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Brazilian Journal of Plant Physiology

versão On-line ISSN 1677-9452

Braz. J. Plant Physiol. v.16 n.2 Londrina maio/ago. 2004 



Effects of changes in the photosynthetic photon flux density on net gas exchange of Citrus limon and Nicotiana tabacum


Oscilação cíclica da condutância estomática em Citrus limon e Nicotiana tabacum



Carlos PimentelI, *; Rafael Vasconcelos RibeiroII; Mauro Guida dos SantosII; Ricardo Ferraz de OliveiraII; Eduardo Caruso MachadoIII

IDepartamento de Fitotecnia, Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, CEP 23851-970, Itaguaí, RJ, Brasil
IIDepartamento de Ciências Biológicas, CP 09, Escola Superior de Agricultura "Luiz de Queiroz"-USP, CEP 13419-110, Piracicaba, SP, Brasil
IIICentro de Pesquisa e Desenvolvimento em Ecofisiologia e Biofísica, CP 28, Instituto Agronômico de Campinas, CEP 13001-970, Campinas, SP, Brasil




The objective of this study was to evaluate the effects of changes in the photosynthetic photon flux density (PPFD) on net gas exchange of Citrus limon, a woody species, and Nicotiana tabacum, an herbaceous species. When PPFD was increased from 50 to 350 mmol.m-2.s-1 and returned to 50 mmol.m-2.s-1 after 60 min, the CO2 assimilation rate (A) increased and stabilized after 15 min in both species. Stomatal conductance (gs), however, continued to increase. After returning to low PPFD, A immediately diminished to a low value for both lemon and tobacco. Stomatal conductance of lemon diminished slowly over 60 min, whereas gs for tobacco took only 15 min to decrease. This difference in behavior is probably due to over-sensitivity of stomata of woody species, such as Citrus, when exposed to high light, retarding stomatal closure on return to low PPFD. Furthermore, when lemon, growing at a PPFD of 300 mmol.m-2.s-1, was submitted to a step increase of 600 mmol.m-2.s-1, there was an oscillatory behavior of A and gs requiring 150 min to stabilize. The causes of this behavior are discussed with respect to improved intrinsic water use efficiency by stomatal closure.

Key words: CO2 assimilation rate, gas exchange, stomata, intrinsic water use efficiency.


O objetivo desse estudo foi avaliar o efeito da variação da densidade de fluxo de fótons fotossintéticos (DFFF) nas trocas gasosas de Citrus limon, uma espécie lenhosa, e Nicotiana tabacum, uma espécie herbácea. Quando a DFFF foi aumentada de 50 para 350 mmol.m-2.s-1 durante 60 min, retornando em seguida a 50 mmol.m-2.s-1, a taxa de assimilação de CO2 (A) aumentou e estabilizou após 15 min, mas a condutância estomática (gs) continuou crescendo, em ambas as espécies. Quando se retornou à baixa DFFF, A diminuiu instantaneamente nas duas espécies, mas a gs do limoeiro diminuiu lentamente durante 60 min, enquanto em tabaco, a gs levou apenas 15 min para decrescer. Essa diferença nas respostas é provavelmente devida à hipersensibilidade dos estômatos de espécies lenhosas, como Citrus, quando submetidas à alta DFFF, retardando o seu fechamento, quando de volta à baixa DFFF. Além disso, quando o limoeiro, crescendo sob DFFF de 300 mmol.m-2.s-1, foi submetido subitamente a 600 mmol.m-2.s-1, iniciou-se uma flutuação de A e gs, levando 150 min para se estabilizar. As causas dessa oscilação são discutidas em função do ajuste da eficiência intrínseca no uso de água, devido ao controle estomático.

Palavras-chave: eficiência intrínseca no uso de água, estômatos, taxa de assimilação de CO2, trocas gasosas.




