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CROP PHYSIOLOGY & METABOLISM

Physiological Limitations to Photosynthetic Carbon Assimilation in Cotton under Water Stress

^a Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602-7272
^b Dep. of Plant Agriculture, Univ. of Guelph, Guelph, ON, Canada N1G 2W1

^* Corresponding author (hjearl{at}uoguelph.ca)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Water stress may reduce leaf net photosynthetic carbon assimilation (A_N) through both stomatal effects, which reduce the leaf internal CO₂ concentration (C_i), and nonstomatal effects, which result in reduced A_N at a given level of C_i. However, the leaf gas exchange techniques used to calculate C_i are susceptible to important artifacts when applied to water-stressed leaves, making such C_i estimates unreliable. As an alternative to C_i, the CO₂ concentration in the chloroplast (C_C) can be calculated from simultaneous measurements of A_N from gas exchange measurements, and the thylakoid electron flux from chlorophyll fluorometry. This permits diffusional effects (stomatal plus mesophyll limitations to CO₂ diffusion) to be differentiated from chloroplast-level effects. We used this method to investigate physiological restrictions to photosynthesis in leaves of water stressed cotton (Gossypium hirsutum L.) plants in a series of greenhouse experiments. A null-balance lysimeter was used to slowly induce four distinct levels of water stress. Combined leaf gas exchange/chlorophyll fluorescence measurements differentiated the treatments more effectively than gas exchange measurements alone. All treatments reduced C_C, but only the two most severe stress treatments significantly increased nondiffusional restrictions, detectable as a reduction in the slope of A_N on C_C. In a second experiment, recovery of leaf photosynthesis was determined 24 and 48 h after relief of a severe stress by rewatering. Recovery of the A_N/C_C relationship was substantial but incomplete after 24 h and did not recover further by 48 h after rewatering, indicating lasting chloroplast-level injury as a result of the stress. Similar experiments should be conducted under field conditions to determine if water stress results in irreversible chloroplast-level injury in field-grown cotton.

Abbreviations: , leaf fractional absorption of incident PPFD • A_N, leaf net CO₂ assimilation rate • A_G, leaf gross CO₂ assimilation rate • A_N-C, leaf net CO₂ assimilation rate corrected for sample chamber leakage • A_N-M, measured leaf net CO₂ assimilation rate (uncorrected for sample chamber leakage) • C_A, ambient CO₂ concentration • C_C, CO₂ concentration at the carboxylation site in the chloroplast • C_i, leaf internal CO₂ concentration • C_S, CO₂ concentration in the sample chamber • DAP, days after planting • _II, quantum efficiency of Photosystem II • _II, fraction of absorbed PPFD absorbed by the antennae of Photosystem II • F_S, steady state chlorophyll fluorescence signal • F^'_M, maximum (light saturated) chlorophyll fluorescence signal • G_L, sample chamber conductance to CO₂ flux (leakage) • G_M, mesophyll conductance to CO₂ in the liquid phase • G_S, stomatal conductance to water vapor • IRGA, infrared gas analyzer • J_e, electron flux through Photosystem II • K_S, CO₂/O₂ specificity ratio of RubisCO • L, leaf area in the sample chamber • LRWC, leaf relative water content • O_C, oxygen concentration at the carboxylation site in the chloroplast • PPFD, photosynthetic photon flux density • R_D, rate of leaf respiration in the dark • RSWC, relative soil water content • v_C, velocity of the carboxylation reaction of RubisCO • v_O, velocity of the oxygenation reaction of RubisCO • W_D, pot plus soil dry weight • W_P, plant fresh weight • W_S, pot plus soil water saturated weight • W_T, pot target weight

