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Keywords:

  • grassland ecosystems;
  • stomata;
  • stomatal limitation of photosynthesis;
  • subambient CO2;
  • water use efficiency

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

An investigation to determine whether stomatal acclimation to [CO2] occurred in C3/C4 grassland plants grown across a range of [CO2] (200–550 µmol mol−1) in the field was carried out. Acclimation was assessed by measuring the response of stomatal conductance (gs) to a range of intercellular CO2 (a gsCi curve) at each growth [CO2] in the third and fourth growing seasons of the treatment. The gsCi response curves for Solanum dimidiatum (C3 perennial forb) differed significantly across [CO2] treatments, suggesting that stomatal acclimation had occurred. Evidence of non–linear stomatal acclimation to [CO2] in this species was also found as maximum gs (gsmax; gs measured at the lowest Ci) increased with decreasing growth [CO2] only below 400 µmol mol−1. The substantial increase in gs at subambient [CO2] for S. dimidiatum was weakly correlated with the maximum velocity of carboxylation (Vcmax; r2 = 0·27) and was not associated with CO2 saturated photosynthesis (Amax). The response of gs to Ci did not vary with growth [CO2] in Bromus japonicus (C3 annual grass) or Bothriochloa ischaemum (C4 perennial grass), suggesting that stomatal acclimation had not occurred in these species. Stomatal density, which increased with rising [CO2] in both C3 species, was not correlated with gs. Larger stomatal size at subambient [CO2], however, may be associated with stomatal acclimation in S. dimidiatum. Incorporating stomatal acclimation into modelling studies could improve the ability to predict changes in ecosystem water fluxes and water availability with rising CO2 and to understand their magnitudes relative to the past.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Stomatal conductance (gs) is generally expected to decline in herbaceous plants with an increase in atmospheric CO2 above the current concentration (Field, Jackson & Mooney 1995; Knapp et al. 1996; Drake, Gonzalez-Meler & Long 1997; Wand et al. 1999). There are many important physiological and ecological implications of such a decline. For example, lower gs may alter ecosystem hydrology by reducing transpiration and could increase the surface temperature through reduced evaporative cooling (Sellers et al. 1996; Jackson et al. 1998; Bounoua et al. 1999). Reduced gs, coupled with increased photosynthesis may also improve the water use efficiency (WUE) of many plants, which is of particular importance for productivity in arid and semi-arid regions (Polley, Johnson & Mayeux 1992; Polley et al. 1993; Sage 1995; Field et al. 1995; Owensby et al. 1999; Smith et al. 2000).

A major factor that could alter stomatal responses to CO2 is the degree to which stomata acclimate to growth CO2 concentration. Stomatal acclimation to CO2 would be ecologically important if it either tempered or enhanced the reduction in gs with rising CO2 (Šantrüček & Sage 1996; Morison 1998). Acclimation is defined here as a change in stomatal function that occurs when plants are grown in contrasting CO2 concentrations (Šantrüček & Sage 1996; Morison 1998). For example, physiological acclimation would be demonstrated if the stomatal behaviour of plants grown at contrasting CO2 concentrations differed when measured at the same CO2 concentration. Stomatal acclimation to CO2 may occur in several ways, including changes in maximum gs and stomatal sensitivity to CO2. Other responses to CO2, such as changes in stomatal morphology (density and size) may also influence gs independently of physiological acclimation. Despite its potential importance for regulating plant water loss at high CO2, stomatal acclimation has only been examined in a few species (Drake et al. 1997; Morison 1998).

The often dramatic response of terrestrial vegetation to elevated CO2 has led to greater interest in plant responses to past CO2 increases (Sage & Coleman 2001). Atmospheric CO2 was as low as 180 µmol mol−1 during the last glacial maximum and has risen by 37% since the 1700s (Barnola et al. 1987; Jouzel et al. 1993). Past CO2 increases have been implicated in shifting global distributions of C3 and C4 plants and increases in ecosystem productivity (Polley et al. 1993; Ehleringer, Cerling & Helliker 1997). Although studies suggest that carbon assimilation may be affected by subambient CO2 (Sage & Reid 1992; Tissue et al. 1995; Anderson et al. 2001; Sage & Coleman 2001), comparatively little is known about stomatal responses to past atmospheric CO2 concentrations and their underlying mechanisms.

