Annals of Botany 89: 183±189, 2002 doi:10.1093/aob/mcf027, available online at www.aob.oupjournals.org Drought-inhibition of Photosynthesis in C3 Plants: Stomatal and Non-stomatal Limitations Revisited J . F L E X A S * and H . M E D R A N O Laboratori de Fisiologia Vegetal, Departament de Biologia, Universitat de les Illes Balears ± Instituto MediterraÂneo de Estudios Avanzados (UIB-IMEDEA), Carretera de Valldemossa Km 7´5, 07071 Palma de Mallorca, Balears, Spain Received: 27 April 2001 Returned for revision: 13 August 2001 Accepted: 22 October 2001 There is a long-standing controversy as to whether drought limits photosynthetic CO2 assimilation through stomatal closure or by metabolic impairment in C3 plants. Comparing results from different studies is dif®cult due to interspeci®c differences in the response of photosynthesis to leaf water potential and/or relative water content (RWC), the most commonly used parameters to assess the severity of drought. Therefore, we have used stomatal conductance (g) as a basis for comparison of metabolic processes in different studies. The logic is that, as there is a strong link between g and photosynthesis (perhaps co-regulation between them), so different relationships between RWC or water potential and photosynthetic rate and changes in metabolism in different species and studies may be `normalized' by relating them to g. Re-analysing data from the literature using light-saturated g as a parameter indicative of water de®cits in plants shows that there is good correspondence between the onset of drought-induced inhibition of different photosynthetic sub-processes and g. Contents of ribulose bisphosphate (RuBP) and adenosine triphosphate (ATP) decrease early in drought development, at still relatively high g (higher than 150 mmol H2O m±2 s±1). This suggests that RuBP regeneration and ATP synthesis are impaired. Decreased photochemistry and Rubisco activity typically occur at lower g (<100 mmol H2O m±2 s±1), whereas permanent photoinhibition is only occasional, occurring at very low g (<50 mmol H2O m±2 s±1). Sub-stomatal CO2 concentration decreases as g becomes smaller, but increases again at small g. The analysis suggests that stomatal closure is the earliest response to drought and the dominant limitation to photosynthesis at mild to moderate drought. However, in parallel, progressive down-regulation or inhibition of metabolic processes leads to decreased RuBP content, which becomes the dominant limitation at severe drought, and thereby inhibits photoã 2002 Annals of Botany Company synthetic CO2 assimilation. Key words: C3 plants, drought, water stress, photosynthesis, stomatal conductance, photochemistry, carboxylation, photophosphorylation, RuBP regeneration, Rubisco. INTRODUCTION There is a long-standing controversy as to whether drought mainly limits photosynthesis through stomatal closure (Sharkey, 1990; Chaves, 1991; Ort et al., 1994; Cornic and Massacci, 1996) or by metabolic impairment (Boyer, 1976; Lawlor, 1995). Evidence that impaired ATP synthesis is the main factor limiting photosynthesis even under mild drought (Boyer, 1976; Tezara et al., 1999) has further stimulated debate (Cornic, 2000; Lawlor and Cornic, 2002). Comparing results from different authors is dif®cult due to interspeci®c differences in the response of photosynthesis to leaf water potential and/or relative water content (RWC), the parameters most commonly used to assess the degree of drought (Tardieu and Simmoneau, 1998). To overcome this, we have exploited the relationship between stomatal conductance (g) and photosynthetic CO2 assimilation (Wong et al., 1979), since an early and progressive effect of drought is stomatal closure (Boyer, 1976; Sharkey, 1990; Chaves, 1991; Ort et al., 1994; Lawlor, 1995; Cornic and Massacci, 1996). We have recently demonstrated (Flexas et al., 2002; Medrano et al., 2002) that, by relating photosynthetic parameters to their corresponding lightsaturated g, a pattern of responses is revealed which is * For correspondence. Fax +34 971 173184, e-mail dbajfs4@ps.uib.es independent of acclimation processes and only slightly dependent on the cultivars and species. For instance, the relationships between different photosynthetic parameters and the absolute values of g in grapevines (Vitis vinifera) and several Mediterranean sclerophyll shrubs were very similar. This applied even when maximum g reached approx. 