JXB Advance Access originally published online on April 23, 2004
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Journal of Experimental Botany, Vol. 55, No. 400, pp. 1157-1166, May 1, 2004
© 2004 Oxford University Press
Limitations to Photosynthetic Performance |
Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of Rubisco
Received 5 November 2003; Accepted 10 February 2004
1 Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia
2 Department of Biological Sciences University of Essex, Colchester CO4 3SQ, UK
* To whom correspondence should be addressed. Fax +61 2 6125 5075. E-mail: Susanne.Caemmerer{at}anu.edu.au
Abbreviations: A, CO2 assimilation rate; Ca, ambient CO2 concentration; Ci, intercellular CO2 concentration, F', steady-state fluorescence during photosynthesis in the light; Fm maximun fluorescence of a dark-adapted leaf after a saturating light pulse; Fm', maximum fluorescence of a light-adapted leaf after a saturating light pulse; Fo, minimal fluorescence yield of a dark-adapted leaf; Fq'/Fm' = (Fm' – F')/Fm, quantum yield of electron flow to PSII; Fq'/Fv' = (Fm'– F')/(Fm – Fo), photochemical quenching; PSII, photosystem II; RuBP, D-ribulose-1,5-bisphosphate; Rubisco, RuBP carboxylase/oxygenase; g, stomatal conductance; I, irradiance; NPQ = (Fm – Fm')/Fm, Stern–Volmer non-photochemical quenching.
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Abstract |
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High-resolution imaging of chlorophyll a fluorescence from intact tobacco leaves was used to compare the quantum yield of PSII electron transport in the chloroplasts of guard cells with that in the underlying mesophyll cells. Transgenic tobacco plants with reduced amounts of Rubisco (anti-Rubisco plants) were compared with wild-type tobacco plants. The quantum yield of PSII in both guard cells and underlying mesophyll cells was less in anti-Rubisco plants than in wild-type plants, but closely matched between the two cell types regardless of genotype. CO2 assimilation rates of anti-Rubisco plants were 4.4 µmol m–2 s–1 compared with 17.3 µmol m–2 s–1 for the wild type, when measured at a photon irradiance of 1000 µmol m–2 s–1 and ambient CO2 of 380 µmol mol–1. Despite the large difference in photosynthetic capacity between the anti-Rubisco and wild-type plants, there was no discernible difference in the rate of stomatal opening, steady-state stomatal conductance or response of stomatal conductance to ambient CO2 concentration. These data demonstrate clearly that the commonly observed correlation between photosynthetic capacity and stomatal conductance can be disrupted in the long term by manipulation of photosynthetic capacity via antisense RNA technology. It was concluded that stomatal conductance is not directly determined by the photosynthetic capacity of guard cells or the leaf mesophyll.
Key words: Chlorophyll fluorescence, guard cell, Rubisco, stomatal conductance, tobacco.
