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Brazilian Journal of Plant Physiology

On-line version ISSN 1677-9452

Braz. J. Plant Physiol. vol.19 no.2 Londrina Apr./June 2007 



Limitations to photosynthesis at different temperatures in the leaves of Citrus limon


Análise in vivo das limitações da fotossíntese, sob diferentes temperaturas, em folhas de Citrus limon



Carlos PimentelI, *; Carl BernacchiII, III; Steve LongIII

IDepartamento de Fitotecnia, Universidade Federal Rural do Rio de Janeiro, Seropédica, 23851-970 Brazil
IICenter for Atmospheric Sciences, Illinois State Water Survey, Champaign, IL, USA
IIIDepartment of Plant Biology, University of Illinois, Urbana, IL 61801, USA




The response of CO2 assimilation rate (A) to the intercellular partial pressure of CO2 (Ci) was measured on intact lemon leaves over a range of temperatures (10 to 40ºC). The A/Ci response shows how change in the leaf temperature alters the activity of ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) and RuBP regeneration via electron transport. The rate of A reached a maximum of 7.9 to 8.9 µmol m-2 s-1 between 25 and 30ºC, while dark respiration (Rd) increased with temperature from 0.4 µmol m-2 s-1 at 10ºC to 1.4 µmol m-2 s-1 at 40ºC. The maximum rates of carboxylation (Vc,max) and the maximum rates of electron transport (Jmax) both increased over this temperature range from 7.5 to 142 µmol m-2 s-1 and from 23.5 to 152 µmol m-2 s-1, respectively. These temperature responses showed that A can be limited by either process depending on the leaf temperature, when Ci or stomatal conductance are not limiting. The decrease in A associated with higher temperatures is in part a response to the greater increase in the rate of oxygenation of RuBP compared with carboxylation and Rd at higher temperatures. Although A can in theory be limited at higher Ci by the rate of triose-phosphate utilization, this limitation was not evident in lemon leaves.

Key words: A/Ci curves, gas exchange, lemon.


A resposta da taxa de assimilação de CO2 (A) à pressão parcial de CO2 (Ci) foi medida em folhas intactas de limão cravo, numa ampla faixa de temperaturas (10 to 40ºC). A variação na curva A/Ci mostrou como as mudanças na temperatura foliar alteram a atividade da ribulose-1,5-bisfosfato (RuBP) carboxilase-oxigenase (Rubisco) e a regeneração da RuBP, via transporte de elétrons. O valor máximo de A obtido foi de 7.9 a 8.9 µmol m-2 s-1, entre 25 e 30ºC, enquanto a respiração mitocondrial (Rd) aumentou com a temperatura, de 0.4 µmol m-2 s-1 a 10ºC até 1.4 µmol m-2 s-1, a 40ºC. A taxa máxima de carboxilação (Vc,max) e a taxa máxima de transporte de elétrons (Jmax) aumentaram naquela faixa de temperatura, de 7.5 a 142 µmol m-2 s-1, e de 23.5 a 152 µmol m-2 s-1, respectivamente. A redução em A associada às altas temperaturas é, em parte, uma resposta ao maior aumento na taxa de oxigenação da RuBP, comparada à taxa de carboxilação, e de Rd sob altas temperaturas. Apesar de A poder ser, em teoria, limitada sob elevada Ci pela taxa de utilização de triose-fosfato, essa limitação não foi evidente nas folhas analisadas.

Palavras-chave: curvas A/Ci, limão cravo, trocas gasosas.