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
SOIL WATER DEFICITS constitute a primary limitation to crop productivity in many regions of the world. An enhanced mechanistic understanding of how water stress affects crop growth could benefit current efforts to create more drought tolerant crop cultivars (Turner, 1997) by identifying those specific traits that determine crop performance under water deficit conditions and which are amenable to alteration either through genetic transformation or conventional breeding approaches. Reduced photosynthetic carbon assimilation (and therefore reduced crop dry matter accumulation) is a principal effect of soil water deficit in cotton and other crops. At the level of the whole canopy, the effects of water deficits on cotton leaf area expansion and absorption of photosynthetically active radiation are easily measured and have been well documented (Turner et al., 1986; Puech-Suanzes et al., 1989; Ball et al., 1994). Water stress also affects the efficiency with which absorbed radiation is used to carry out carbon fixation at the leaf level, and the mechanisms by which this occurs have been the subject of many studies in a variety of species over the past three decades. There is now a broad consensus that stomatal closure and the consequent reduction in leaf internal CO₂ concentration (C_i) are the major reason for reduced leaf photosynthetic rates under mild or moderate water stress (reviewed by Chaves, 1991; Cornic, 2000; Flexas et al., 2004). However, under severe water stress photosynthesis may also be restricted by nonstomatal effects, that is, inhibition or downregulation of photosynthesis at the level of the chloroplast, such that the net C assimilation rate (A_N) is reduced at a given C_i. Since the early 1980s, many researchers have measured leaf gas exchange parameters and used some variation of the method developed by von Caemmerer and Farquhar (1981) to calculate C_i. In field studies with cotton, water stress has often been found to alter the relationship of A_N to C_i, thus implicating nonstomatal effects in the suppression of photosynthesis (e.g., Ephrath et al., 1993; Faver et al., 1996). However, this method has been called into question because of important artifacts that can invalidate the gas exchange-based estimates of C_i in drought stressed leaves. First, the gas exchange equations used to calculate C_i rely on the assumptions that the diffusive pathways for water vapor and CO₂ are the same, and that essentially all such diffusion occurs through the stomata. In water stressed leaves with very low stomatal conductance (G_s), these assumptions are not met, since under these conditions a significant fraction of total water vapor exchange may occur directly through the cuticle, and resistance to diffusion through the epidermal–cuticular pathway is typically more than an order of magnitude greater for CO₂ than for water vapor (Boyer et al., 1997). Since the gas exchange equations are seldom corrected for cuticular transpiration, C_i can be greatly overestimated when G_s is low (Kirschbaum and Pearcy, 1988; Meyer and Genty, 1998). Second, overestimation of C_i also occurs if water stress results in heterogeneous ("patchy") stomatal closure (Downton et al., 1988; Meyer and Genty, 1998). In either case, overestimation of C_i may lead to the erroneous conclusion that water stress has resulted in increased nonstomatal limitations to photosynthesis.

More recently, combined measurements of leaf gas exchange and chlorophyll fluorescence have been used to investigate the involvement of nonstomatal effects in limiting photosynthesis under water stress. Dai et al. (1992) and Sánchez-Rodríguez et al. (1999) found that the ratio of Photosystem II efficiency (_II, from chlorophyll fluorometry) to A_N increased as C_i decreased. They established the relationship between C_i and _II:A_N under water-replete conditions (where gas exchange-based estimates of C_i are reliable), and then used it to estimate C_i in water stressed leaves from measurements of A_N and _II alone. In both of these studies, putative increases in nonstomatal limitations were indicated by the traditional analysis of the A_N/C_i relationship but not when C_i was determined from the _II/A_N ratio. A related approach is to calculate the CO₂ concentration at the carboxylation site in the chloroplast (C_C) from (i) estimates of the thylakoid electron flux (J_e) from chlorophyll fluorescence and (ii) gas exchange-based measurements of A_N and dark respiration (R_D) (Epron et al., 1995; Lal et al., 1996; Flexas et al., 2002; see Materials and Methods for relevant theory). Again, such studies have generally found a stable relationship between A_N and C_C, indicating little or no involvement of increased limitations at the chloroplast level in restricting photosynthesis under water stress, even when the A_N/C_i relationship suggests that such effects are present (Lal et al., 1996; Flexas et al., 2002; Cornic and Fresneau, 2002). Only under very severe stress (A_N reduced by more than 80%) are chloroplast-level effects typically observed (Flexas et al., 2004).