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Introduction |
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Guard cells are located in the epidermis of plant leaves and, in pairs, form stomatal pores. Variations in stomatal aperture control both the influx of CO2 and water loss from plants through transpiration to the atmosphere. A large number of environmental factors that affect stomatal aperture have been described and investigated (Farquhar and Sharkey, 1982; Morison, 1987; Mansfield et al., 1990). In brief, stomata open in response to low CO2 concentrations, high light, and high humidity, whereas closure is promoted by high CO2 concentrations, darkness, low humidity, and/or high temperature and drought. However, understanding of the responses of stomata to these environmental stimuli is incomplete and no model has emerged to link them with guard cell metabolism. Early observations showed that guard cells, in isolated epidermal peels, are able to respond to environmental factors such as light, CO2, and humidity, suggesting that part of the mechanism(s) responsible for sensing these environmental factors must be located in the epidermis (Willmer and Fricker, 1996; Frechilla et al., 2002). In intact photosynthesizing leaves, stomatal conductance is co-ordinated with the CO2 requirement of the mesophyll, such that the ratio of intercellular to ambient CO2 concentration, Ci/Ca, is maintained almost constant with variation in irradiance and CO2 (Wong et al., 1985; Sharkey and Raschke, 1981). There is also a striking correlation between photosynthetic capacity and stomatal conductance which maintains the Ci/Ca ratio constant when photosynthetic capacity is modulated in the longer term by growth conditions such as nitrogen nutrition, light environment, and elevated CO2 concentration (Wong et al., 1979, 1985). This led to the hypotheses that guard cells may be able to respond directly to the photosynthetic capacity of the mesophyll via some signal from the mesophyll, or that the guard cell photosynthesis may act as a signal via a photosynthetic metabolite (Wong et al., 1979). They suggested that there were several suitable metabolites, such as ATP, NADPH, and RuBP, the concentrations of which decrease with an increase in the rate of carboxylation associated with an increase in Ci, that could serve as signals, causing the conservation of Ci/Ca with varying Ca. Farquhar and Wong formulated an empirical model relating stomatal conductance to the chloroplast ATP pool and photosynthetic capacity of leaves (Farquhar and Wong, 1984; Buckley et al., 2003). Similarly Zeiger and Zhu (1998) and Zhu et al. (1998) demonstrated a close correlation between guard cell zeaxanthin content and stomatal aperture and suggested that guard cell zeaxanthin may be the signal metabolite. Several other empirical models of stomatal function have built on the close link between photosynthetic capacity and stomatal conductance (Ball et al., 1987; Jarvis and Davies, 1998).
Most guard cells have chloroplasts, but there had been doubt as to whether a complete photosynthetic carbon reduction (PCR) and photosynthetic carbon oxidation (PCO) cycle operated in the guard cells (for reviews see Zeiger et al., 2002; Outlaw, 2003). The presence of Rubisco in guard cells has been verified with immunocytochemical localization (Zemmel and Gepstein, 1985; Ueno, 2001). Measurements of chlorophyll fluorescence in guard cell chloroplasts of intact leaves under different CO2 and O2 partial pressures have demonstrated that Rubisco acts as a significant sink for ATP and NADPH in guard cells (Cardon and Berry, 1992; Baker et al., 2001; Lawson et al., 2002, 2003). However, the role guard cell photosynthesis plays in stomatal function in vivo, through provision of ATP, starch, or sucrose and whether it participates in environmental sensing and signalling systems remains to be elucidated (Zeiger et al., 2002; Outlaw, 2003; Ritte and Raschke, 2003). Talbott and Zeiger (1998) have suggested that three osmoregulatory pathways coexist in guard cells and that the relative importance of these different pathways may change depending upon the time of day and/or growth conditions. Uptake of K+ and Cl– from the apoplast and the synthesis of malate from carbon skeletons derived from starch are believed to be involved in early morning opening and under blue light. In addition, sucrose from starch hydrolysis is also thought to have a role under these conditions. Interestingly, the model put forward by Talbott and Zeiger suggests that, in the afternoon, it is sucrose, supplied directly from photosynthetic carbon fixation in the guard cells that is important to maintain stomatal opening. Several studies have demonstrated that mesophyll derived sucrose accumulates in the guard cell apoplast and suggested that this may also be an important regulator of stomatal conductance (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001).
Although a correlation between stomatal conductance and photosynthetic capacity has been observed in many studies (Wong et al., 1979, 1985; Hetherington and Woodward, 2003), it has been shown that this relationship can be broken in the short term in the intact leaf (Sharkey and Raschke, 1981; Jarvis and Morison, 1981; Morison, 1987). In transgenic plants with impaired photosynthesis, stomatal conductance appeared largely unaffected (Quick et al., 1991; Stitt et al., 1991; Hudson et al., 1992; Lauerer et al., 1993; Evans et al., 1994; Price et al., 1995; von Caemmerer et al., 1997; Muschak et al., 1999), thereby breaking the correlation between photosynthetic capacity and stomatal conductance under high light, in intact plants over the longer term. Here results obtained on stomatal function in transgenic plants with reduced Rubisco are reviewed and new data about guard cell chloroplast electron transport in transgenic tobacco with reduced Rubisco are presented.