Recent empirical and modelling studies suggest that plant and ecosystem responses to increasing CO2 may be non-linear (Ackerly & Bazzaz 1995; Luo, Sims & Griffin 1998; Luo & Reynolds 1999; Anderson et al. 2001). However, a majority of studies have examined plant responses to step increases in atmospheric CO2, generally comparing ambient to twice ambient manipulations (Drake et al. 1997; Wand et al. 1999). Because atmospheric CO2 is increasing gradually, results from step change experiments cannot be easily interpolated to intermediate CO2 concentrations, and threshold or non-linear responses to CO2 may go undetected (Luo & Reynolds 1999). Observations of plants grown across a range of CO2 (e.g. Polley et al. 1993) may therefore help refine predictions of ecosystem responses to future CO2 increases (Ackerly & Bazzaz 1995) and determine the magnitude of changes that have occurred since the start of the Industrial Revolution.

In this study, the influence of atmospheric CO2 on stomatal physiology was examined using experimental chambers that maintain a continuous gradient of CO2 from 200 to 550 µmol mol−1 (Johnson, Polley & Whitis 2000). A previous study in this C3/C4 grassland ecosystem reported 40–80% declines in gs along the gradient for several species (Anderson et al. 2001). Stomatal acclimation was examined in three dominant species of this grassland. First, we tested whether stomatal acclimation to CO2 occurred and examined whether there was a threshold or non-linear response to the CO2 gradient. Second, the widespread occurrence of photosynthetic acclimation to rising CO2 suggests that stomata may also acclimate to maintain the tight coupling between photosynthesis and conductance. We therefore determined whether stomatal acclimation was associated with changes in photosynthetic acclimation (e.g. Jarvis, Mansfield & Davies 1999). Finally, we examined the implications of variation in stomatal behaviour for carbon gain by assessing its effects on relative stomatal limitation of photosynthesis (Jones 1985) and intrinsic WUE.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Study site and experimental system

The measurements were conducted in a grassland area near the USDA-ARS Grassland, Soil and Water Research Laboratory in Temple, TX (31°05′ N, 97°20′ W). The site has been managed as grassland for the last 50 years and was last grazed by cattle in 1992. Soils are in the Austin black soil series, classified as a fine-silty, carbonatic, thermic Udorthentic Haplustoll with 35–55% clay in the top 40 cm (Johnson et al. 2000). To determine the potential for interspecific variation in responses to CO2, the study focused on three abundant species with different growth forms and photosynthetic pathways: Solanum dimidiatum Raf., a C3 perennial forb; Bromus japonicus L., a C3 annual grass; and Bothriochloa ischaemum (L.) Keng, a C4 perennial grass. Other dominant species at the site include Solidago canadensis L. and Ratibida columnaris (Sims) D. Don. Mean annual precipitation (1913–99) is 877 mm and the mean minimum and maximum annual temperatures are 13·2 and 25·9 °C, respectively. C3 species are mostly active early in the growing season, and C4 species dominate by mid-summer.

The experimental system that was used consisted of two elongated chambers over parallel and adjacent plots of grassland, each 60 m in length, 1 m wide, and 1 m tall. Air was introduced into one end of each chamber and was progressively depleted of CO2 by photosynthesis as it was moved down the chamber by a blower. The desired CO2 concentrations were maintained by automatically varying the rate of air flow. During daytime, the CO2 concentration gradients in the subambient and superambient chambers ranged from 360 to 200 µmol mol−1 and from 550 to 360 µmol mol−1, respectively. At night, CO2 gradients were maintained at 150 µmol mol−1 above daytime levels by reversing air flow and using respiratory CO2 releases to create the gradient. Treatments began in May 1997 and operated each growing season (mid-February to mid-November) up to and including 2000.

The chambers were divided into 10 sections, each 5 m in length, with chilled-water cooling coils between sections to control temperature and humidity. Each section was enclosed in polyethylene film (which transmitted 85–95% of incident PPFD), and a rubber-coated barrier extended 1 m deep into the soil along the sides of each chamber. To maintain environmental conditions at ambient levels in chambers, air temperature and humidity were controlled by cooling and dehumidifying air before it entered each 5 m section of chamber. Irrigation was applied equally to each 5 m section to match ambient rainfall through July 1999. Thereafter, water was applied such that soil water content in sections matched that of adjacent grassland exposed to ambient CO2 as measured by neutron attenuation. During the drought years of 1999 and 2000, the total water applied to the chambers was 349 and 381 mm, respectively. There were no consistent effects of CO2 treatment on soil water content during these years (Polley et al. unpublished results). Therefore, stomatal responses to CO2 were not confounded by CO2-induced variation in soil water availability along the gradient. However, it should be noted that interspecific differences might have been affected by water availability because each species was measured at a different time of the year. Additional details of design, construction and operation of these chambers can be found in Johnson et al. (2000).