500 mmol H2O m±2 s±1 in grapevines, and only 200 mmol H2O m±2 s±1 in sclerophyll shrubs (Medrano et al., 2002). The relationship between different photosynthetic parameters and g was not observed with relative water content or leaf water potential, i.e. decreased photosynthesis caused by drought occurred at different leaf water status in different species, albeit at similar stomatal conductance. Based on these previous ®ndings and using data from the literature, we have analysed at what values of gÐand thus at different severity of droughtÐsome photosynthetic metabolic processes are impaired. MATERIALS AND METHODS In order to see if g, relative water content or water potential provide a clearer basis or reference for the effects of drought on photosynthetic response to drought, we analysed the literature cited in Table 1. ã 2002 Annals of Botany Company 184 Flexas and Medrano Ð Regulation of Photosynthesis of C3 Plants Under Drought TA B L E 1. References used for the analysis of each photosynthetic sub-process and in the construction of Fig. 2) Photosynthetic sub-process References Species RuBP availability Flexas, 2000 GimeÂnez et al., 1992 Gunasekera and Berkowitz, 1993 Santakumary and Berkowitz, 1991 Sharkey and Badger, 1982 Sharkey and Seeman, 1989 Stuhlfaulth et al., 1990 Tezara et al., 1999 Vu et al., 1987 Wingler et al., 1999 Havaux et al., 1987 Lawlor, 1983 Meyer and de Kouchkovsky, 1992 Sharkey and Badger, 1982 Tezara et al., 1999 Younis et al., 1979 BjoÈrkman and Powles, 1984 Brestic et al., 1995 Damesin and Rambal, 1995 Demmig et al., 1988 Faria et al., 1998 Flexas, 2000; Flexas et al., 1998, 1999a, 1999b Lal et al., 1996 Meyer and Genty, 1999 MunneÂ-Bosch and Alegre, 2000 MunneÂ-Bosch et al., 1999 Pankovic et al., 1999 Wingler et al., 1999 AntolõÂn and SaÂnchez-DõÂaz, 1993 Castrillo and Calcagno, 1989 Holaday et al., 1992 Lal et al., 1996 Medrano et al., 1997 Pankovic et al., 1999 Plaut and Federman, 1991 Tezara et al., 1999 Vu and Yelenosky, 1988 Vu et al., 1987 Wingler et al., 1999 Angelopoulos et al., 1996 Brodribb, 1996 Faria et al., 1998 Flexas et al., 1998; Flexas, 2000 MeÂthy et al., 1996 Ramanjulu et al., 1998 Valladares and Pearcy, 1997 Brodribb, 1996 Epron and Dreyer, 1993 Faver et al., 1996 Flexas, 2000 GimeÂnez et al., 1992 Jensen et al., 1996 Johnson et al., 1987 Lal et al., 1996 Luo, 1991 Martin and Ruiz-Torres, 1992 Nicolodi et al., 1988 Ramanjulu et al., 1998 Shangguan et al., 1999 Vitis vinifera Helianthus annuus Nicotiana tabacum Spinacia oleracea Xanthium strumarium Phaseolus vulgaris Digitalis lanata Helianthus annuus Glycine max Hordeum vulgare Nicotiana tabacum Triticum aestivum Lupinus albus Xanthium strumarium Helianthus annuus Spinacia oleracea Nerium oleander Phaseolus vulgaris Quercus pubescens Nerium oleander Quercus ilex, Q. suber, Olea europaea, Eucalyptus globulus Vitis vinifera Vicia faba, Hordeum vulgare Rosa rubiginosa Melissa of®cinalis Lavandula stoechas, Rosmarinus of®cinalis Helianthus annuus Hordeum vulgare Medicago sativa Lycopersicon esculentum Triticum aestivum Vicia faba, Hordeum vulgare Trifolium subterraneum Helianthus annuus Gossypum hirsutum Helianthus annuus Citrus sinensis Glycine max Hordeum vulgare Olea europaea Acacia melanoxylon, Eucalyptus tenuiramis, Podocarpus lawrencii Quercus ilex, Q. suber, Olea europaea, Eucalyptus globulus Vitis vinifera Quercus pubescens Morus alba Heteromeles arbutifolia Acacia melanoxylon, Eucalyptus tenuiramis, Podocarpus lawrencii Quercus robur, Q. petraea Gossypum hirsutum Vitis vinifera Helianthus annuus Brassica napus Triticum ssp. Vicia faba, Hordeum vulgare Abutilon theophrasti Triticum aestivum Medicago sativa Morus alba Triticum aestivum ATP synthesis Photochemistry Rubisco activity Permanent photoinhibition Ci in¯exion point The species analysed in every reference are indicated in the right-hand column. Photosynthetic metabolism was divided into ®ve subprocesses implicated as important sites of inhibition of photosynthetic metabolism under drought. The sub-processes were: (1) ribulose 1,5-bisphosphate (RuBP) regener- ation capacity (GimeÂnez et al., 1992; Gunasekera and Berkowitz, 1993) as indicated by the RuBP content in leaves; (2) ATP synthesis (Younis et al., 1979; Meyer and de Kouchkovsky, 1992; Tezara et al., 1999) as indicated by Flexas and Medrano Ð Regulation of Photosynthesis of C3 Plants Under Drought F I G . 