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Materials and methods |
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Plant material and growth conditions
Transgenic tobacco with reduced amounts of Rubisco was grown from seed collected from selfed T2 progeny of a tobacco (Nicotiana tabacum cv. W38) transformant with an antisense gene directed against the Rubisco small subunit driven by the CaMV35S promoter (Hudson et al., 1992). Seedlings were homozygous with two copies of the rbcS antisense gene and typically had 10–15% of wild-type Rubisco content. Untransfromed cv. W38 tobaccos were used as controls. Plants were grown at Essex University, UK in controlled environmental cabinets at a photon irradiance of 300 µmol m–2 s–1 and an 18 h photoperiod at 25/18 °C day/night temperatures. CO2 concentration inside the cabinet was maintained at 800 µmol mol–1. These growth conditions were chosen to ensure that plants had similar photosynthetic rates under the growth conditions and resulted in anti-Rubisco plants, which looked similar to wild-type plants and grew at similar rates. Fluorescence images of whole plants were taken at the seedling stage. Plants were then transplanted into 1.0 l pots. These plants were used for gas exchange and guard cell fluorescence measurements once they had reached the six leaf stage.
Plants were also grown at the Australian National University, Canberra ACT, Australia in controlled environmental cabinets under similar conditions at a photon irradiance of 300 µmol m–2 s–1 with an 18 h photoperiod at 25/18 °C day/night temperatures and CO2 concentration of 800 µmol mol–1. Plants were grown in 1.0 l pots with 2.5–4 g Osmocote l–1 soil (15/4.8/10.8/1.2 by vol. N/P/K/Mg + trace elements: B, Cu, Fe, Mn, Mo, Zn, Scotts Australia Pty Ltd., Castle Hill, Australia) added to the soil. These plants were used for gas exchange measurements only, at the same plant age as above.
Fluorescence imaging of seedlings
Images of chlorophyll fluorescence parameters were obtained essentially as described by Barbagallo et al. (2003) using a CI Imager chlorophyll fluorescence imaging system (Technologica Ltd, Colchester, UK). Seedlings were dark-adapted for 15 min before Fo, the minimal fluorescence was measured using weak measuring pulses. Then Fm, the maximal fluorescence was measured during an 800 ms exposure to a photon irradiance of 4800 µmol m–2 s–1. The photon irradiance was then increased to 300 µmol quanta m–2 s–1 for 15 min and F' was continuously monitored while Fm', the maximal fluorescence from leaves in actinic light was monitored at 5 min intervals by applying saturating light pulses. This was repeated at a photon irradiance of 500 µmol m–2 s–1 before the seedlings were; Returned to darkness and Fm and Fo were measured again after 15 min dark-adaptation.
Fluorescence imaging of guard cell chloroplasts
Guard cell fluorescence was imaged from the underside of leaves of six transgenic tobacco plants with reduced Rubisco and six wild-type plants of tobacco when plants were at the six leaf stage. The protocol and the microscope imaging system used were as described by Lawson et al. (2003). The purpose-designed microscope cuvette was attached to a portable gas exchange system (CIRAS 2, PP Systems, Hitchin, Hertsforshire, UK). CO2 and water vapour in the leaf chamber were maintained at 380 µmol mol–1 and 23 mmol mol–1, respectively.
