PhotosynthesisResearch 42:217-225, 1994. © 1994KluwerAcademicPublishers. Printedin theNetherlands. Regular paper
Changes in the photosynthetic light response curve during leaf development of field grown maize with implications for modelling canopy photosynthesis C.M Stirling1, C. Aguilera, N. R. Baker & S. P. Long Department of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, C04 3SQ, UK; 1present address and address for correspondence: Institute of Terrestrial Ecology, Bangor Research Unit, University of Wales, Deiniol Road, Bangor, Gwynedd, LL57 2UP, UK Received8 February1994;acceptedin revisedform 17 October1994
Key words: photosynthesis, leaf age, maize
Abstract Changes in the photosynthetic light-response curve during leaf development were determined for the fourth leaf of maize crops sown on 23 April and 10 June. Temperatures were unusually mild during late spring/early summer and neither crop experienced chilling damage. The concept of thermal time was used to take into account the effects of different temperature regimes on developmental stage, thereby enabling photosynthetic light-response data to be combined for both crops to describe the general response. Large variations in the upper asymptote (Asat) and convexity (O) of the light-response curve occurred during leaf development, but the maximum quantum yield of CO2 assimilation remained relatively constant throughout. Dark respiration rates showed a small but significant decrease with leaf age and generally ranged between 5 and 10% of Asat. A simple mathematical model was developed to assess the sensitivity of daily leaf photosynthesis (AL) to reductions in the Asat, O and the initial slope (~) of the light-response curve at different stages of leaf development. On bright sunny days, and at all developmental stages, AL was ca. twice as sensitive to reductions in Asat than to reductions in • and e . In overcast conditions, however, all three parameters contributed significantly to reductions in leaf photosynthesis, although the contribution of tb was greatest during early leaf growth, while older leaves were most sensitive to depressions in Asat. The implications of these results for modelling the sensitivity of canopy photosynthesis to chill-induced photoinhibition of the light-response curve are discussed.
Introduction Developmental stage has been shown to have a marked effect on the extent of chilling damage to photosynthesis, with mature leaves most sensitive to chill-induced photoinhibition (Ireland et al. 1985; Long et al. 1989), while immature leaves are susceptible to disruption in chloroplast development at low temperatures (Nie and Baker 1991; Stirling et al. 1991; Robertson et al. 1993). Surprisingly, in view of the known interactions between developmental stage and stress-induced damage to photosynthesis, few workers have quantified the underlying changes in photosynthesis with leaf development under field conditions. Where developmental effects on photosynthesis have been quantified,
measurements have generally been limited to instantaneous or light-saturated rates of C O 2 uptake (Thiagarajah et al. 1979; Constable and Rawson 1980; Dwyer and Stewart 1986; Wullshleger and Oosterhuis 1990), despite evidence that photosynthetic performance at low light intensities is a major determinant of crop growth (Baker et al. 1988; Farage and Long 1987; Stirling et al. 1993). On the few occasions where detailed studies of the whole light-response curve have been undertaken, measurements did not encompass the early stages of leaf growth (Marshall and Biscoe 1980). To construct a meaningful model for the prediction of temperature effects on maize photosynthesis during early growth, account must be taken of how photosynthesis varies with leaf development. In their
218 natural environment, leaves will experience wide fluctuations in incident photosynthetically active photon flux density (PPFD) and so it is important to characterise age-related changes in the whole of the lightresponse curve. The photosynthetic light-response curve (PLRC) can be defined by three parameters: (i) the maximum quantum yield of CO2 assimilation (~), derived from the slope of the initial linear response of CO2 uptake to PPFD, (ii) the upper asymptote, representing the light-saturated rate of CO2 assimilation (Asat) and (iii) the convexity coefficient (O) describing the curvature of the response between (i) and (ii). Most models of canopy photosynthesis are based on the assumption that the parameters defining the PLRC are the same for all leaves and that variation in the rates of leaf photosynthesis with canopy depth arise solely from differences in incident PPFD. Clearly this is an over simplification, since factors such as developmental stage and light acclimation of the PLRC will also contribute significantly to variation in leaf photosynthesis, although these two factors will be compounded under field conditions. Since most age-related studies of the PLRC have been restricted to unshaded leaves at the top of the canopy or to isolated plants grown under controlled environment conditions, it is difficult to extrapolate the results to natural canopies where developmental changes in the PLRC will be modified by the radiation microclimate within the canopy. The work reported here provides the first detailed analysis of in situ age-related changes in the PLRC of field grown maize. A simple mathematical model is used to assess the effects of observed changes in the shape of the PLRC with leaf development on daily leaf photosynthesis and its sensitivity to step reductions in Asat, ~bandO.