It is possible for C_C to be reduced by water stress even when C_i is not affected, since recent evidence suggests that the resistance to diffusion of CO₂ in the mesophyll (i.e., in the liquid phase from the substomatal cavity to the carboxylation site in the chloroplast) may also be increased by severe water stress (Flexas et al., 2004). Examination of the A_N/C_i relationship (when C_i estimates are reliable) permits differentiation between stomatal and nonstomatal limitations, while examination of the A_N/C_C relationship permits differentiation between total diffusional (stomatal plus mesophyll) and nondiffusional (chloroplast-level) limitations.

The first objective of the present work was to use combined leaf gas exchange and chlorophyll fluorescence measurements to quantify chloroplast-level limitations to photosynthesis in cotton leaves under four different levels of water stress. Small differences in photosynthetic activity under very severe water stress are not likely to result in important differences in crop productivity, since only a minor fraction of total seasonal C assimilation typically occurs under severe stress conditions. However, the extent of recovery of photosynthetic capacity following relief of a severe soil water deficit may be an important determinant of subsequent growth and productivity. Accordingly, in a second experiment, a severe water stress treatment was imposed, and then the extent of recovery of the resulting limitations to photosynthesis was determined 24 and 48 h following relief of the stress by rewatering.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plant Material
Experiments were conducted in a greenhouse in Athens, GA, USA (34°N, 84°W). Cotton (cv. Delta Pearl) plants were grown in 2.5-L plastic food containers (Berry Plastics Corp., Evansville, IN) with drainage holes added. The soil was a Pacolet sandy loam (a member of the clayey, Kaolinitic, thermic family of Typic Hapludults) amended with sand to a texture of 800 g kg^–1 sand, 120 g kg^–1 silt, and 80 g kg^–1 clay. The pots were filled with 3300 g of soil. Seeds were sown five to a pot in February 2003 (Exp. 1) or April 2003 (Exp. 2) and fertilized with 50 mL of a 0.8% (w/v) solution of 20-20-20 (%N, P and K as N, P₂O₅ and K₂O equivalents) fertilizer plus micronutrients (Miller Greenhouse Special, Miller Chemical and Fertilizer Co. Corp., Hanover, PA). Cotyledons were expanded and horizontal at 10 to 12 d after planting (DAP); at this time, plants were thinned to one per pot, and an additional 50 mL of fertilizer solution was added twice weekly thereafter. Air temperatures were maintained at 27 ± 4°C during the day and 20 ± 2°C during the night. Photoperiod was extended to 16 h with overhead 400-W metal halide lamps that produced a supplemental photosynthetic photon flux density (PPFD) of approximately 230 µmol m^–2 s^–1 at the tops of the plants. To prevent water stress during the initial growth stages, water was maintained at between 55 and 85% of soil water holding capacity by daily weighing and watering of the pots until water stress treatments were begun at between 35 and 45 DAP.

Relative Soil Water Content and Soil Water Holding Capacity
Before planting, soil water holding capacity was determined by watering two extra pots to excess, capping them with plastic lids, then allowing them to drain until reaching a constant weight (the saturated weight, W_S). The soil was then dried in an 80°C forced air dryer until it had reached constant weight, and the pot dry weight (W_D) was taken as the sum of the soil dry weight, the pot weight and the lid weight. Relative soil water content (RSWC) was calculated as:

[1]

where W_P is an estimate of plant fresh weight from destructive harvest of roots and shoots of two extra plants per experiment just before beginning the water stress treatments.

Water Stress Treatments
Water stress treatments were imposed with a 16-balance gravimetric lysimeter and computer controlled watering system, which is described in detail by Earl (2003). Each pot sat on an electronic balance, and its weight was monitored continuously by a computer. Whenever plant transpiration caused the weight of a pot to decline by 15 g below the target weight for that pot at that point in the experiment, the computer activated a solenoid valve, allowing water to flow to that pot via vinyl tubing until the pot weight was 15 g above its target weight. The target weight (W_T) for each balance was calculated by the computer software as:

[2]

where RSWC is the target relative soil water content expressed as a fraction between 0 and 1. New target RSWC values for each balance were entered manually on a daily basis as required by the experimental protocol.