Gas exchange measurements
Gas exchange measurements were made on young plants, which had six leaves or less with a portable gas exchange system (Li-Cor 6400, Li-Cor Lincoln, Nebraska). Light was provided by a Red/Blue LED light source (Li-Cor 6400-02B). Leaves were first equilibrated at a photon irradiance of 100 µmol quanta m–2 s–1 for 20 min. The photon irradiance was then increased to 1000 µmol m–2 s–1 for 30–40 min and then returned to 100 µmol m–2 s–1. During the experimental run, leaf chamber CO2 and humidity were maintained at 380 µmol mol–1 and 23 mmol mol–1 and leaf temperature was maintained at 25 °C. This resulted in constant leaf to air vapour pressure of approximately 10 mbar. The experiments were repeated at different ambient CO2 concentrations. As above, the leaves were allowed to stabilize at a photon irradiance of 100 µmol m–2 s–1 at the chosen CO2 concentration, before this was increased to 1000 µmol m–2 s–1.
Measurements of stomatal numbers
After gas exchange measurements, silicone rubber impressions were taken of the abaxial leaf surface with dental impression material (Xantopren VL plus, Heraeus Kulzer, Dormagen, Germany) in accordance with the method described by Weyers and Johanson (1985). The impressions were divided into three 10x10 mm areas per leaf from which positives were made with clear nail varnish spread onto microscope slides. Guard cell numbers per 10x10 mm area were counted in nine different fields of view per area following the systematic sampling strategy outlined by Poole and Kürschner (1999) using a microscope and eye area graticule.
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Results |
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Chlorophyll fluorescence images of wild-type and anti-Rubisco tobacco seedlings
Images of the quantum yield of PSII (Fq'/Fm') and non-photochemical quenching (NPQ), are shown in Fig. 1. These images illustrate the differences in photosynthetic characteristics between wild-type and anti-Rubisco tobacco seedlings. Fq'/Fm' is proportional to chloroplast electron transport rate (Genty et al., 1989) and is much higher in the wild type compared with the anti-Rubisco seedling, showing that a reduction in Rubisco reduces chloroplast electron transport. It is now widely accepted that NPQ is stimulated by acidification of the thylakoid lumen. Consequently, the higher values of NPQ in the anti-Rubisco plants, compared with wild-type seedlings, may reflect decreased utilization of ATP, which would be expected to decrease lumen pH. A summary of replicate fluorescence measurements made in the sequence described in Fig. 1B is given in Table 1. Low values of Fq'/Fm' and constitutively high NPQ have been observed in several transgenic plants with impaired photosynthesis. Examples include potato and tobacco plants with reduced fructose-1,6-bisphosphatase (FBPase) activity (Bilger et al., 1995; Fisahn et al., 1995), transgenic tobacco with reduced activity of phosphoribulokinase (Habash et al., 1996), tobacco, and rice with reduced amounts of Rubisco (Quick et al., 1991; Lauerer et al., 1993; Ruuska et al., 2000a; Ushio et al., 2003); and tobacco with reduced glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity (Price et al., 1995; Ruuska et al., 2000a).
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Reduced quantum yield of PSII in guard cell chloroplasts of anti-Rubisco plants
Fluorescence of guard cell chloroplasts was imaged when plants were at approximately the six-leaf stage. In the anti-Rubisco plants, the transgene construct was driven by the CaMV 35S promoter and, therefore, it was expected that the antisense gene would be expressed in all cell types including the guard cells (Hudson et al., 1992). Fluorescence images from guard cells of wild-type and anti-Rubisco plants revealed that Fq'/Fm' was reduced in the antisense plants (Fig. 2). There were no apparent anatomical differences in guard cells of transgenic and wild-type plants and anti-Rubisco plants had similar numbers of stomata per leaf area (Fig. 3).Values of Fq'/Fm' of guard cell chloroplasts were lower in anti-Rubisco compared with wild-type plants, at all but one photon irradiance measured (Fig. 4A). As observed previously by Lawson et al. (2002, 2003), there was a good correlation between guard cell and mesophyll values of Fq'/Fm' (Fig. 4B).