Temperature is one of the most variable environmental factors, which can suppress photosynthesis both at high and low values. Under global warming scenarios, the study of temperature effects on photosynthesis is essential to predict crop production in the future (Long, 1991). To examine the biochemistry of photosynthesis in leaves, measurement of CO2 assimilation rate (A) in relation to chloroplast CO2 partial pressures (Cc) would be ideal as this is the CO2 pressure determining the Rubisco carboxylation. However, measuring Cc is difficult. Therefore, it has become a common practice to calculate the CO2 partial pressure in substomatal cavities (intercellular CO2 partial pressure, Ci), based on measurements of gas exchange under different ambient CO2 partial pressures (von Caemmerer, 2000; Long and Bernacchi, 2003). The response of A to Ci under different temperatures can be interpreted in terms of the biochemical processes controlling the response of A (Sage, 1994).

The group of evergreen fruit trees includes numerous horticulturally and economically important crops, as Citrus spp., which are cultivated throughout most tropical and subtropical areas of the world. Although citrus tree thrive in hot, dry environments, leaf photosynthesis has a relatively low temperature optimum of 25ºC to 30ºC (Goldschimidt and Koch, 1996). The term evergreen relates to the nondeciduous nature of leaves and, as such, has immediate consequences for leaf longevity and photosynthesis. Broadleaf evergreen citrus leaves are relatively thick with a small proportion of leaf volume occupied by intercellular air space. They have a shiny waxy cuticle particularly on the adaxial surface and stomata are located almost exclusively on the abaxial surface (Goldschimidt and Koch, 1996). Therefore, citrus leaves have low rates of A (4 to 8 µmol CO2 m-2 s-1 seem realistic under optimal conditions), and low stomatal and mesophyll conductances (Lloyd et al., 1992). In addition, its leaves act as a carbohydrate storage organ with slow rates of assimilates export, which in turn can feedback to reduce A (Syvertsen and Lloyd, 1994). Wullschleger (1993) made a retrospective analysis of the A/Ci curves of 109 C3 species and concluded that the maximum rate of carboxylation (Vc,max) and the light-saturated rate of electron transport (Jmax) were in general higher for herbaceous annuals plants than for woody perennials. Therefore, the aim of this study was to characterize the A/Ci response and the in vivo calculated photosynthetic parameters in Citrus limon, an evergreen plant with low A values even for a C3 plant, estimated from the A/Ci response curves over a range of temperature.



Three individuals of Citrus limon L. were germinated and grown in environmentally-controlled greenhouses located at the University of Illinois, Urbana, USA. Plants were grown in a soil-less growth medium (Sunshine Mix #1, SunGro Horticulture, Bellevue, USA) and were watered regularly. Nutrient additions were given weekly in the form of 300 µL L-1 of NPK 15:5:15 (Peters Excel, The Scotts Co., Marysville, USA) to pot saturation. Greenhouse temperature levels were set at 25ºC for the 16-h photoperiod and 18ºC for night.

Leaf gas exchange rates were measured using an open gas exchange system with independent [CO2] control using a 6 cm2 clamp-on leaf cuvette (LI 6400, LI-COR, Lincoln, USA). The gas-exchange system was zeroed daily using CO2-free air, and leakage of CO2 into and out of the chamber, with a Citrus leaf inside, was determined for the range of CO2 concentrations used in this study and used to correct measured leaf fluxes. The chamber was modified by replacing the peltier external heat sink with a metal block containing water channels, which in turn were connected to a heating/cooling circulating water bath (Endocal RTE-100, Neslab Instruments, Newington, USA). The modified heating/cooling blocks, used in conjunction with the peltier temperature controls, provided leaf temperature control at any preset value between 10-40ºC. Leaf temperature was measured using a chromal-constantin thermocouple pressed to the lower leaf surface. The temperatures reported by this particular thermocouple were cross-checked against standard mercury-in-glass thermometers in a controlled temperature chamber and found to be within ±0.4ºC (Bernacchi et al., 2003).