Steady-state gas exchange and stomatal acclimation

Steady-state leaf gas-exchange was measured at saturating irradiance with an open gas-exchange system (LI-6400; Li-Cor Inc., Lincoln, NE, USA) between 0900 and 1500 h central standard time. Incident irradiance during all measurements was provided by red-blue light-emitting diodes. Measurements were taken when each species was at its peak abundance, which was June 2000 for S. dimidiatum, April 2000 for Br. japonicus, and August 1999 for Bo. ischaemum. The youngest fully expanded leaf was sampled on three to four plants per section at a minimum of six treatment CO2 concentrations (six chamber sections). Stomatal conductance (gs) was measured at saturating light levels of 1200, 1600, and 2000 µmol m−2 s−1 for S. dimidiatum, Br. japonicus and Bo. ischaemum, respectively. A Peltier cooling module maintained leaf temperatures at 20–23, 27–30 and 29–32 °C for Br. japonicus, S. dimidiatum and Bo. ischaemum, respectively, matching the approximate ambient conditions at the time of the measurement. Leaf-to-air vapour pressure deficits (LAVPD) were maintained at levels that permitted the measurement of maximum gs, which were 0·9–1·1 kPa for S. dimidiatum and Br. japonicus and 1·4–1·6 kPa for Bo. ischaemum. To calculate gs and intercellular CO2 concentration (Ci) a boundary layer conductance of 4·86 mol m−2 s−1 was used for the grasses and 1·42 mol m−2 s−1 was used for S. dimidiatum. Boundary layer conductance was calculated on the basis of leaf area and fan speed using the energy balance algorithms of the LI- 6400. Leaf area was measured with a portable leaf area meter (LI-3100; Li-Cor Inc.) or from leaf dimensions, depending on sample morphology. To examine long-term consequences of CO2 for stomatal function, measurements were made during the third and fourth growing season of the experiment.

To examine stomatal acclimation to growth CO2 concentrations, we measured the response of gs and the Ci/Ca ratio to a manipulation of Ci (by changing the external CO2 concentration, Ca) within the leaf cuvette. Stomatal conductance versus Ci (gsCi) curves were measured on each species during the same period as steady-state gas exchange. Once clamped in the cuvette, leaves were exposed to the light, LAVPD, and temperature levels described above and the respective growth CO2. After steady-state conditions were achieved, the first measurement was taken, and Ci was then reduced to the CO2 compensation point and raised in steps (Šantrüček & Sage 1996). Stomatal conductance was recorded after steady-state conditions were re-established at each CO2 level. Steady state was determined when the coefficient of variation of change in water vapour was < 0·05% (generally after 30–50 min).

To measure stomatal density (the number of stomates per mm2) and the size of stomata, casts were made of leaves sampled in the field during April 2000. Stomatal size was defined as the length in micrometres between junctions of the guard cells at each end of the stomate (Malone et al. 1993), and was therefore related to the maximum potential opening of the stomatal pore, and not the amount of opening that occurs at a given Ci. Measurements of stomatal density and stomatal size were made on leaf casts (e.g. Williams & Green 1988) for at least six individuals per species at each of six CO2 concentrations (a total of at least 36 plants per species). For each impression, a leaf section located 2–3 cm from the petiole was pressed onto a microscope slide covered with polyvinylsiloxane dental impression material (‘Extrude’ Medium; Kerr Manufacturing Co., Orange, CA, USA). After the polymer hardened (approximately 5 min), the leaf was removed and the resulting leaf mould was later used as a cast for clear nail polish. Each impression was analysed at 400× (for S. dimidiatum and Bo. ischaemum) or 100× (for Br. japonicus) using a light microscope interfaced with a solid-state TV camera (Model CCD-72-SX; DAGE-MTI Inc., Michigan City, IN, USA) using NIH Image 1·58 (U.S. National Institutes of Health; http:rsb.info.nih.govnih-image). The stomatal density and stomatal size were sampled on three to six fields-of-view per slide, depending on the variation in the counts, and averaged for each slide.