Oscillatory transpiration is a rhythmic fluctuation that has been described in several species (Barrs, 1971), with an average cycle periodicity of 15 to 120 min (Kramer and Boyer, 1995). Stomatal cycling can be triggered by environmental signals such as changes in photosynthetic photon flux density (PPFD) (Zipperlen and Press, 1997) or changes in xylem water potential and hydraulic conductivity (Lp) (Rose and Rose, 1994). Stomatal cycling is common in Citrus under field conditions, when the PPFD, vapor pressure deficit (VPD) of air or soil water deficit increase. This cycle has a period of about one hour, and increases in amplitude and decreases in duration as the daytime progresses (Levy and Kaufmann, 1976). Stomatal closure can also be induced by a non-hydraulic root signal from roots, usually ABA (Kramer and Boyer, 1995), or changes in Lp, a hydraulic signal, especially under high transpiration rates (Steudle, 2001). This change in Lp is caused, among others factors, by an apoplastic water movement through the endodermis cell wall in addition to that passing along the cellular pathway under high transpiration rates (Hartung et al., 2002). In maize under high transpiration rates, despite the increase in radial water flow, the ABA concentration in the xylem remained constant or even increased (Hose et al., 2001). This is due to transport of the root apoplastic and symplastic ABA by solvent drag (Hartung et al., 2002).

Low CO2 assimilation rate (A) and leaf diffusive conductance (gs) often characterize leaves of perennial species, as in Citrus. Although most agricultural and herbaceous C3 plants have A in the range 20-40 mmol CO2.m-2.s-1, in mature leaves of Citrus, under near optimal environmental conditions, A greater than 12 mmol CO2.m-2.s-1 is rarely observed (Lloyd et al., 1992). Although gs of Citrus are also relatively low (generally less than 0.2 mol.m-2.s-1), under optimal or near optimal conditions intercellular CO2 partial pressure (Ci) is typically in the range of 200-250 mmol.mol-1 (Lloyd et al., 1992), while for herbaceous plants Ci can vary from 250 to 350 mmol.mol-1 (Long and Hällgren, 1993). Thus, the stomatal limitation of A in woody species can be higher than 20%, as reported by Jones (1998) for herbaceous plants.

Citrus and others woody species have a low maximum rate of carboxylation (Vcmax) because of the low chloroplastic CO2 partial pressure (Lloyd et al., 1992), resulting from a low mesophyll conductance to CO2 diffusion (Syvertsen and Lloyd, 1994). In Citrus, Rubisco accounts for only 10% of the nitrogen in the leaf (Lloyd et al., 1987). Therefore, for long-lived leaves of woody shade-tolerant rain forest species exposed to light, the mean time to attain 90% full photosynthetic induction fell in the range of 11-36 min, as opposed to 1-3 min for short-lived species (Zipperlen and Press, 1997).

Presently in Brazil there is an increased interest in gas exchange studies with Citrus, especially after the incidence of citrus variegated chlorosis (CVC), a vascular disease caused by the bacterium Xylella fastidiosa, which causes reduction in photosynthesis and productivity of Citrus (Ribeiro et al., 2003). However, gas exchange measurements need to be carefully done to avoid environmental changes during measurements, which induce stomatal oscillations.

Thus, the aim of this study was to evaluate the different effects of changing PPFD on A and gs behavior of lemon, an evergreen woody plant with low mesophyll conductance to CO2 diffusion, compared to an annual herbaceous plant, specifically tobacco, with its higher mesophyll conductance to CO2 diffusion.



The plants of Citrus limon Burm. f. (two years old) and of Nicotiana tabacum cv. W38 (45 days old) germinated and grown in a greenhouse, in 1.5 L and 5 L pots, for tobacco and lemon, respectively, with a soil-less growth medium (Sunshine Mix #1, SunGro Horticulture, Inc., Bellevue, WA USA), at the University of Illinois, IL, USA (40º01"N, 88º16"W). The plants were watered regularly and were fertilized weekly with approximately 300 mL.L-1 of NPK 15:5:15 (Peters Excel, The Scotts Co., Marysville, OH USA) to pot saturation. Greenhouse air temperature was set at 25ºC for the 16 h complemented photoperiod and 18ºC for night. One week before measurements three plants were transferred to a cabinet (E15, Conviron, Winnipeg, Manitoba, Canada), with a PPFD of 300 mmol.m-2.s-1 at canopy height, a photoperiod of 14 h, a day/night air temperature of 25/20ºC, and a VPD of 0.7 kPa.