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Stomatal conductance and rate of stomatal opening
Both the rate of stomatal opening and stomatal conductance were measured after 30 min at high photon irradiance (1000 µmol quanta m–2 s–1) on the same plants on which the guard cell fluorescence measurements were made. The gas exchange measurements were also repeated on a second set of plants grown at the Australian National University (Fig. 5; Tables 2, 3). Figure 5 shows the experimental protocol. Leaves were allowed to stabilize at a photon irradiance of 100 µmol m–2 s–1; after which photon irradiance was increased to 1000 µmol m–2 s–1 while ambient CO2, and humidity were maintained constant. In the wild-type plants, CO2 assimilation rate increased rapidly after the irradiance was increased to between 16.5–18 µmol m–2 s–1. By contrast, in the anti-Rubisco plants, only a very small stimulation in photosynthesis was observed when the photon irradiance was increased, up to a maximum of 5 µmol m–2 s–1. The rate of stomatal opening was measured in parallel with photosynthesis and an initial fast, brief phase of opening was observed followed by a slower, prolonged opening phase (Fig. 5B). This was similar to the pattern observed by Cardon et al. (1994) for Zea mays. No significant difference was found in either the rate of stomatal opening (approximated by linear regression after the first rapid phase of opening) or stomatal conductance at 30 min between wild-type and anti-Rubisco plants (Fig. 5; Tables 2, 3). Because of the much lower CO2 assimilation rates in the anti-Rubisco plants, these plants operated at much higher intercellular CO2 concentrations (Fig. 5C).
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Response of stomatal conductance and rate of stomatal opening to CO2 concentration
To gain further insight into the relationship between CO2 concentration and stomatal opening, the experimental protocol described above was repeated at different ambient CO2 concentrations (Figs 6, 7). Both the rate of stomatal opening and stomatal conductance at 30 min were similar between wild-type and anti-Rubisco plants and both decreased with increasing ambient CO2 concentration (Fig. 6), although CO2 assimilation rates and the ratio of intercellular CO2 to ambient CO2 concentrations were very different (Fig. 7).
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Discussion |
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Guard cell photosynthesis is reduced in anti-Rubisco plants
Many studies have characterized the effect of antisense RNA suppression of Rubisco content on CO2 assimilation rate in leaves (Quick et al., 1991; Stitt et al., 1991; Hudson et al., 1992; Lauerer et al., 1993; Krapp et al., 1994; Stitt and Schulze, 1994; Evans et al., 1994; von Caemmerer et al., 1994; Ruuska et al., 1998, 2000a, b; Makino et al., 2000; Ushio et al., 2003). The decrease of Rubisco activity via these molecular techniques results in an imbalance between the capacity of the chloroplast electron transport to regenerate RuBP and the PCR cycle’s capacity to fix CO2. This is evident in the altered characteristics of the CO2 response of leaf CO2 assimilation (Hudson et al., 1992). This imbalance, in turn, leads to an increase in metabolite concentrations such as RuBP and ATP (Quick et al., 1991; Lauerer et al., 1993) and increased conversion of the xanthophyll cycle pigments to zeaxanthin (Ruuska et al., 2000a; Ushio et al., 2003). The decrease in Rubisco content also leads to decreased rates of chloroplast electron transport as shown by the reduced Fq'/Fm' values in Fig. 1 and Table 1 with a concomitant increase of non-photochemical quenching. Ruuska et al. (2000b) showed that this was not accompanied by an increase in chloroplast electron transport to other acceptors, such as O2, for example.
The decrease in Fq'/Fm' values from guard cells of anti-Rubisco compared with wild-type plants seen in Fig. 4, provides the first evidence that a decrease in Rubisco content suppresses guard cell photosynthesis. This contributes further to the mounting evidence that Rubisco acts as a significant sink for ATP and NADPH in guard cell chloroplasts. Although there is no direct evidence, it is likely that the lower content of Rubisco in guard cell chloroplasts has led to a similar imbalance between chloroplast electron transport and PCR and PCO cycle capacity, and that this may also lead to increased levels of concentrations of metabolites, such as RuBP and ATP, as well as zeaxanthin content.