In Exp. 1, four pots were randomly assigned to each of four treatments: well watered (75% RSWC), mild water stress (25% RSWC), moderate water stress (15% RSWC), and severe water stress (5% RSWC). The automated watering system was programmed to permit soil water to decline by a maximum of 10% RSWC per day because of transpiration; once a pot weight had declined by this amount in a single day, its weight was maintained within 15 g (approximately 2.5% RSWC) of the target weight for the rest of the day. Similarly, when a pot had reached its target final weight range, the watering system maintained it within 15 g of that weight (Fig. 1) . Once the "severe water stress" pot reached its final weight (usually actually about 10% RSWC, near the lower limit of transpirable soil water) and had been maintained there for one day, physiological parameters were measured for all four treatments in that replication.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Relative soil water content vs. time for the control treatment (75% RSWC) and three water stress treatments in Exp. 1 (top) and for the control, stress, and two recovery treatments in Exp. 2 (bottom). Each trace represents typical data for a single pot.

For both Exp. 1 and Exp. 2, the drought stress protocols were initiated sequentially for the different replications so that physiological measurements were made on only one replication per day. The measurement day was between 44 and 47 DAP in Exp. 1 and between 49 and 58 DAP in Exp. 2, depending on the replication. The four pots within a replication were also grouped together on four adjacent lysimeter balances, in a randomized complete block design.

Gas Exchange and Fluorescence Measurements
Fluorescence measurements were made on the second youngest fully expanded leaves with two Portable Photosynthesis Systems (Model LI-6400, LI-COR Inc., Lincoln NE) each fitted with a Leaf Chamber Fluorometer (Model LI-6400-40). At the beginning of each measuring day, the infrared gas analyzers (IRGAs) of the LI-6400 were calibrated as per the manufacturer's instructions, and the fluorometer signal offset was zeroed. The flow rate of air through the sample chamber was set at 250 µmol s^–1, and leaf temperature was maintained at 27 ± 0.8°C by the chamber thermoelectric coolers. The sample chamber CO₂ concentration was adjusted to 360 µL L^–1 with the system's CO₂ injector (Model 6400-01, LI-COR).

The modulation frequency of the fluorometer measuring light was 10 kHz under actinic illumination and increased to 20 kHz during saturating pulses. The measuring light intensity was set to Level 10, and the gain setting was 20. For determination of the steady state fluorescence signal under actinic illumination (F_S), the sampling rate was 0.5 Hz, but this was increased to 20 Hz for determination of the maximum fluorescence signal during saturating pulses. Pulse duration was 0.8 s, which was determined in preliminary experiments to be adequate to achieve the apparent F^'_M.

Each leaf was dark adapted for approximately 45 min before introducing it into the measurement chamber. Once inside the chamber additional time was allowed so that leaf temperature, stomatal conductance, and leaf CO₂ exchange reached steady state. At this point, net CO₂ efflux from the leaf was recorded as an estimate of dark respiration (R_D), and then incident PPFD was set to 1500 µmol m^–2 s^–1, with approximately 90% of the actinic light provided by the fluorometer's red LEDs and 10% by the blue LEDs. The leaf was allowed to achieve steady state G_S and A_N at a chamber CO₂ concentration of 360 µL L^–1 (standard conditions) before making the first fluorescence measurements. Then, measurements were made at different chamber CO₂ concentration [produced by using reference side (incoming) CO₂ concentrations of 50, 100, 200, 400, 500, 700, 1000, 1500, 2000, and 2400 µL L^–1]. At each level of CO₂ concentration, sufficient time was allowed for the leaf to reach a steady state A_N, then leaf gas exchange data were recorded and F_S and F^'_M were determined three times, using three saturating pulse intensity settings (4, 6, and 10) applied in random order. Three pulse intensities were used because previous work had shown that apparent F^'_M determined with a single pulse of intensity 10 underestimated the true F^'_M with this instrumentation under similar experimental conditions (Earl and Ennahli, 2004). A linear regression of apparent F^'_M on 1/PPFD during the saturating pulse provided an estimate of the true F^'_M for each measurement (Fig. 2) (Markgraf and Berry, 1990; Earl and Ennahli, 2004). Simultaneously with leaf gas exchange measurements, the CO₂ concentration of the air in the vicinity of the measurement chamber was determined with another IRGA (Model 6200, LI-COR). The entire work area was well ventilated with an electric fan to prevent large fluctuations in CO₂ concentration external to the chamber.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Determining the true light saturated F'M by the three-pulse method. Apparent F'M was determined for three 0.8-s pulses of different PPFD, then the light-saturated F'M (at infinite PPFD) was estimated as the intercept of the linear regression of apparent F'M on the inverse of PPFD during the pulse. Example data are shown for leaves from each of the three water stress treatments in Exp. 1. Actinic PPFD during the measurement was 1500 µmol m–2 s–1 in every case.