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, NE USA). The gas-exchange system was calibrated for CO2 with a source of air containing a precisely known concentration of CO2 in the range to be analyzed, and for H2O vapor with a dew point generator (LI-610, LI-COR, Lincoln, NE USA), as proposed by Long and Hällgren (1993). It was zeroed daily using CO2-free air, and the VPD in the cuvette was maintained between 0.5 and 1.0 kPa to prevent stomatal closure. All the measurements were carried out at 25ºC and 360 mL.L-1 of CO2 on the youngest fully expanded leaf prior to stem elongation.

The lemon response of CO2 assimilation rate (A) to PPFD (the A/Q curve, as stated by Long and Hällgren, 1993) was conducted on plants from the cabinet, using an artificial quartz halide light source (LI-6400-02 LED light source, LI-COR, Lincoln, NE USA) controlled with a quantum sensor located inside the leaf cuvette. The A/Q curve was obtained after one hour at the higher PPFD used. It was done diminishing the PPFD by 100 mmol.m-2.s-1 after 5 min at each value of PPFD, from 1,500 to 500 mmol.m-2.s-1, so as to avoid photoinhibition, and by 50 mmol.m-2.s-1 from 500 to zero mmol.m-2.s-1, to obtain the curve inflexion. On the other hand, lemon and tobacco responses of A, gs, Ci and Intrinsic Water Use Efficiency (IWUE = were evaluated under a change of PPFD from 50 to 350 mmol.m-2.s-1, after stabilizing under 50 mmol.m-2.s-1 (data shown for the last 10 min). The PPFD was lowered again to 50 mmol.m-2.s-1 after 60 min under 350 mmol.m-2.s-1, as used by Barradas and Jones (1996) on Phaseolus vulgaris, an annual herbaceous plant. Finally, the cycling of A and gs measurements in lemon were obtained after gas exchange parameters were stabilized (after at least 2 h) at 300 mmol.m-2.s-1 (data showed for the last 20 min), and then increasing the PPFD to 600 mmol.m-2.s-1 during 130 min.

The measurements were all carried out with one leaf using three replicate plants in a completely randomized statistical design. Data were subjected to analysis of variance.



Typical of a C3 plant, the photosynthetic light response curve (A/Q curve) of C. limon (figure 1) produced a saturation of A at 600 to 800 mmol.m-2.s-1, which represents 25-30% of full sunlight (Long and Hällgren, 1993). Consequently, high light intensities over 600 to 800 mmol.m-2.s-1 may cause photoinhibition reducing A (Bolhàr-Nordenkampf and Öquist, 1993), and therefore, for environmental-controlled assays with Citrus, the ideal light intensities for maximal A are in this range (Bernacchi et al., 2001). An increase of 20% of the integrated daily A was obtained with nursery trees of Citrus sinensis, by using a reflective net over the greenhouse during midday to avoid photoinhibition by high PPFD (Medina et al., 2002).



The response of A, gs and Ci of lemon to changing PPFD from 50 to 350 and returning to 50 mmol.m-2.s-1 (figure 2), produced values in the same range as those for bean (Barradas and Jones, 1996). After 15 min of changing PPFD from 50 to 350 mmol.m-2.s-1, A stabilized around 10.0 mmol CO2.m-2.s-1 (figure 2A). On the other hand, the Citrus gs values increased constantly when changing the PPFD to 350 mmol.m-2.s-1 reaching a value of 0.17 mol.m-2.s-1 at 60 min (figure 2B). However, when returning the PPFD from 350 to 50 mmol.m-2.s-1, A decreased instantly to 2.5 mmol CO2.m-2.s-1 and stabilized at this value. However, gs diminished slowly, from 0.17 to 0.054 mol.m-2.s-1, tending to stabilize only after 60 min. These results are in agreement with those obtained with long-lived leaves (Zipperlen and Press, 1997), but contrast data for bean, an herbaceous plant, where a more rapid decrease of gs was recorded when returning to 50 mmol.m-2.s-1 (Barradas and Jones, 1996). The Ci values for lemon increased as for A when changing to a higher PPFD (figure 2C), but when returning to a low PPFD, the Ci values diminished more slowly than A. In addition, the lemon Ci values, after stabilization at low or higher PPFD, were low, around 250 mmol.mol-1, in agreement with Lloyd et al. (1992). On the other hand, the IWUE shows a step increase when changing to a higher PPFD (figure 2D) because the increase of A is greater than that of gs. After 60 min under high light, it diminished to values close to those under low light due to the slower increase of gs compared to A. When returning to low light, the IWUE diminished rapidly but returned to initial values after the slow decrease of gs.