The relative stomatal limitation of photosynthesis (lg) was calculated, based on the response of net photosynthesis (A) to variation in Ci (ACi curves). These curves were recorded concurrently with gsCi curves on sampled plants and fitted with a non-linear regression model describing an exponential rise to a maximum:

inline image 1

where c is the y intercept, 1/b is the rate constant, and a+c is CO2 saturated A (Amax). This model provided a good fit to our data (r2 > 0·97) and has been used previously for herbaceous plants (Jacob, Greitner & Drake 1995; Reid & Fiscus 1998). The relative stomatal limitation (lg) was calculated using the differential method of Jones (1985):

inline image 2

where rg is the gas-phase resistance to CO2 uptake (the supply function) and r* is the slope of the ACi curve (demand function). We calculated r* as the first derivative of Eqn 1 at the operating Ci and calculated rg as (Ca − Ci)/A at the operating Ci (Jones 1985). This model was also used to calculate lg for the C4 species because A was not saturated at high CO2 (550 µmol mol−1; Anderson et al. 2001), permitting the calculation of a positive slope for the ACi curve at the operating Ci.

To examine whether variation in stomatal conductance was associated with the maximum velocity of carboxylation (Vcmax) and photosynthetic capacity (Jarvis et al. 1999), we compared maximum gs (gsmax), measured at the lowest CO2 concentration to Vcmax and Amax (Eqn 1). Vcmax was estimated from the biochemical model of von Caemmerer & Farquhar (1981):

inline image 3

where the constants were KC = 43·5 Pa, KO = 23·3 kPa, and Γ* (CO2 compensation point in the absence of mitochondrial respiration in the light) = 4·44 Pa at a temperature of 26 °C (Harley, Webber & Gates 1985; Reid & Fiscus 1998) and an [O2] of 21 kPa. Carboxylation efficiency (CE), which is the first derivative of Eqn 1 at the CO2 compensation point, was calculated as

inline image 4

where Ci = (1/b) ln[a/(a − A + c)] at A = 0 (Reid & Fiscus 1998).

We note that because the ACi curves were measured during the construction of the gsCi curves, our calculations of Vcmax may be influenced by changes in the activation state of Rubisco over the course of the measurement. However, our observations of Vcmax using the ‘slow’ approach were similar to values derived from standard ACi curves measured on these species previously (Anderson et al. 2001).

Statistical analyses

To examine whether CO2 treatments influenced gsCi and (Ci/Ca)–Ci curves, we used the analysis of repeated measures (anovar) for physiological response curves (e.g. Potvin, Lechowicz & Tardif 1990) in SPSS 10·0 for Windows (SPSS Inc., Chicago, IL, USA). Ci was the within subjects factor whereas growth CO2 was the between subjects factor. The statistical significance of relationships between measured variables and growth CO2 was determined using linear, hyperbolic and power functions in SPSS 10·0. As multiple measurements (three to five) were taken in each treatment section, there was more than one Y for each X (where X = growth CO2). Although means (± 1 SE) are presented in figures, all analyses were carried out using individual plants.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Steady-state gs declined strongly with increasing growth CO2 for all species (Fig. 1). The decline was non-linear for S. dimidiatum{[y = a + (b/x2) where a = 191·99 and b = 9·27 × 106]; r2 = 0·94; P < 0·001}, decreasing by 81% from 200 to 550 µmol mol−1 CO2. Three-quarters of this decline occurred from subambient to ambient growth CO2. The decline in gs with growth CO2 was linear for Br. japonicus (r2 = 0·74; P < 0·0001; a 49% decline from 215 to 550 µmol mol−1) and weakly non-linear for Bo. ischaemum (y = axb, where a = −0·64 and b = 8·30 × 103; r2 = 0·76; P < 0·001; a 46% decline from 215 to 540 µmol mol−1). Solanum dimidiatum had the highest overall gs among the three species, followed by Br. japonicus and Bo. ischaemum (Fig. 1).

image

Figure 1. Mean (± 1 SE) values of steady-state stomatal conductance (gs) for S. dimidiatum (A), Br. japonicus and Bo. ischaemum (B) plants grown across a range of atmospheric CO2. Note the difference in scales on the Y-axis between (A) and (B).