[3]

An additional five 1-cm diameter leaf disks per leaf were extracted and used to determine leaf water potential according to Brown and Bartos (1982) with thermocouple psychrometers (Wescor Inc., Logan, UT).

Leaf absorption of incident PPFD () was determined for each leaf with an LI-1800-12S external integrating sphere (LICOR) connected to a reflectance spectrometer (Unispec, PP Systems, Haverhill, MA). Leaf transmittance and reflectance measurements were made at 3.6-nm intervals between 400 and 700 nm, and of the actinic light in the LI-6400 chamber was calculated as described by Earl and Tollenaar (1997).

Calculations and Data Analysis
Measurements of A_N were corrected for leakage of CO₂ across the leaf chamber gasket as

[4]

where A_{N – C} and A_{N – M} are the leakage corrected and apparent (measured) leaf net CO₂ assimilation rates, respectively, with units of µmol CO₂ m^–2 s^–1, G_L is a CO₂ leakage coefficient for the chamber determined in preliminary experiments (2.5 x 10^–7 mol s^–1, data not shown), L is the leaf area in the chamber (2.0 x 10^–4 m²), and C_S and C_A are the sample chamber and external (ambient) CO₂ concentrations, respectively, with units of µmol CO₂ mol^–1 air. This correction was minor when A_N was high or when C_S – C_A was small. However, it constituted a substantial fraction of apparent A_N for water stressed leaves when C_S was very high or very low compared with C_A. Values of C_i were also recalculated from the leakage corrected values of A_N. Hereafter all mention of A_N, R_D, and C_i refers to leakage corrected estimates.

Mitochondrial respiration of illuminated leaves was assumed to be equal to R_D, and so gross CO₂ assimilation (A_G) was estimated as A_N + R_D. Quantum efficiency of Photosystem II (_II) was estimated according to Genty et al. (1989) as

[5]

and thylakoid linear electron flux (J_e) was then calculated as

[6]

where _II is the fraction of absorbed PPFD absorbed by the antennae of Photosystem II (Loreto et al., 1994). A value of 0.45 was used for _II (Earl and Ennahli, 2004). Assuming that four electrons are consumed per carboxylation or oxygenation of RuBP by RubisCO and that electron flux to alternate sinks is negligible,

[7]

where v_C and v_O are the rates of carboxylation and oxygenation, respectively. A release of 0.5 CO₂ is expected per oxygenation event, so

[8]

Combining these last two equations shows that v_C/v_O may be estimated from measurements of A_G and J_e as

[9]

The value of C_C was then calculated following Lal et al. (1996) as

[10]

where O_C is the partial pressure of oxygen at the carboxylation site, assuming a negligible O₂ concentration gradient between the atmosphere and the carboxylation site (Gerbaud and André, 1987), and K_S is the CO₂/O₂ specificity of RubisCO, adjusted for leaf temperature (Woodrow and Berry, 1988). This method considers both O_C and C_C in gas-phase equivalents, rather than the actual dissolved concentrations at the carboxylation site, thus making the C_C values more conveniently comparable to C_i values. That is, the temperature correction of K_S adjusts for temperature effects on both RubisCO CO₂/O₂ specificity and CO₂ and O₂ solubility.