1. Analysis of Rubisco activity under drought. The y-axis shows the percentage (%) of the studies from the literature in which the activity of Rubisco ®rst decreased in relation to intervals of (A) light-saturated stomatal conductance, (B) of relative water content (RWC) and (C) of leaf water potential. the ATP content of leaves or ATP synthase activity (photophosphorylation) or the amount of ATP synthase; (3) leaf photochemistry (Cornic and Massacci, 1996; Flexas et al., 1999a, b) as indicated by chlorophyll a ¯uorescence; (4) ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco) activity (Castrillo and Calcagno, 1989; Medrano et al., 1997; Tezara et al, 1999); and (5) permanent photoinhibition (BjoÈrkman and Powles, 1984; Valladares and Pearcy, 1997). In addition, the change in sub-stomatal CO2 concentration (Ci) with progressive drought was also analysed as an indicator of the predominance of stomatal or non-stomatal limitations to photosynthesis (Ort et al., 1994; Cornic and Massacci, 1996). We related the Ci in¯exion point between decreasing and increasing Ci to the value of g. The data were grouped according to the change in each of these ®ve sub-processes (Table 1), irrespective of the methods used to assess the effects of drought in each experiment (usually gas exchange or photoacoustic meas- 185 urements, coupled with determinations of chlorophyll a ¯uorescence, on leaves, followed by destructive sampling and biochemical analyses). Changes in Rubisco activity and RuBP regeneration derived from CO2-response curves of photosynthesis (A/Ci curves) were not considered, since they assume that regulation under non-stressed conditions is applicable to stressed. In addition, they are dif®cult to compare with biochemical assessments (Medrano et al., 2002). For each study and sub-process, the threshold of g below which the sub-processes was impaired by the drought treatment (i.e. the value of g at which each process started to become inhibited) was estimated. When g was not given, it was derived from the relationship between g and leaf water potential obtained for the same species under similar conditions either by the same or other authors. When there were uncertainties about the values of g, these studies were not included in the analysis. Finally, for simplicity and because only approximate g values were usually available (or impossible, for example, to determine accurately from the ®gures given), the occurrence of inhibition of each sub-process (expressed as a percentage of the total number of cases analysed) was related to four discrete intervals of g. These were: g > 150 mmol H2O m±2 s±1 (i.e. control plants to mild drought); 150 mmol H2O m±2 s±1 > g > 100 mmol H2O m±2 s±1 (i.e. moderate drought); 100 mmol H2O m±2 s±1 > g > 50 mmol H2O m±2 s±1 (i.e. severe drought); g < 50 mmol H2O m±2 s±1 (i.e. very severe drought). When the data were available, results were also related to discrete intervals of relative water content and leaf water potential. This method determines the onset of changes in metabolism with progressive drought, by comparison with unstressed plants (the control). If the changes in a particular process occur with only small increase in stress, they appear in the range of g > 150 mmol H2O m±2 s±1 (i.e. control plants to mild drought). This is because the g values of the control plants are not distinguished from mildly stressed plants. It means that the onset of metabolic changes occurs with very limited drought as g starts to decrease. R E SU L T S A N D D IS C U SS IO N Using different values of stomatal conductance, g, as a reference to analyse the effects of drought on photosynthetic metabolism provides a clearer pattern of the changes in different parts of metabolism in response to drought