Photosynthesis was measured after acclimation of the leaf to temperature (until A was steady-stated and total CV was lower than 0.3, at least after 2 h at each new temperature) at photosynthetic photon flux density (PPFD) between 600 and 800 µmol m-2 s-1, which was light-saturating for this species. Photosynthetic photon flux density was controlled using an artificial quartz halide light source controlled with a quantum sensor located inside the leaf cuvette. The vapor pressure deficit in the cuvette was maintained between 0.5 and 2.0 kPa to prevent stomatal closure by passing the air entering the gas-exchange system through either anhydrous calcium carbonate (Drierite, W.A. Hammond Drierite Company, Xenia, USA) at lower temperatures when humidity was high or by bubbling the air through water for the higher temperatures. Values for A and Ci were calculated using the equations of von Caemmerer and Farquhar (1981). A protocol commonly used in determining this A versus Ci response is: firstly, induce photosynthesis at the growth CO2 concentration (36 to 380 µmol mol-1) and saturating PPFD (between 600 and 800 µmol m-2 s-1) until A is steady-stated (over a 5-min period). Values of A and Ci are recorded and then ambient CO2 partial pressure (Ca) is decreased to 300, 250, 200, 150, 100 and 50 µmol mol-1. Upon completion of this sequence, Ca is returned to growth CO2 concentration to check that the original A can be restored and then is increased stepwise to 450, 550, 650, 800, 1000 µmol mol-1. Steady-state photosynthesis needs to be obtained at each step (with a total CV lower than 0.3, at least after 5 min for each step).

Three replicate measurements of A/Ci curves (Figure 1: A/Ci curve at 25ºC) were measured on different plants, at 5ºC intervals between 10 and 40ºC. The parameters Vc,max, Jmax and Rd were estimated using regression analysis of the curves (Figure 2) based on the equations presented in the appendix (Long and Bernacchi, 2003). The temperature responses of Vc,max, Jmax, and Rd were plotted from the results of the regression analysis at each measurement temperature from 10 to 40ºC (e.g., Bernacchi et al., 2001). Data for photosynthesis measured at a CO2 concentration of 370 µmol mol-1 was extracted from the curves and plotted as a function of temperature. Using the equations presented by Farquhar et al. (1980), based on A/Ci measurements (Figure 1) and thus the calculated response of Vc,max (Figure 2) at the complete range of temperatures, estimations of the temperature response of photosynthesis under non-RuBP limiting conditions was also determined.





Data were subjected to analysis of variance (ANOVA) for temperature effects and means were compared by Student-Newman-Keuls test at 0.05 of probability, when significance was detected.



Under light saturating conditions photosynthesis for C3 plants is limited by Rubisco capacity, the Rubisco limited phase, as shown in Figure 1. As Ci increases above typical levels for this specie, photosynthesis will typically become limited by RuBP regeneration via electron transport, the RuBP limited phase, and by triose-phosphate utilization (TPU) at substantially higher Ci, the TPU limited phase (Sage, 1994; von Caemmerer, 2000).

This last limitation, however, was not observed in this experiment with lemon (Figure 1), as is frequent in field-based measurements (Adams et al., 2000). During the electron transport limitation, the RuBP limited phase, CO2 uptake still increases because CO2 out-competes O2 for the available RuBP, but during the triose limitation photosynthesis is no more CO2 dependent (von Caemmerer, 2000).

The results obtained with lemon under temperatures from 10ºC to 40ºC showed a maximum values of A between 25 and 30ºC, with 8.9 and 7.9 mmol m-2 s-1, respectively, while Rd increased significantly with temperature from 0.4 to 1.5-1.4 µmol m-2 s-1 (obtained as in Figure 2). The range of temperature for maximal A measured (Figure 3A) is in agreement with Golschmidt and Koch (1996), who stated that the genus Citrus, which originated in tropical and semitropical regions, have an optimal temperature between 25ºC to 30ºC. The highest value of A (8.9 mmol m-2 s-1; Figure 3A) was obtained at 25ºC and A decreased both above and below this temperature. This is in contrast to Rd, which rose with temperature (Figure 3B). The low value of A for the perennial woody Citrus spp. when compared to annual herbaceous plants (Wullschleger, 1993), but also to other perennials fruit crops as Prunus persica, might be attributed to a lower mesophyll conductance (gm) or/and low leaf nitrogen present as Rubisco (Lloyd et al., 1992), which can in turn reduce Vc,max (Long, 1991).