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There was strong evidence for stomatal acclimation in S. dimidiatum. The gsCi response curves for this species differed significantly across CO2 treatments. Maximum gs (gsmax; gs measured at the lowest Ci), increased and the gsCi curves were progressively steeper for growth CO2 concentrations below ambient levels (P < 0·05, anovar, Fig. 2A). We also found evidence of a non-linear response of stomatal acclimation to CO2 as the gsCi curves did not differ among plants exposed to growth CO2 > 400 µmol mol−1. These differences in gsCi curves also indicated that stomatal sensitivity to Ci was greater in plants grown at less than 400 µmol mol−1 CO2. Although the (Ci/Ca)–Ci curves did not differ significantly (P = 0·10) among CO2 treatments (Fig. 2B), average Ci/Ca was generally higher in subambient CO2-grown plants measured at Ci < 400 µmol mol−1. This trend is consistent with stomatal acclimation observed in Fig. 2A. Modification of stomatal behaviour in S. dimidiatum was not strongly associated with the maximum velocity of carboxylation (Vcmax). The gsmax was weakly, although significantly, correlated with Vcmax (r2 = 0·27; P = 0·05, Fig. 3A). Variation in CO2 saturated photosynthesis (Amax), however, was not correlated with gsmax (r2 = 0·05; P = 0·32; Fig. 3B). In contrast to S. dimidiatum, there was no evidence of stomatal acclimation in Br. japonicus or Bo. ischaemum, as the gsCi curves (Figs 4A & B) and (Ci/Ca)–Ci curves (data not shown) were statistically indistinguishable across growth CO2 concentrations.

image

Figure 2. The response of mean (± 1 SE) gs to Ci (gsCi response curve; (A) and Ci/Ca to Ci[(Ci/Ca)–Ci response curve; (B) for S. dimidiatum plants grown across a range of atmospheric CO2. For gs responses, a different letter next to each curve indicates that it is significantly different (P < 0·05, anovar) from the other curves. Symbols correspond to different growth CO2 concentrations (µmol mol−1).

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image

Figure 3. The relationship between maximum stomatal conductance (gsmax; measured at the lowest Ci) and Vcmax (A) and Amax (B) as determined from gsCi response curves. The gsmax was significantly associated with Vcmax (r2 = 0·27; P = 0·05), but was not correlated with Amax (P > 0·05).

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image

Figure 4. The response of mean (± 1 SE) gs to manipulation of Ci (gsCi response curve) for Br. japonicus (A) and Bo. ischaemum (B) plants grown across a range of atmospheric CO2. Symbols correspond to different growth CO2 concentrations (µmol mol−1).

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Interestingly, the stomatal density (Fig. 5A) increased linearly with growth CO2 in S. dimidiatum (r2 = 0·30; P < 0·001) and Br. japonicus (r2 = 0·41; P < 0·0001), but decreased linearly with growth CO2 in Bo. ischaemum (r2 = 0·15; P < 0·05). Stomatal size (Fig. 5B), measured as the length between junctions of the guard cells at each end of the stomate, decreased linearly with growth CO2 in S. dimidiatum (r2 = 0·49; P < 0·0001). In contrast, stomatal size increased weakly with growth CO2 in Br. japonicus (r2 = 0·14; P < 0·05) and was not associated with growth CO2 in Bo. ischaemum (r2 = 0·06; P = 0·13).

image

Figure 5. The response of mean (± 1 SE) stomatal density (A) and stomatal size (B) to a range of atmospheric CO2 for the three study species. Statistically significant differences (P < 0·05) in the magnitude of each variable among species are indicated by different letters. A * next to each letter indicates that the slope of the relationship is significantly (P < 0·05) different from zero.

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Despite higher gs, plants grown at lower CO2 concentrations had greater relative stomatal limitation of photosynthesis (lg) than plants grown at higher CO2 concentrations (Fig. 6). The strongest effect was for Br. japonicus, in which the relationship was non-linear (second-order polynomial, r2 = 0·82; P < 0·0001). Stomatal limitation of photosynthesis decreased linearly with increasing growth CO2 for S. dimidiatum (r2 = 0·51; P < 0·001) and Bo. ischaemum (r2 = 0·26; P = 0·03). Although S. dimidiatum and Br. japonicus had similar lg at elevated growth CO2, they diverged at subambient CO2. Among species, stomatal limitation of photosynthesis was inversely proportional to gs, being greatest in Bo. ischaemum, followed by Br. japonicus and S. dimidiatum.

image

Figure 6. The response of mean (± 1 SE) relative stomatal limitation of photosynthesis (lg) to increasing growth CO2 for C3 (A) and C4 (B) species. Note the difference in scales on the Y-axis between (A) and (B).