than using relative water content or leaf water potential. This is illustrated in Fig. 1 for Rubisco activity. When plotted as a function of g intervals, Rubisco activity starts to decrease when g drops below 100 mmol H2O m±2 s±1 (Fig. 1A). However, when plotted as a function of the RWC intervals proposed by Lawlor (1995) to re¯ect different stages of drought effects on photosynthesis, no clear pattern was observed (Fig. 1B). Rubisco activity decreased in 65 % of studies at RWC between 80 and 50 %, but in a substantial proportion (35 %) of cases, Rubisco activity was lost at very high RWC (90±100 %). With leaf water potential as a reference (Fig. 1C), the pattern of response was even less clear, with Rubisco activity inhibited over a range of water 186 Flexas and Medrano Ð Regulation of Photosynthesis of C3 Plants Under Drought F I G . 2. Occurrence of the onset of drought-induced decrease of metabolic processes as a function of the corresponding light saturated stomatal conductance (g), from the literature (Table 1). The y-axis shows the percentage (%) of the studies (the number is shown as n) in which the decrease occurred at different intervals of g. For simplicity, those studies in which no effect of drought on metabolism occurred are not included but are mentioned in the text. The effects on metabolism are represented by: A, RuBP content (RuBP regeneration, n = 10); B, ATP content (ATP synthesis, n = 6); C, Photochemistry (n = 14); D, Rubisco activity (n = 13); E, Permanent photoinhibition (n = 10); F, Appearance of the Ci in¯exion point (n = 17). potentials. Other photosynthetic processes showed similar responses to g, RWC and leaf water potential (not shown). Using RWC or water potential as references, only photochemistry and permanent photoinhibition showed a degree of correspondence similar to that observed when using g. Photochemistry decreased mainly between 80 and 50 % RWC with leaf water potentials below ±1 MPa. However, permanent photoinhibition occurred at RWC between 80 and 50 % as well, but at leaf water potentials only below ±1´5 MPa. Following this primary evaluation, we used g as a reference parameter to analyse the literature. The results of this analysis are given in Fig. 2. Clearly, decreased RuBP (Fig. 2A) and impaired ATP synthesis (Fig. 2B) have been most frequently reported to occur in early phases of drought, when g is still relatively large (>150 mmol H2O m±2 s±1). Important exceptions are the studies of Sharkey and Seeman (1989), in which RuBP content of Phaseolus vulgaris was unaffected at g around 100 mmol H2O m±2 s±1, and of Ortiz-LoÂpez et al. (1991), in which inhibition of ATPase in Helianthus annuus did not occur even at very low g (approx. 50 mmol H2O m±2 s±1). Decreased photochemistry (Fig. 2C) and Rubisco activity (Fig. 2D) are commonly reported to occur at severe stress, and in our analysis this corresponded to g < 100 mmol H2O m±2 s±1. Only in the study of MunneÂ-Bosch et al. (1999) in Rosmarinus of®cinalis, were electron transport rates un- Flexas and Medrano Ð Regulation of Photosynthesis of C3 Plants Under Drought affected even when g dropped to 75 mmol H2O m±2 s±1. We found only two reports, both using Phaseolus vulgaris, showing unaltered Rubisco activity at g < 100 mmol H2O m±2 s±1 (Sharkey and Seeman, 1989; Brestic et al., 1995). Permanent photoinhibition (Fig. 2E) was only occasional. Indeed, in about half the references analysed permanent photoinhibition did not occur; when it did, it was at very low g (<50 mmol H2O m±2 s±1) (see Epron and Dreyer, 1993; Faria et al., 1998; Flexas and Medrano, 1998). As stomata close, the CO2 inside the leaf, Ci, initially declines with increasing stress and then increases as drought becomes more severe (Lawlor, 1995). According to Cornic and Massacci (1996), Ci estimated from ¯uorescence decreases to compensation point under drought and can be estimated accurately. If Ci is high, this re¯ects the inaccuracies in the Ci calculation under drought (i.e. heterogeneous stomatal closure, cuticular conductance, etc.), which tend to overestimate Ci. The decrease in Ci indicates that stomatal limitations dominate, with moderate drought, irrespective of