Potentially 50 to 70% of carbon assimilated in plant biomass is released back to the atmosphere as CO2 during subsequent plant respiration (Baldocchi and Amthor, 2001). The response of A to temperature is parabolic and its decrease at high temperature occurs through numerous potential processes, including increases in Rd, decrease in membrane stability, decrease in the specificity factor of Rubisco and an accumulation of carbohydrates (Baldocchi and Amthor, 2001). In addition, the limitation of A imposed by an increase in gm with temperature suggests that the dominant process(es) determining gm is not physical, but probably protein-mediated, possibly involving a carbonic anhydrase or aquaporins (Bernacchi et al., 2002; Long and Bernacchi, 2003).

Under non-limiting environmental conditions, in vitro Rubisco activity (Vc,max) for the activated enzyme extracted from citrus leaves is generally in the range of 300 to 400 µmol CO2 mg chlorophyll-1 h-1 (Vu and Yelenovsky, 1988). These authors equate a Vc,max value of about 75 µmol CO2 mg chlorophyll -1 h-1 for leaves of "Valencia" orange (Citrus sinensis [L.] Osbeck) having a maximum A of 8 mmol CO2 m-2 s-1. On the other hand, from an A/Ci plot, Syvertsen and Lloyd (1994) obtained a value for Vc,max varying from 75 to 106 µmol m-2 s-1, and a value for Jmax varying from 130 to 140 µmol m-2 for "Marsh" and "Ruby Red" grapefruit at 25ºC, respectively. The in vivo values of Vc,max and Jmax for C. limon at 25ºC were 55 µmol m-2 s-1 and 87 µmol m-2 s-1 (Figure 4A,B), respectively. These values of Vc,max and Jmax at 25ºC are close to the mean values obtained by Wullschleger (1993) for perennials species, i.e. 44 and 97 µmol CO2 m-2 s-1, respectively, whereas the mean values of these parameters for herbaceous annuals plants are 75 and 154 mmol CO2 m-2 s-1, respectively.



In this study with lemon, the in vivo values of Vc,max and Jmax increased significantly with temperature from 7.5 to 142 µmol m-2 s-1 (Figure 4A) and from 23.5 to 152 µmol m-2 s-1 (Figure 4B), respectively. In addition, the increase in Vc,max with temperature is greater than Vo,max (Figure 4A,C), and Vo,max values varied from 2.4 to 35.7 µmol m-2 s-1 (Figure 4C). From our measurements, we found that Vo,max /Vc,max declines with temperature. However, due to the differential effect of temperature on the velocity of carboxylation relative to oxygenation (Vc /Vo), observed A values will actually decline with increasing temperatures due to increased photorespiration (Long et al., 2004). Therefore, the ratio of Vo,max/Vc,max is reduced (Figure 4D) at high temperature and there is a greater increases in Vc,max compared to Vo,max, as stated by Bernacchi et al. (2001) and, thus, the proportion of potential carbon uptake lost to photorespiration increases (Long, 1991).

Therefore, depending on temperature A casn be limited by very different processes. The amount and activation state of the photosynthetic enzymes, each representing a different limiting process to overall CO2 assimilation, are integral for determining the temperature optimum of photosynthesis.



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Received: 14 May 2007; Returned for revision: 02 July 2007; Accepted: 06 August 2007.