Tobacco, with higher values of A, gs and Ci than lemon, produced the same response patterns to PPFD as shown for bean (Barradas and Jones, 1996). After 15-20 min under 350 mmol.m-2.s-1, A was stabilized around 15 mmol CO2.m-2.s-1 (figure 3A), but gs continued to increase reaching a value of 0.576 mol.m-2.s-1 at 60 min (figure 3B). When returning to 50 mmol.m-2.s-1, A diminished to 2.8 mmol CO2.m-2.s-1 immediately and gs took only 15 min to decrease to 0.307 mol.m-2.s-1, thereafter decreasing slowly to 0.255 mol.m-2.s-1 over 70 min at 50 mmol.m-2.s-1. On the other hand, the Ci values for tobacco (figure 3C) were between 250 to 350 mmol.mol-1 after stabilizing at low or high PPFD, and on return to low PPFD, stabilized as for gs. As seen for lemon, the IWUE produced a step increase under high light (figure 3D), and then diminished gradually during the 60 min under this condition, but it stabilized at a value greater than those under low light. On return to low light, it decreased suddenly but after a while, it returned to the same initial values. On the other hand, values of A, gs and Ci for tobacco were much higher than for lemon, whereas IWUE was much lower than for lemon.



Thus, for tobacco, as for bean (Barradas and Jones, 1996), the changes of A in response to either increasing or decreasing PPFD were significantly faster than changes in gs. However, this is not the case for a woody plant such as lemon, at least when changing the PPFD from a high to a low value showing that the changes in gs of lemon are slower and the transpiration rates higher than for herbaceous plants. The slower reduction of gs in Citrus, compared to tobacco, when reducing the PPFD is probably due to over sensitivity of the stomatal response to light for woody species (Zipperlen and Press, 1997), which did not occur in tobacco or bean, both herbaceous plants. In Citrus, as for other woody species, the CO2 mesophyll conductance is considerably lower (1.1 to 2.2 mmol CO2.m-2.s-1.Pa -1) than those reported for unstressed herbaceous plants (Lloyd et al., 1992). However, the low mesophyll conductance of Citrus can be due to the lower Ci values, which is due to the lower gs values of lemon, because the Ci values are proportional to gs values (Long and Hällgren, 1993). Thus, for C. limon gs increases can affect A probably because of the low mesophillic conductance and the need of very high PCO2 for saturation of A (Lloyd et al., 1992). As a result, the response of A and gs, and consequently of IWUE, to changing PPFD from 50 to 350 and back to 50 mmol.m-2.s-1 in Citrus (figure 2), a perennial woody species, was quite different from tobacco or bean (Barradas and Jones, 1996). The A and gs values for lemon varied from 3 to 10 mmol CO2.m-2.s-1 and from 0.04 to 0.17 mol.m-2.s-1, respectively (figures 2A and 2B), whereas for tobacco, these values varied from 2.8 to 15 mmol CO2 m-2.s-1 and from 0.19 to 0.61 mol.m-2.s-1 respectively (figures 3A and 3B). By contrast, the IWUE values for lemon varied from 15 to 170 mmol CO2.mol-1H2O (figure 2D), while for tobacco, these values varied from 5 to 55 mmol CO2.mol-1H2O (figure 3D). Therefore, in woody perennial species such as Citrus, with much lower gs values than annual herbaceous species such as tobacco, the gs control of A will be more important (Lloyd et al.,1992), especially when reducing the PPFD, improving their IWUE. Also, the IWUE of lemon was always higher than for tobacco, and under high light it stabilizes at values close to those under low light, but for tobacco, the IWUE values under high light are greater than that under low light.