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Because plants were measured at the same LAVPD across treatments, the intrinsic water use efficiency (A/gs) is representative of the water cost of photosynthesis in contrasting growth CO2 environments. To illustrate the influence of stomatal acclimation to CO2 on stomatal optimization of water loss relative to carbon gain, we plotted A/gs of all plants measured at a Ca of 200 µmol mol−1 (where the strongest evidence for acclimation was observed, Fig. 2A) as a function of their growth CO2 concentration (Fig. 7A–C). In S. dimidiatum, A/gs increased significantly with growth CO2 (r2 = 0·59; P < 0·0001). Thus, when measured at 200 µmol mol−1 CO2, A/gs for plants grown at 550 µmol mol−1 was twice that of plants grown at 250 µmol mol−1 CO2. In contrast, growth CO2 concentration had no effect on A/gs measured at a common Ca for either grass species (Figs 7B & C).

image

Figure 7. Mean (± 1 SE) intrinsic leaf water use efficiency (A/gs) measured at 200 µmol mol−1Ca for each species in each growth CO2 treatment. A/gs increased significantly with growth CO2 only in S. dimidiatum. Note the difference in scales on the Y-axis between (A), (B) and (C).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We found clear evidence for stomatal acclimation to growth CO2 in Solanum dimidiatum, a C3 perennial forb, based on comparisons of the gsCi curves among plants grown across a range of CO2 concentrations. The strongest response of gs to Ci occurred in plants grown at 250 and 294 µmol mol−1 CO2, followed by plants grown at 354 µmol mol−1 CO2 (Fig. 2A). In contrast, gsCi curves did not differ for plants grown at greater than 400 µmol mol−1 CO2. Although other studies have observed stomatal acclimation from ambient to elevated CO2 (Morgan et al. 1994; Tuba, Szente & Koch 1994; Chen, Begonia & Hesketh 1995; Šantrüček & Sage 1996; Bunce 2001; Lodge et al. 2001), no evidence of such a pattern was found for any species in the present study (Figs 2A & 4). The results indicate that in S. dimidiatum, stomatal acclimation to growth CO2 occurred non-linearly, below an apparent threshold of 350–400 µmol mol−1 CO2. To our knowledge, this is the first study to document stomatal acclimation to subambient CO2 in the field.

Neither grass species, Br. japonicus or Bo. ischaemum, showed evidence of stomatal acclimation to growth CO2; there were no differences in the gsCi response curves for either species across growth CO2 treatments (Fig. 4A & B). This result was surprising, given systematic differences in absolute photosynthetic rates (Anderson et al. 2001) and gs (Fig. 1) between the C3 annual Br. japonicus and the C4 perennial Bo. ischaemum. Our observations for Br. japonicus and Bo. ischaemum therefore suggest that stomatal responses to CO2 in C3 and C4 grass species, like growth responses, may be quite similar (Wand et al. 1999). In contrast, stark differences in stomatal acclimation to CO2 between S. dimidiatum and Br. japonicus indicate that species with similar photosynthetic pathways may have vastly different stomatal responses to CO2.

Previously, we observed significant up-regulation of the maximum velocity of carboxylation (Vcmax) at subambient growth CO2 in S. dimidiatum (Anderson et al. 2001). The observation that gs is often coupled to photosynthetic capacity (e.g. Wong, Cowan & Farquhar 1979) raises the possibility that stomatal acclimation in S. dimidiatum represents a response to photosynthetic acclimation rather than a direct response to growth CO2. A significant positive relationship was observed between gsmax and Vcmax (Fig. 3A), but variation in Vcmax could only account for 27% of the variation in gsmax. The CO2 saturated photosynthetic rate (Amax), in contrast, was not related to gsmax (Fig. 3B). Although the mechanism for stomatal acclimation to atmospheric CO2 concentration is not known, the present results suggest that stomatal acclimation to CO2 may not be driven solely by adjustments in photosynthetic biochemistry (e.g. Bunce 2001; Lodge et al. 2001; but see Jarvis et al. 1999).