Preliminary analysis revealed that the relationship between A_N and C_C remained linear up to a C_C value of 150 µL L^–1 for all treatments. The initial slope of this relationship was determined for each leaf via regression analysis, making use only of data for which C_C was below 150 µL L^–1. Analysis of variance was used to determine significant (P < 0.05) treatment effects on LRWC, leaf water potential, the initial slope of the A_N/C_C curve, R_D, leaf absorption of PAR, and (at standard conditions and steady state), G_S, A_N, C_i, _II, J_e, v_C/v_O, and C_C. Treatment means were separated by a protected LSD test at the P = 0.05 probability level.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Exp. 1, both leaf water potential and LRWC declined as RSWC decreased, and in the case of leaf water potential, all four RSWC treatments differed significantly from one another (Table 1). There was a strong linear relationship between LRWC and leaf water potential across the four treatment means (R² = 0.97; regression not shown). Relative to the control treatment (75% RSWC), mild water stress (25% RSWC) did not produce statistically significant decreases in A_N and G_S, but the more severe stresses (15 and 5% RSWC) reduced both A_N and G_S to near zero (Table 1). The mild stress also did not produce statistically significant changes in _II and J_e, but when J_e and A_N were used to calculate v_C/v_O, this ratio was significantly lower in the mild stress treatment than in the control. The two most severe stress treatments greatly suppressed _II, J_e, and v_C/v_O relative to the control and mild stress treatments, and _II and J_e were significantly lower in the 5% RSWC treatment than the 15% RSWC treatment (Table 1). R_D and averaged –1.4 µmol m^–2 s^–1 and 0.89, respectively, and were not significantly affected by the treatments (data not shown).

View this table: [in this window] [in a new window]   Table 1. Effect of relative soil water content (RSWC) on leaf water potential (l), leaf relative water content (LRWC), net CO2 assimilation rate (AN), stomatal conductance (GS), calculated leaf internal CO2 concentration (Ci), quantum efficiency of Photosystem II (II), electron flux through Photosystem II (Je), the ratio of carboxylation to oxygenation of ribulose bisphosphate by RubisCO (vC/vO), the calculated CO2 concentration at the carboxylation site (CC), and the slope of AN on CC in Exp. 1. Measurements were made between 44 and 47 d after planting, depending on the replication.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Examples of the relationship between AN and Ci (top) and AN and CC (bottom) for each of the four water stress treatments in Exp. 1.

In general, leaf photosynthetic activity of control plants, as measured by A_N, J_e, and the slope of A_N/C_C was higher in Exp. 2 than in Exp. 1 (compare Tables 1 and 2). Also, the 5% RSWC treatment seemed to be somewhat less severe in Exp. 2, as evidenced by the slightly larger values of A_N and G_S. As in Exp. 1, the 5% RSWC treatment in Exp. 2 produced highly variable estimates of C_i, and significantly reduced _II, J_e, v_C/v_O, C_C, and the initial slope of A_N/C_C. Rewatering produced substantial recovery of most parameters after 24 h, but A_N, _II, J_e, and A_N/C_C remained significantly depressed relative to control plants. By contrast, C_C and v_C/v_O did not differ from controls 24 h after rewatering. There was no significant change in any measured parameter between 24 and 48 h after rewatering (Table 2). As in Exp. 1, R_D and were unaffected by the treatments in Exp. 2 (average values of –1.6 µmol m^–2 s^–1 and 0.89, respectively; data not shown).

View this table: [in this window] [in a new window]   Table 2. Net CO2 assimilation rate (AN), stomatal conductance (GS), calculated leaf internal CO2 concentration (Ci), quantum efficiency of Photosystem II (II), electron flux through Photosystem II (Je), the ratio of carboxylation to oxygenation of ribulose bisphosphate by RubisCO (vC/vO), the calculated CO2 concentration at the carboxylation site (CC), and the slope of AN on CC under control conditions (75% RSWC), severe water stress (5% RSWC) and 24 and 48 h after rewatering severely water stressed plants in Exp. 2. Measurements were made between 49 and 58 d after planting, depending on the replication.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In total, the results of Exp. 1 are consistent with the model suggested by Medrano et al. (2002), where mild stress reduces A_N through its effects on C_C, resulting in increased photorespiration (decreased v_C/v_O) but no reduction in J_e or carboxylation efficiency (A_N/C_C). As stress becomes more severe and C_C declines further, J_e and carboxylation efficiency are also suppressed (Medrano et al., 2002).