It has been suggested that the PCR and PCO cycle consume a smaller fraction of the photochemical energy in guard cells than in mesophyll cells, with the remainder being used for ion pumping and phosphoenolpyruvate carboxylation (Shimazaki et al., 1989; Tominaga et al., 2001). However, the close correlation between Fq'/Fm' in guard and mesophyll cells in both wild-type and anti-Rubisco plants suggests that the antisense reduction in Rubisco had a similar effect on chloroplast electron transport in guard cells and mesophyll cells. Whether the altered biochemistry in the guard cell chloroplasts of the anti-Rubisco plants is, in part, responsible for the altered relationship between stomatal conductance and photosynthetic capacity is discussed below.
The correlation between photosynthetic capacity and stomatal conductance
The reduction in Rubisco by antisense RNA here had no apparent effect on stomatal conductance, despite the severe reduction in rates of CO2 assimilation (Fig. 5; Tables 2, 3). Measurements of stomatal conductance in Rubisco antisense plants by Stitt et al. (1991) and Hudson et al. (1992) were the first results to show that the commonly observed correlation between stomatal conductance and photosynthetic capacity is disrupted by these molecular manipulations of photosynthetic capacity. The lack of stomatal response to a decrease in photosynthetic capacity has been observed frequently in transgenic plants with photosynthetic impairments due to antisense constructs to PCR cycle enzymes (Stitt et al., 1991; Lauerer et al., 1993; Evans et al., 1994; Price et al., 1995; Paul et al., 1995; von Caemmerer et al., 1997; Fisahn et al., 1995; Muschak et al., 1999). It was also observed in tobacco plants with an antisense construct to the chloroplastic cytochrome b6f complex in all but the plants with the lowest levels of b6f complex (Price et al., 1998). It was also found that the altered guard cell photosynthesis did not affect the rate of stomatal opening under these experimental conditions (Fig. 5; Tables 2, 3). This suggests that guard cells are not strongly reliant on guard cell photosynthesis or photosynthetic electron transport for the maintenance of stomatal conductance.
These results raise important questions about how the observed correlation between stomatal conductance and photosynthetic capacity is co-ordinated when photosynthetic capacity is altered through variation in growth environments (Wong et al., 1979; Morison 1987; Hetherington and Woodward, 2003). Similarities between mesophyll and guard cell photosynthesis in anti-Rubisco plants would suggest that metabolite pools of ATP, RuBP or zeaxanthin in guard cells may be increased in these transgenic plants at all CO2 concentrations. Under these conditions the empirical model of stomatal function by Farquhar and Wong (1984) would predict higher stomatal conductance for the anti-Rubisco plants. The model would also predict a reduced CO2 sensitivity of stomatal conductance, however, neither of these predictions were observed in the anti-Rubisco plants (Fig. 6; Hudson et al., 1992). The lack of a relationship between photosynthetic capacity and stomatal conductance at high light in transgenic plants with impaired photosynthesis suggests that there may not be a direct mechanism linking the two processes. This could mean that co-ordination of both processes is fortuitous and the result of co-evolution of independent processes.