Material and methods
Experimental site and plant material To impose a range of temperatures during leaf growth, maize crops were planted on two dates, 23 April and 10 June, at a site in N.E. Essex (57052 ' N, 0°5W E). Caryopses were sown in three randomised blocks and at intervals of 0.20 m along rows spaced 0.35 m apart to give a final population of ca. 14 plants m -2. Environmental conditions were monitored by a weather station (Delta-T Devices Ltd, Newmarket, UK) situated within 5 m of the experimental site. Screen temperature, and soil temperature at a depth
of 5 cm, were measured at 10 min intervals and hourly means recorded.
Sampling strategy Measurements of CO2 uptake were confined to the fourth leaf of plants sown on 23 April and 10 June. To quantify changes in the PLRC curve during leaf growth, and to avoid confounding effects arising from the developmental gradient that exists along the maize leaf (Miranda et al. 1981b), gas exchange measurements were restricted to a single region of the fourth leaf, ca. 3 cm in length and 5 cm removed from the leaf tip. This region of leaf 4 was marked with Tip-Ex as it unfolded from the main stem. Approximately 50 plants within the central 3 m 2 of each plot were tagged in this manner, selecting only plants in which the fourth leaf emerged on the same calender date, i.e. 12 May in the early- and 29 June in the late-sown crop.
Gas exchange measurements The protocol for measuring the rate of CO2 uptake has been described previously (Stirling et al. 1991). Three gas exchange systems were operated simultaneously to obtain replicate light-response curves. Net CO2 uptake of detached leaves were measured in a temperature controlled leaf disc chamber, using an infrared CO2 analyser (Type 225-Mk3, Analytical Development Co., Hoddeson, UK). Leaf temperature was maintained at 20 °C + 1 °C by circulating water from a heater/circulator (C-400, Techne Ltd, Duxford, UK) and flow-through cooler (EN-350 Neslab, Portsmouth, NH, USA). Air was drawn from the roof of the laboratory and passed through two 30 dm 3 vessels to minimise short-term fluctuations in the atmospheric CO2 concentration. The chamber was illuminated with a 250-W quartz-iodide source (Scholly Fiberoptik, GmbH, Germany) and photosynthetic photon flux density (PPFD) within the range 0-2000/zmol m -2 s- 1 was obtained at the leaf surface by attenuating the beam with thin sheets of glass fibre mounted in front of the chamber window. PPFD incident at the leaf surface was determined using a quantum sensor (SKP 215, Skye Instruments Ltd, Powys, UK), placed below the chamber window in the position normally occupied by the leaf. Whole shoots were excised from the field between 0830 and 0900 h and the cut end placed in distilled water for transport to the laboratory where they were dark-adapted for ca. 30 min. Leaf sections from the
219 fourth leaf were then cut under distilled water, surface water removed and the section placed in the chamber ensuring that the cut surfaces were continuously irrigated with distilled water circulated by a peristaltic pump (MHRE 200, Watson-Marlow Ltd, Falmouth, UK). Following gas exchange measurements, the area of each leaf section was measured and its absorptivity determined in a Taylor integrating sphere (S0143-000, Varian Associates, Palo Alto, CA, USA).
Leaf area expansion A 1 m length of row was removed from each of the three replicate plots at approximate weekly intervals between 20 and 60 days after sowing. Growth analysis followed the procedures previously described (Stirling et al. 1991), with two plants per plot taken as a sub-sample for measurements of individual leaf area, using an area meter (Delta-T Devices Ltd, Newmarket, UK).
Thermal time analysis The concept of thermal time was used to take into account the effects of different temperature regimes on the developmental stage of crops sown on 23 April and 10 June. The calculation of thermal time is described by Stirling et al. (1991) and is based on the linear relationship between developmental rate and temperature, 0t = (T - Tb)t
(1)
where 0t is the thermal time (degree days, °C d), Tb and T are the minimum and mean dally temperatures (°C), respectively, and t is the time (d). Thermal time was calculated using soil temperature at a depth of 5 cm (Peacock 1975) and a Tb of 100 C.