Thus, there were both diffusional and nondiffusional components to the suppression of photosynthesis under severe stress in Exp. 1, and this was observed again in the 5% RSWC treatment in Exp. 2 (Table 2); the 5% RSWC treatment significantly reduced both C_C and A_N/C_C. Upon rewatering, only the nondiffusional component remained, since C_C recovered completely, but A_N/C_C was still significantly suppressed (Table 2). The reduction in A_N/C_C under water stress should not necessarily be considered indicative of injury to the photosynthetic apparatus, since it might simply represent downregulation of photosynthetic capacity that is appropriate given the reduced availability of CO₂ in the chloroplast (Escalona et al., 1999). However, the lasting effect of water stress on A_N/C_C observed even 48 h after rewatering (Table 2) can only be seen as loss of photosynthetic function at the chloroplast level, although the exact nature of this apparent injury is not clear. Souza et al. (2004) found that water stress promoted accumulation of soluble sugars in Vigna unguiculata (L.) Walp. leaves, suggesting the possibility of an end product inhibition of photosynthesis. However, that inhibition was completely relieved within a few days of rewatering, unlike what was observed in the present work. It would be of interest to make similar measurements of A_N/C_C on field-grown cotton to see if levels of water stress in the field comparable to those in the present work (near zero G_S, severe leaf wilting, LWRC below 70%) would also result in a lasting inhibition of A_N and A_N/C_C or if recovery would be complete on rewatering.

As discussed above, estimates of C_i based on leaf gas exchange measurements are generally considered to be unreliable under conditions of severe water stress when G_S is very low. However, it should be noted that a relatively high C_i concurrently with a low C_C is not always indicative of a poor C_i estimate. A large C_i:C_C ratio can also arise when the conductance to diffusion of CO₂ in the mesophyll from the substomatal cavity to the carboxylation site in the chloroplast (G_M) is very low. Recent results suggest that G_M is determined by an enzymatic process (Bernacchi et al., 2002) and can be reduced by water stress (Flexas et al., 2004). Such a decrease in conductance to CO₂ diffusion in the mesophyll would constitute a nonstomatal limitation to photosynthesis but would not directly affect the A_N/C_C slope. The reduced A_N/C_C slope observed in the present work indicates a chloroplast level limitation to photosynthesis, not an increased resistance to diffusion in the mesophyll.

We observed that A_N/C_i curves tended to be erratic under water stress, while A_N/C_C curves were more consistent (e.g., Fig. 3). Besides the two different artifacts already discussed that can cause C_i to be overestimated under water stress, calculated C_i is also very sensitive to small errors in the estimate of A_N; when A_N is understimated (for instance, because of random IRGA noise), the gas exchange calculations produce an overestimate of C_i and vice versa. This amplification of the original error further reduces the quality of the data and increases error variance in the A_N/C_i curve. By contrast, calculated values of C_C are increased by overestimates of A_N and decreased by underestimates of A_N. The effect is that A_N/C_C data tend to be "self correcting," producing artificially smooth curves.

In conclusion, combined measurements of leaf gas exchange and chlorophyll fluorescence revealed that levels of water stress that appeared to be similar on the basis of leaf gas exchange measurements only were actually physiologically distinct. Mild stress produced measurable reductions in v_C/v_O and C_C, even though A_N was not significantly affected. This was consistent with a reduced availability of CO₂ at the carboxylation site because of reduced G_S (and possibly G_M); there was no evidence of inhibition of photosynthesis at the level of the chloroplast. By contrast, under severe stress both diffusive limitations and chloroplast-level limitations were increased, as evidenced by a reduced C_C and a reduced A_N/C_C slope, respectively. Rewatering of severely stressed plants completely reversed the diffusive limitation (C_C returned to control levels), but A_N/C_C did not recover completely and A_N continued to be significantly reduced relative to control plants because of a lasting chloroplast-level inhibition.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This research was funded by the USDA NRI Plant Responses to the Environment Program, and by state and Hatch funds allocated to the Georgia Agric. Exp. Stn.

Received for publication February 15, 2005.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

• Ball, R.A., D.M. Oosterhuis, and A. Mauromoustakos. 1994. Growth dynamics of the cotton plant during water-deficit stress. Agron. J. 86:788–795.[Abstract/Free Full Text]
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