* Corresponding author:




Model theory

Farquhar et al. (1980) presented a model of leaf level photosynthesis with two rate limiting steps with a third added by Harley and Sharkey (1991). This model states that at any given internal concentration of CO2, photosynthesis is limited by the slower of three processes: 1) the maximum rate of Rubisco-catalyzed carboxylation (Rubisco-limited A); 2) the regeneration of RuBP controlled by electron transport rate (electron transport-limited A); or 3) the regeneration of RuBPcontrolled by the rate of triose-phosphate utilization (TPU-limited A). Both CO2 and O2 compete for the Rubisco binding site in the processes known as carboxylation and oxygenation, respectively (Farquhar et al., 1980). To account for the competitive inhibition between CO2 and O2, A is mathematically expressed as:

where vc and vo are the rates of carboxylation and oxygenation, respectively, and Rd is the mitochondrial respiration (Farquhar et al., 1980).

When A is Rubisco-limited (Wc) the velocity of carboxylation can be expressed as:

where Vc,max is the maximum rate of carboxylation, O is the oxygen concentration, and Kc and Ko are the Michaelis-Menten constants for CO2 and O2, respectively (Farquhar et al., 1980).

The velocity of carboxylation when limited by the rate of electron transport (Wj) is expressed as stated by von Caemmerer (2000):

where J is the potential rate of electron transport and can be expressed as a function of light saturated rate of electron transport (Jmax), as stated by von Caemmerer (2000):

where f is the fraction of light not absorbed by functional photosynthetic pigments and I is the photon flux hitting the leaf.

Triose phosphate utilization limited photosynthesis (Wp) was not a limitation in this study but can occur at low temperatures or high levels of CO2 for others species and is expressed as:

where VTPU is the velocity of triose phosphate utilization, which is multiplied by three to represent three mol CO2 that can be fixed for every mol of triose-phosphate made available (Harley and Sharkey, 1991).

Incorporating the three rate limiting steps into equation 1 yields:

where the term G* is the CO2 compensation point in the absence of Rd. The term [1- G*/Ci] represents photorespiration and is derived from the equation:

where Vo,max is the maximum rate of oxygenation (Farquhar et al., 1980; von Caemmerer 2000). Photosynthesis limited by Wp is insensitive to changes in CO2 or O2 and thus the term representing photoinhibition is removed (von Caemmerer, 2000).

From the Rubisco limited portion of the A/Ci curve (integrating equation 2 into equation 1), below the inflection point of the curve (obtained by the interception of the adjusted curves for Rubisco limited and RuBP limited phases), the values of Vc,max and Rd can be calculated from the equation:

where G*= 42.05 µbar; O= 20.9 µbar; Kc= 404.9 µbar; and Ko= 278.4 mbar at 25ºC (Long and Bernacchi 2003). For other temperatures, G*, Kc and Ko are adjusted by the equation parameter = exp(c- DHa/RTk), where c and DHa values for each parameter are presented in Bernacchi et al. (2001). The two unknowns Vc,max and Rd can be solved, as shown by Long and Bernacchi (2003), by plotting A (below the inflection point: Rubisco limited) as a linear function of f' (Figure 2):


In this linear function, Vc,max is the slope and Rd the intercept (Figure 2).

On the other hand, from the RuBP limited portion of the A/Ci curve (using equation 3 into equation 1) above the inflection point of the curve (obtained by the interception of the adjusted curves for Rubisco limited and RuBP limited phases), Jmax can be calculated from the equation stated by Long and Bernacchi (2003):

Similarly for Vc,max, Jmax can be obtained by plotting A (above the inflection point where it is RuBP limited) as a linear function of Ci (i.e. g'), but fixing the Rd value obtained from Vc,max calculations (equation [9]) to avoid large errors in estimated Rd from a linear regression (A x Ci, i.e. g'), due to small errors in the higher rates of A (RuBP limited). Thus, after solving for Rd from the Rubisco limited portion (together with Vc,max), the value for Rd can be used in a linear regression (Long and Bernacchi, 2003), together with the high values of A (from RuBP limited portion) in a linear function of g' (Figure 2):


where Jmax is the slope in this linear function (Figure 2).

Finally, Vo,max can be solved by the equation:

where Vc,max , Ko , G* , Kc and O are either known or solved using previous equations.

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