any metabolic impairment. However, at a certain stage of water stress, shown by a threshold value of g, Ci frequently increases, indicating the predominance of non-stomatal limitations to photosynthesis. In most cases the point at which Ci starts to increase, which we call the Ci in¯exion point, occurs at g around 50 mmol H2O m±2 s±1. Only Nicolodi et al. (1988) in Medicago sativa and Luo (1991) in Abutilon theophrasti observed the Ci in¯exion point at higher g. The results of this literature survey analysing the effects of drought on photosynthesis are consistent with a gradual pattern of response of photosynthesis to water stress similar to that proposed by Lawlor (1995). After an early partial closure of stomata, metabolic limitation, caused by either damage (i.e. permanent) or adjustment (i.e. reversible `down-regulation') occurs. The limitation at large g, when drought is mild, is often impaired ATP synthesis and thus ATP-limited regeneration of RuBP. Further reduction of g as drought increases leads to reduced photochemical activity. The analysis shows that, as it is the Rubisco activity, this loss is more progressive with increasing drought than sometimes suggested (Lawlor, 1995; Lawlor and Cornic, 2002). Photoinhibition eventually occurs under conditions of very severe drought and almost complete stomata closure. The Ci in¯exion point is also observed predominantly at low g. This pattern of metabolic changes supports the assertion by Cornic (2000) that stomatal closure is the primary cause of the reduction in photosynthetic rate under mild drought, but shows that metabolic damage or down-regulationÐthis analysis cannot distinguish between themÐis progressive and commences with small changes in g under mild drought. In particular, decreased ATP content, implying impaired synthesis [and thus supporting the observations of Younis et al. (1979) and Tezara et al. (1999) of impaired photophosphorylation and loss of ATP synthase, respectively] is important. To our knowledge, only one reference (Ortiz-LoÂpez et al., 1991) reported no inhibition of ATPase under mild to moderate drought. A major consequence of loss of ATP would be limited RuBP regeneration under mild drought, shown clearly as an early effect of drought by our 187 analysis. Nevertheless, despite the decreased capacity of these metabolic processes, decreased Ci con®rms the predominance of stomatal limitation in restricting photosynthetic rate in the early phase of water loss. However, the metabolic changes are responsible for loss of photosynthetic potential during this phase (Lawlor and Cornic, 2002). Our analysis does not include the effects of drought on nitrate reductase and sucrose phosphate synthase, enzymes shown in a number of studies to be inhibited under water stress. This is because too few analyses with information on g are available. The activities of these enzymes can be restored by placing the water-stressed plant in high CO2 for a number of hours (Sharkey, 1990; Cornic and Massacci, 1996). This strongly suggests that CO2 availability in the chloroplast, mainly regulated by g, may serve as a signal to trigger metabolic adjustments in the leaf in response to water de®cit. This would be consistent with the observed response of the different photosynthetic processes to g. ATP synthesis is probably not restored by elevated CO2 (Tang et al., 2002), suggesting that the enzyme is not impaired, directly or indirectly, by low CO2 concentration. Instead, increased magnesium concentration has been shown to inhibit ATP synthase (Tang et al., 2002). Alternatively, inhibition of ATP synthesis, and not lowered Ci, may be responsible for impairments to metabolism, which cannot be regulated by adjustments in metabolism. One of the major goals for future research on drought effects on photosynthesis should be to con®rm how general are the responses that have been identi®ed (Lawlor, 1995; Lawlor and Cornic, 2002). From an analysis of the literature over the widest range of drought and for a number of species with different responses to drought, we have shown that changes in metabolism occur despite stomatal closure. 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