The CO2 response of stomatal conductance
Stomatal conductance of most leaves responds to changes in CO2 concentration. In general, stomata open when CO2 concentrations are lowered and close when CO2 concentrations are increased. The question about how guard cells sense CO2 has not been resolved and a number of different hypotheses exist (for reviews see Mansfield et al., 1990; Assmann, 1999; Cousson, 2000). It is generally thought that guard cells sense Ci, rather than Ca, or perhaps an intermediate concentration in the stomatal pore. This is an attractive proposition because ambient CO2 does not usually vary greatly and sensing Ci would allow guard cells to respond to the photosynthetic requirements of the mesophyll. Stomatal responses to Ci are typically determined by varying Ca and this results in co-variation of Ci and Ca. Mott (1988), in an elegant set of experiments, used a gas-exchange system which controlled conditions on the two sides of the leaf independently to produce changes in Ci in amphistomatous leaves of Xanthium strumarium and Helianthus annuus while keeping CO2 concentrations at the leaf surface constant. Mott concluded that these leaves responded to changes in Ci rather than Ca. The molecular manipulations of photosynthetic capacity via antisense RNA technology can be viewed as a similar experiment. The artificial decrease in photosynthetic capacity results in an increase in Ci at constant Ca that does not, however, appear to be perceived by guard cells, as stomatal conductance is similar to the wild type and Ci/Ca is increased (Figs 5, 7; Tables 2, 3). Experiments that have examined transgenic plants with varying decreases in photosynthetic capacity show little change in stomatal conductance, but increasing Ci/Ca ratios as photosynthetic capacity is reduced (see for example Quick et al., 1991; Stitt et al., 1991; Hudson et al., 1992; Lauerer et al., 1993; Price et al., 1995, 1998; von Caemmerer et al., 1997). Variation in photosynthetic rate in C3 leaves through variations in ambient O2 concentrations are also not perceived by guard cells. A reduction in O2 concentration increases photosynthetic rate, thus resulting in reduced ratios of Ci/Ca with no change in stomatal conductance (Gauhl and Bjorkman, 1969; Farquhar and Wong, 1984). Perhaps the evolution of C4 photosynthesis can be viewed in the same light. The correlation between photosynthetic capacity and stomatal conductance has also been altered through increased photosynthetic capacity, resulting in lower ratios of Ci/Ca (Farquhar et al., 1978; Wong et al., 1985). Interestingly, Lawson et al. (2003) showed that the response of guard cell fluorescence to CO2 and O2 concentrations in the C4 species, Amaranthus caudatus, was C3-like, suggesting that guard cell chloroplasts of C4 species operate a C3 photosynthetic pathway. This is supported by immunogold labelling studies on Amaranthus viridis, which showed substantial amounts of Rubisco in the guard cells, but not in mesophyll cells (Ueno, 2001).
Although the anti-Rubisco plants operate at different ratios of Ci/Ca compared with wild type (Tables 2, 3), stomatal conductance did respond to ambient CO2 and the stomatal response was similar between wild-type and anti-Rubisco plants (Figs 6, 7). This had also been observed by Hudson et al. (1992), for anti-Rubisco plants, and by Muschak et al. (1999) for potato plants with reduced FPBase activity. By contrast, Stitt et al. (1991) reported lower stomatal conductance at low CO2 concentrations in transgenic tobacco with the low Rubisco content. Hudson et al. (1992) reported that stomata appeared to respond to CO2 in such a way that Ci/Ca was maintained almost constant across CO2 concentrations, albeit at a different ratio of Ci/Ca. This was also observed here (Fig. 7) and the maintenance of the ratio of Ci/Ca fits with the observations of Wong et al. (1979). Since the guard cells of isolated epidermis are able to respond to CO2, the sensor for the stomatal response to CO2 must be contained within the epidermis (for a review, see Assmann, 1999; Frechilla et al., 2002). From the similarity of the CO2 response of stomatal conductance of wild-type and transgenic plants, it is tempting to conclude that either Ca or CO2 in the stomatal pore is sensed, rather than Ci. Furthermore, the results confirm that the stomatal CO2 response is not related to the CO2 response of guard cell or mesophyll photosynthesis in this study (Lawson et al., 2002).
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Conclusions |
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It has been shown that antisense decrease of Rubisco content reduced both guard cell and mesophyll photosynthesis with no apparent effect on stomatal conductance at high photon irradiance, the rate of stomatal opening or the response of stomatal conductance to Ca. It was concluded that the commonly observed correlation between photosynthetic capacity and stomatal conductance can be disrupted by the manipulation of photosynthetic capacity via antisense RNA technology, and that stomatal conductance is not tightly linked to the photosynthetic capacity of guard cells or of the leaf mesophyll.
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Acknowledgement |
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Dr S von Caemmerer’s visit to Essex University was supported by a Leverhulme Visiting Professorship.
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References |
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