Increasing the light intensity to 600 mmol.m-2.s-1, for plants growing at 300 mmol.m-2.s-1, induce an oscillatory stomatal behavior in lemon (figure 4). The gs values increased from 0.07 to a maximal gs value of 0.1 mol.m-2.s-1 in 10 min at 600 mmol.m-2.s-1, while A did not change and was maintained around 7.5 mmol CO2.m-2.s-1. This response is in agreement with data obtained for long-lived leaves, where a faster increase for gs than for A was found, which takes 11-36 min to reach 90% of full photosynthetic induction with increasing light intensity. This contrasts the 1-3 min for the species with short-lived leaves. In the former, gs increased at a much greater rate than photosynthesis due to a low mesophyll conductance and thus a slower full photosynthetic induction (Zipperlen and Press, 1997). However, a high gs value means a high transpiration rate, and if there is no parallel increase in A (figure 4A), it tends to diminish the IWUE (figure 4D). Afterwards, gs decreased (figure 4B), due to excessive transpiration, and consequently A will also decrease, and the minimal A and gs values were 3.0 mmol CO2.m-2.s-1 and 0.04 mol.m-2.s-1, respectively, after 60 min of saturating light intensity. The rapid initial increases in gs at 600 mmol CO2.m-2.s-1, without changes in A, lowers the IWUE because gs increased (figure 4D). Then, there is an induction of stomatal closure and increase in IWUE, probably due to the increased amount of ABA carried by the increased transpiration stream (Hartung et al., 2002). Thus, the reduction in gs, and consequently in transpiration rate, will improve the IWUE and prevent wilting. However, A became too low and gs began to increase again because stomatal movement favours maximum possible A values with lower transpiration rates (Kramer and Boyer, 1995). Then, A and gs stabilized at values of 7.1 mmol CO2.m-2.s-1 and 0.07 mol.m-2.s-1, respectively, and the total oscillatory cycle in C. limon took two and half hours.



The temporal dynamics of the circadian control of stomatal movements indicate that it may benefit the plant by increasing IWUE (Kramer and Boyer, 1995). The extra carbon obtained during peaks can outweigh the reduced carbon gain during troughs, resulting in greater net carbon assimilation with a lower loss of water (Webb, 1998) than would be obtained during a steady rise to a maximum, where transpiration would increase without an increase in A (Zipperlen and Press, 1997). The patterns of stomatal oscillations, obtained with potted Citrus trees in these assays, are in agreement with the observations in field-grown trees of C. sinensis (Levy and Kaufmann, 1976). This oscillation can occur in water-stressed plants and is initiated by a sudden shock such as water shortage in the soil, a short period of darkness, cooling of the soil, or changes in humidity and temperature (Kramer and Boyer, 1995).

Therefore, the results presented here for different gs and Ci values and behavior when changing the PPFD, in the woody species Citrus as compared to the herbaceous species tobacco, highlight the necessity of great care when carrying out gas exchange measurements, especially with woody species. To avoid confusing cyclical plant response with random noise in measurements of an average gas exchange rate of a group of plants, especially when growing in pots, frequently determined by taking just one measurement per plant, repetitive measurements at 10 min intervals can determine whether transpiration is stable or oscillating when environmental conditions are varying (Rose and Rose, 1994). Also, simultaneous measurements of gas exchange with light adapted fluorescence analysis (the measurement of the efficiency of Photosystem II photochemistry - FPSII) can also be used because fluorescence can vary faster than gas exchange, from static to oscillating, indicating stomatal oscillation and or patchy stomatal closure, or some environmental stress (Cardon et al., 1994).