As in previous studies in grasslands, increasing growth CO2 reduced steady-state gs in all species (Fig. 1; Johnson, Polley & Mayeux 1993; Jackson et al. 1994; Knapp et al. 1996; Niklaus, Spinnler & Körner 1998; Lee et al. 2001). Our results suggest that stomatal acclimation to CO2 in S. dimidiatum was responsible for driving both the large (81%) and non-linear decrease in steady-state gs along the CO2 gradient (Fig. 1A). For example, steady-state gs at a CO2 concentration of 200 µmol mol−1 was 75% higher than it would have been if acclimation had not occurred (based on a comparison of the gsCi curves of plants grown at 250 µmol mol−1 CO2 versus those grown at greater than 400 µmol mol−1 CO2; Fig. 2A). In contrast, gs declined linearly and more modestly (46–49%) with CO2 in the two grass species in which stomatal acclimation was not observed.

Some studies suggest that decreased stomatal density is a mechanism for reducing gs in response to rising CO2 (Woodward 1987; Beerling & Woodward 1993; Kurschner et al. 1997). Our results from a C3/C4 grassland provide little support for this pattern, as the species showing the strongest decline in gs with rising CO2 (S. dimidiatum,Fig. 1A), actually increased stomatal density with increasing growth CO2 (Fig. 5A). Similarly, the relationship between stomatal density and growth CO2 was very different between Br. japonicus and Bo. ischaemum, species that had nearly identical declines in gs in response to increased CO2 along the gradient. Of the three species measured only Bo. ischaemum decreased stomatal density in response to rising CO2 (Fig. 5A), but the strength of this relationship was relatively weak (r2 = 0·15). These results suggest that gs responses to CO2 need not correlate in magnitude or direction with stomatal density responses to CO2. However, it should be noted that measurements of gs and stomatal density were not made at the same time; therefore we cannot rule out the possibility that phenological variation in stomatal morphology also contributed to the lack of correlation between stomatal density and gs. Nevertheless, our results suggest that the response of stomatal density to CO2 is not necessarily generalizable across species or growth forms (e.g. Knapp et al. 1994).

Stomates were significantly larger in S. dimidiatum plants grown at subambient CO2 (Fig. 5B). As a potential developmental response to CO2 starvation, the increased stomatal size facilitates CO2 diffusion into the leaf (Parkhurst 1994) because conductance is proportional to the square of the effective radius of the stomatal pore (Nobel 1991). Therefore, larger stomatal size at subambient CO2 may contribute to the substantial increases in gs that were observed in S. dimidiatum (Fig. 1A). Similarly, small stomatal size at elevated CO2 could constrain gs upon exposure to subambient CO2, as was observed in our study (Fig. 2A). Large stomatal size in S. dimidiatum, however, does not appear to prevent guard cell control of water loss, as gs measured at high Ci was the same for all plants regardless of growth CO2 (Fig. 2A). A reduction in gs for elevated CO2-grown S. dimidiatum, despite an increase in stomatal density, also suggests that increased stomatal size may be the primary morphological adjustment implicated in stomatal acclimation to CO2. The observation that stomatal size varied weakly with CO2 treatment in Br. japonicus and Bo. ischaemum (e.g. Malone et al. 1993), where stomatal acclimation did not occur (Fig. 4), is consistent with this view.

To determine whether stomatal acclimation could influence CO2 diffusion and carbon assimilation, relative stomatal limitation of photosynthesis (lg; Jones 1985) was calculated from ACi curves measured concurrently with gsCi curves (Fig. 6). In general, the magnitude of lg was inversely proportional to gs among species (S. dimidiatum < Br. japonicus < Bo. ischaemum). Stomatal closure at higher growth CO2, however, did not lead to an increase in lg, suggesting that the stimulation of photosynthesis by CO2 more than compensated for any diffusional limits imposed by stomata (Tissue et al. 1995; Drake et al. 1997).The value of lg was approximately similar for both C3 species at elevated CO2 (approximately 10–15%) but lg in Br. japonicus increased disproportionately relative to S. dimidiatum at subambient growth CO2 concentrations. These results suggest that stomatal acclimation may have a significant role in reducing lg in S. dimidiatum at subambient CO2 when compared with the non-acclimating Br. japonicus.