Analysis of the light-response curve for C02 assimilation A quadratic equation, describing a non-rectangular hyperbolic response of CO2 assimilation (Thornley and Johnson 1990) was fitted to data using a statistical package (STATGRAPHICS, Statistical Graphics Corporation, USA). This equation was solved for its positive root, A + Rd = {(I)Q + Asat - x/[OQ + Asat) 2
-4OQAsatO]}/(20)
(2)
where A is the net rate of CO2 assimilation, Rd is the rate of dark respiration, Q is the absorbed photon flux density, O, O and Asat are the initial slope, convexity and upper asymptote of the light-response curve. Note that the parameter qb is not analogous to the maximum quantum yield of CO2 assimilation (~b), determined empirically from the initial linear region of the lightresponse curve, since Eq. (2) describes a non-linear response at low Q when O is less than 1.0. Therefore, to obtain accurate estimates of the maximum quantum yield of CO2 assimilation, ~ was calculated separately using linear regression analyses of the linear region of the relation between CO2 uptake and Q, where Q ranged between 40 and 150 #mol m -2 s -1.
Leaf photosynthesis model A simple model was developed to assess the relative sensitivity of individual leaf photosynthesis to reductions in the parameters Asat, ~ and O at different stages of leaf development. Diurnal incoming radiation and ratios of diffuse to direct radiation were determined as a function of solar angle, daylength, latitude, time of year and atmospheric transmittance (Eqs. 15, Appendix) in accordance with Long (1991) after Norman (1980) and Forseth and Norman (1991). Simulations were for 21 June at a latitude 52.50 N and for clear sky and overcast conditions with an atmospheric transmissivity of 0.75 and 0.37, respectively, and total dally PPFD of 28 and 14 mol m -2 d - l . Daily net CO2 uptake per unit leaf area (AL) was calculated for a single leaf within the canopy, assuming a leaf inclination angle (7) of 450 (Ross 1981). Incident radiation was modelled for the upper leaf surface only, with the leaf receiving both direct and diffuse radiation when the solar angle (0) exceeded the leaf inclination angle (i.e. O > 45 °) but diffuse radiation only at low solar angles when O < 450 (Stirling et al. 1994). Instantaneous net photosynthetic rates (AL) were calculated from Eq. (2) and daily total net photosynthesis was then computed by integrating over the day.
Results
Leaf area expansion The crop sown on 23 April was exposed to unusually mild temperatures at the beginning of the growing season. For example, during the first 20 days after sowing, air temperature fell below 5 °C on only one occasion,
220 40
(a)
6O
g-.
30
(9 v
40
o
20
-
Changes in the photosynthetic light response curve during leaf development of field grown maize with implications for modelling canopy photosynthesis C.M Stirling1, C. Aguilera, N. R. Baker & S. P. Long Department of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, C04 3SQ, UK; 1present address and address for correspondence: Institute of Terrestrial Ecology, Bangor Research Unit, University of Wales, Deiniol Road, Bangor, Gwynedd, LL57 2UP, UK Received8 February1994;acceptedin revisedform 17 October1994
Key words: photosynthesis, leaf age, maize
Abstract Changes in the photosynthetic light-response curve during leaf development were determined for the fourth leaf of maize crops sown on 23 April and 10 June. Temperatures were unusually mild during late spring/early summer and neither crop experienced chilling damage. The concept of thermal time was used to take into account the effects of different temperature regimes on developmental stage, thereby enabling photosynthetic light-response data to be combined for both crops to describe the general response. Large variations in the upper asymptote (Asat) and convexity (O) of the light-response curve occurred during leaf development, but the maximum quantum yield of CO2 assimilation remained relatively constant throughout. Dark respiration rates showed a small but significant decrease with leaf age and generally ranged between 5 and 10% of Asat. A simple mathematical model was developed to assess the sensitivity of daily leaf photosynthesis (AL) to reductions in the Asat, O and the initial slope (~) of the light-response curve at different stages of leaf development. On bright sunny days, and at all developmental stages, AL was ca. twice as sensitive to reductions in Asat than to reductions in • and e . In overcast conditions, however, all three parameters contributed significantly to reductions in leaf photosynthesis, although the contribution of tb was greatest during early leaf growth, while older leaves were most sensitive to depressions in Asat. The implications of these results for modelling the sensitivity of canopy photosynthesis to chill-induced photoinhibition of the light-response curve are discussed.
