limited (or lack of) response of P_n to elevated CO₂, at mild and severe water stress may be related to decreased Rubisco activity in this experiment rather than to impaired ATP synthesis and RuBP limitation.

Water deficiency decreased electron flux, J, through PSII as expected from the decrease in P_n, but much less than the decrease in P_n, due to higher photorespiration at low _w (Lawlor and Cornic, 2002). Thus _PSII was decreased, particularly when measured in ambient CO₂; the reduction was small in elevated CO₂, suggesting that there was an increased sink for electrons. This was not P_n, which decreased despite the large C_a (Fig. 2) and operating C_i, which would have decreased photorespiration. The reason for the lack of an effect of low _w on _PSII, at elevated CO₂ is not known. Water deficits decreased q_P, showing that the reduction state of the acceptor Q_A was increased, and increased q_NP so a greater proportion of the energy was thermally dissipated at low RWC. Such effects have been frequently observed (Lawlor and Cornic, 2002). In droughted Quercus ilex, elevated CO₂ decreased photochemical activity and increased photoinhibition (Scarascia-Mugnozza et al., 1996). However, in sunflower there was no evidence of photoinhibition as F_v/F_m was unaffected by conditions, as earlier reported by Tezara et al. (1999).

It was concluded that in sunflower, elevated CO₂ increased P_n but led to acclimation, with decreased Rubisco protein but increased activity; water deficits decreased P_n with metabolic inhibition involving loss of Rubisco activity; there were negligible interactions between CO₂ concentration during growth and water supply, on photosynthetic processes. Slightly increased P_n in elevated CO₂ under water deficit may have partially compensated for inhibition by mild stress. However, there was no evidence that growth in elevated CO₂ fundamentally altered the photosynthetic metabolism of sunflower under water stress.

	Acknowledgements

 
We thank Drs M Paul and D Habash for their help with techniques, BID-CONICIT (Venezuela) for a scholarship to Wilmer Tezara and Rothamsted International for additional support, and Dr Ana Herrera and MD Fernández for critically reading the manuscript. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Research Council.

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