Stomatal acclimation, by increasing water loss from the leaf surface, could alter intrinsic WUE (defined here as A/gs). Although rising CO2 increases WUE in this grassland (Anderson et al. 2001), stomatal acclimation appears to modify the trajectory of this response in S. dimidiatum. The influence of stomatal acclimation on WUE was apparent in the strong effect that growth CO2 concentration had on A/gs even when plants were measured at the same Ca. For example, A/gs increased significantly with growth CO2 in S. dimidiatum measured at 200 µmol mol−1 (Fig. 7A). In contrast, A/gs measured at a common 200 µmol mol−1Ca did not vary with growth CO2 in either of the non-acclimating grass species (Fig. 7B & C).

The observation that A/gs was lower in S. dimidiatum than it would have been without acclimation suggests that, in the past, this phenotype could have had a negative impact on plant productivity in water-limited grasslands (Polley et al. 1993; Sage 1995; Hsiao & Jackson 1999). Therefore, what is the ecological significance of stomatal acclimation to subambient CO2? One possibility is that maximizing leaf A/gs is not necessarily an adaptive response to variation in CO2. For example, maintaining a high A/gs may be a disadvantage if conserved water that is stored in soil is lost to competitors or through soil evaporation (DeLucia & Schlesinger 1991; Jones 1993). It is also likely that the strength of stomatal regulation of water loss in a given species is dependent on other correlated physiological and morphological traits (Givnish 1986). For example, weak stomatal control of water loss may be associated with species that have access to stable sources of water via deep roots or that possess xylem that is resistant to drought-induced cavitation (Jones 1993; Jackson, Sperry & Dawson 2000). Given our limited understanding of the whole-plant context of stomatal responses to CO2 and the mechanisms by which these responses occur, the adaptive significance of stomatal acclimation to subambient CO2 remains uncertain.

It is not known to what degree comparisons of extant plants grown in subambient and elevated CO2 concentrations are representative of actual changes in plant function from the past to the present and future. As we have no information on the genotypes of past populations, it is not known if our study species have evolved in response to rising CO2. We observed strong evidence of stomatal acclimation in S. dimidiatum at growth CO2 concentrations as high 294 µmol mol−1, a level which occurred relatively recently (around 1900 AD). Although a step change in atmospheric CO2 can act as a selective agent on plant populations (Ward et al. 2000), the relatively short time span involved suggests that populations of a perennial species such as S. dimidiatum may not have evolved substantially in response to the gradual increase in CO2. In consequence, our results suggest that the greatest stomatal response to CO2 (approximately 76%) in S. dimidiatum has already taken place. Further reductions in gs will likely occur as atmospheric CO2 increases, but will be of much smaller magnitude. In contrast, the absence of stomatal acclimation in Br. japonicus and Bo. ischaemum suggests that future stomatal responses to rising CO2 may mimic those of the past in these species.

Our results have implications for modelling efforts aimed at predicting how stomatal responses to CO2 may feedback on ecosystem hydrology and climate (Henderson-Sellers, McGuffie & Gross 1995; Pollard & Thompson 1995; Sellers et al. 1996; Jackson et al. 1998). When stomatal acclimation is included in these models, it is usually assumed to occur linearly with CO2 (e.g. Sellers et al. 1996). In contrast to these assumptions, we observed stomatal acclimation only at subambient CO2, a phenomenon that caused reductions in gs from subambient to ambient CO2 to be greater than those observed from ambient to elevated CO2. The occurrence of stomatal acclimation and substantial reductions in gs from pre-industrial to current ambient CO2 concentration suggest that large changes in evapotranspiration and soil drainage in grassland ecosystems may have already occurred. Incorporating these physiological perspectives into modelling studies could improve our ability to predict changes in ecosystem water fluxes and water availability with rising CO2 and to understand their magnitudes relative to the past.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank R. P. Whitis for operating and maintaining the experimental CO2 chambers and C. W. Cook and A. Gibson for assistance. We thank L. J. Anderson, C. M. Caruso, D. S. Ellsworth, R. A. Gill, H. J. Schenk and J. Herrick for helpful comments on previous versions of this paper. This research was funded by the National Institute for Global Environmental Change through the US Department of Energy (Cooperative Agreement No. DE-FC03-90ER61010). Any opinions, findings and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the DOE, NIGEC or USDA.

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  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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