Introduction Developmental stage has been shown to have a marked effect on the extent of chilling damage to photosynthesis, with mature leaves most sensitive to chill-induced photoinhibition (Ireland et al. 1985; Long et al. 1989), while immature leaves are susceptible to disruption in chloroplast development at low temperatures (Nie and Baker 1991; Stirling et al. 1991; Robertson et al. 1993). Surprisingly, in view of the known interactions between developmental stage and stress-induced damage to photosynthesis, few workers have quantified the underlying changes in photosynthesis with leaf development under field conditions. Where developmental effects on photosynthesis have been quantified,
measurements have generally been limited to instantaneous or light-saturated rates of C O 2 uptake (Thiagarajah et al. 1979; Constable and Rawson 1980; Dwyer and Stewart 1986; Wullshleger and Oosterhuis 1990), despite evidence that photosynthetic performance at low light intensities is a major determinant of crop growth (Baker et al. 1988; Farage and Long 1987; Stirling et al. 1993). On the few occasions where detailed studies of the whole light-response curve have been undertaken, measurements did not encompass the early stages of leaf growth (Marshall and Biscoe 1980). To construct a meaningful model for the prediction of temperature effects on maize photosynthesis during early growth, account must be taken of how photosynthesis varies with leaf development. In their
218 natural environment, leaves will experience wide fluctuations in incident photosynthetically active photon flux density (PPFD) and so it is important to characterise age-related changes in the whole of the lightresponse curve. The photosynthetic light-response curve (PLRC) can be defined by three parameters: (i) the maximum quantum yield of CO2 assimilation (~), derived from the slope of the initial linear response of CO2 uptake to PPFD, (ii) the upper asymptote, representing the light-saturated rate of CO2 assimilation (Asat) and (iii) the convexity coefficient (O) describing the curvature of the response between (i) and (ii). Most models of canopy photosynthesis are based on the assumption that the parameters defining the PLRC are the same for all leaves and that variation in the rates of leaf photosynthesis with canopy depth arise solely from differences in incident PPFD. Clearly this is an over simplification, since factors such as developmental stage and light acclimation of the PLRC will also contribute significantly to variation in leaf photosynthesis, although these two factors will be compounded under field conditions. Since most age-related studies of the PLRC have been restricted to unshaded leaves at the top of the canopy or to isolated plants grown under controlled environment conditions, it is difficult to extrapolate the results to natural canopies where developmental changes in the PLRC will be modified by the radiation microclimate within the canopy. The work reported here provides the first detailed analysis of in situ age-related changes in the PLRC of field grown maize. A simple mathematical model is used to assess the effects of observed changes in the shape of the PLRC with leaf development on daily leaf photosynthesis and its sensitivity to step reductions in Asat, ~bandO.
Material and methods
Experimental site and plant material To impose a range of temperatures during leaf growth, maize crops were planted on two dates, 23 April and 10 June, at a site in N.E. Essex (57052 ' N, 0°5W E). Caryopses were sown in three randomised blocks and at intervals of 0.20 m along rows spaced 0.35 m apart to give a final population of ca. 14 plants m -2. Environmental conditions were monitored by a weather station (Delta-T Devices Ltd, Newmarket, UK) situated within 5 m of the experimental site. Screen temperature, and soil temperature at a depth
of 5 cm, were measured at 10 min intervals and hourly means recorded.
Sampling strategy Measurements of CO2 uptake were confined to the fourth leaf of plants sown on 23 April and 10 June. To quantify changes in the PLRC curve during leaf growth, and to avoid confounding effects arising from the developmental gradient that exists along the maize leaf (Miranda et al. 1981b), gas exchange measurements were restricted to a single region of the fourth leaf, ca. 3 cm in length and 5 cm removed from the leaf tip. This region of leaf 4 was marked with Tip-Ex as it unfolded from the main stem. Approximately 50 plants within the central 3 m 2 of each plot were tagged in this manner, selecting only plants in which the fourth leaf emerged on the same calender date, i.e. 12 May in the early- and 29 June in the late-sown crop.