Barradas VL, Jones HG (1996) Response of CO2 assimilation to changes in irradiance: laboratory and field data and a model for beans (Phaseolus vulgaris L.). J. Exp. Bot. 47:639-645.         [ Links ]

Barrs HD (1971) Cyclic variations in stomatal aperture, transpiration, and leaf water potential under constant environmental conditions. Annu. Rev. Plant Physiol. 22:223-236.         [ Links ]

Bernacchi, CJ, Singsaas, C, Pimentel, C, Portis Jr AR, Long, SP (2001) Improved temperature response functions for models of Rubisco-limited photosynthesis. Plant Cell Environ. 24:253-259.         [ Links ]

Bolhàr-Nordenkampf HR, Öquist GO (1993) Chlorophyll fluorescence as a tool in photosynthesis research. In: Hall DO, Scurlock JMO, Bolhàr-Nordenkampf HR, Leegood RC, Long SP (eds), Photosynthesis and production in a changing environment. A field and laboratory manual, pp.193-206. Chapman & Hall, London, England.         [ Links ]

Cardon ZG, Mott KA, Berry JA (1994) Dynamics of patchy stomatal movements, and their contribution to steady-state and oscillating stomatal conductance calculated using gas-exchange techniques. Plant Cell Environ. 17:995-1007.         [ Links ]

Hartung W, Sauter A, Hose E (2002) Abscisic acid in the xylem: where does it come from, where it go to? J. Exp. Bot. 53: 27-32.         [ Links ]

Hose E, Clarkson DT, Steudle E, Schreiber L, Hartung W (2001) The exodermis: a variable apoplastic barrier. J. Exp. Bot. 52:2245-2264.         [ Links ]

Jones HG (1998) Stomatal control of photosynthesis and transpiration. J. Exp. Bot. 49:387-398.         [ Links ]

Kramer PJ, Boyer JS (1995) Water relations of plants and soils. Academic Press, San Diego, USA.         [ Links ]

Levy Y, Kaufmann MR (1976) Cycling of leaf conductance in citrus exposed to natural and controlled environments. Can. J. Bot. 54:2215-2218.         [ Links ]

Lloyd J, Kriedemann PE, Syvertsen JP (1987) Gas exchange, water relations and ion concentrations of leaves on salt stressed Valencia orange,Citrus sinensis (L.) Osbeck. Aust. J. Plant Physiol. 14:387-396.         [ Links ]

Lloyd J, Syvertsen JP, Kriedemann PE, Farquhar GD (1992) Low conductances for CO2 diffusion from stomata to the sites of carboxylation in leaves of woody species. Plant Cell Environ. 15:873-899.         [ Links ]

Long SP, Hällgren JE (1993) Measurements of CO2 assimilation by plants in the field and the laboratory. In: Hall DO, Scurlock JMO, Bolhàr-Nordenkampf HR, Leegood RC, Long SP (eds) Photosynthesis and production in a changing environment. A field and laboratory manual, pp.129-167. Chapman & Hall, London, England.         [ Links ]

Medina CL, Souza RP, Machado EC, Ribeiro RV, Silva JAB (2002) Photosynthetic response of citrus grown under reflective aluminized polypropylene shading nets. Sci. Hort. 96:115-125.         [ Links ]

Ribeiro RV, Machado EC, Oliveira RF, Pimentel C (2003) High temperature effects on the response of photosynthesis to light in sweet orange plants infected with Xylella fastidiosa. Braz. J. Plant Physiol. 15:89-97.         [ Links ]

Rose MA, Rose MA (1994) Oscillatory transpiration may complicate stomatal conductance and gas-exchange measurements. HortScience 29:693-694.         [ Links ]

Steudle E (2001) The cohesion-tension mechanism and the acquisition of water by plant roots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:847-875.         [ Links ]

Syvertsen JP, Lloyd JJ (1994) Citrus. In: Schaffer B, Andersen PC (eds) Handbook of environmental physiology of fruit crops - subtropical and tropical crops, pp.65-100. CRC Press, Boca Raton, USA.         [ Links ]

Webb AAR (1998) Stomatal rhythms. In: Lumsden PJ, Millar AJ (eds) Biological rhythms and photoperiodism in plants, pp.69-79. BIOS Scientific Publishers, Washington, USA.         [ Links ]

Zipperlen SW, Press MC (1997) Photosynthetic induction and stomatal oscillations in relation to the light environment of two dipterocarp rain forest tree species. J. Ecol. 85:491-503.        [ Links ]



Received: 01/11/2003, Accepted: 28/03/2004



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