Gas exchange measurements The protocol for measuring the rate of CO2 uptake has been described previously (Stirling et al. 1991). Three gas exchange systems were operated simultaneously to obtain replicate light-response curves. Net CO2 uptake of detached leaves were measured in a temperature controlled leaf disc chamber, using an infrared CO2 analyser (Type 225-Mk3, Analytical Development Co., Hoddeson, UK). Leaf temperature was maintained at 20 °C + 1 °C by circulating water from a heater/circulator (C-400, Techne Ltd, Duxford, UK) and flow-through cooler (EN-350 Neslab, Portsmouth, NH, USA). Air was drawn from the roof of the laboratory and passed through two 30 dm 3 vessels to minimise short-term fluctuations in the atmospheric CO2 concentration. The chamber was illuminated with a 250-W quartz-iodide source (Scholly Fiberoptik, GmbH, Germany) and photosynthetic photon flux density (PPFD) within the range 0-2000/zmol m -2 s- 1 was obtained at the leaf surface by attenuating the beam with thin sheets of glass fibre mounted in front of the chamber window. PPFD incident at the leaf surface was determined using a quantum sensor (SKP 215, Skye Instruments Ltd, Powys, UK), placed below the chamber window in the position normally occupied by the leaf. Whole shoots were excised from the field between 0830 and 0900 h and the cut end placed in distilled water for transport to the laboratory where they were dark-adapted for ca. 30 min. Leaf sections from the
219 fourth leaf were then cut under distilled water, surface water removed and the section placed in the chamber ensuring that the cut surfaces were continuously irrigated with distilled water circulated by a peristaltic pump (MHRE 200, Watson-Marlow Ltd, Falmouth, UK). Following gas exchange measurements, the area of each leaf section was measured and its absorptivity determined in a Taylor integrating sphere (S0143-000, Varian Associates, Palo Alto, CA, USA).
Leaf area expansion A 1 m length of row was removed from each of the three replicate plots at approximate weekly intervals between 20 and 60 days after sowing. Growth analysis followed the procedures previously described (Stirling et al. 1991), with two plants per plot taken as a sub-sample for measurements of individual leaf area, using an area meter (Delta-T Devices Ltd, Newmarket, UK).
Thermal time analysis The concept of thermal time was used to take into account the effects of different temperature regimes on the developmental stage of crops sown on 23 April and 10 June. The calculation of thermal time is described by Stirling et al. (1991) and is based on the linear relationship between developmental rate and temperature, 0t = (T - Tb)t
(1)
where 0t is the thermal time (degree days, °C d), Tb and T are the minimum and mean dally temperatures (°C), respectively, and t is the time (d). Thermal time was calculated using soil temperature at a depth of 5 cm (Peacock 1975) and a Tb of 100 C.
Analysis of the light-response curve for C02 assimilation A quadratic equation, describing a non-rectangular hyperbolic response of CO2 assimilation (Thornley and Johnson 1990) was fitted to data using a statistical package (STATGRAPHICS, Statistical Graphics Corporation, USA). This equation was solved for its positive root, A + Rd = {(I)Q + Asat - x/[OQ + Asat) 2
-4OQAsatO]}/(20)
(2)
where A is the net rate of CO2 assimilation, Rd is the rate of dark respiration, Q is the absorbed photon flux density, O, O and Asat are the initial slope, convexity and upper asymptote of the light-response curve. Note that the parameter qb is not analogous to the maximum quantum yield of CO2 assimilation (~b), determined empirically from the initial linear region of the lightresponse curve, since Eq. (2) describes a non-linear response at low Q when O is less than 1.0. Therefore, to obtain accurate estimates of the maximum quantum yield of CO2 assimilation, ~ was calculated separately using linear regression analyses of the linear region of the relation between CO2 uptake and Q, where Q ranged between 40 and 150 #mol m -2 s -1.
Leaf photosynthesis model A simple model was developed to assess the relative sensitivity of individual leaf photosynthesis to reductions in the parameters Asat, ~ and O at different stages of leaf development. Diurnal incoming radiation and ratios of diffuse to direct radiation were determined as a function of solar angle, daylength, latitude, time of year and atmospheric transmittance (Eqs. 15, Appendix) in accordance with Long (1991) after Norman (1980) and Forseth and Norman (1991). Simulations were for 21 June at a latitude 52.50 N and for clear sky and overcast conditions with an atmospheric transmissivity of 0.75 and 0.37, respectively, and total dally PPFD of 28 and 14 mol m -2 d - l . Daily net CO2 uptake per unit leaf area (AL) was calculated for a single leaf within the canopy, assuming a leaf inclination angle (7) of 450 (Ross 1981). Incident radiation was modelled for the upper leaf surface only, with the leaf receiving both direct and diffuse radiation when the solar angle (0) exceeded the leaf inclination angle (i.e. O > 45 °) but diffuse radiation only at low solar angles when O < 450 (Stirling et al. 1994). Instantaneous net photosynthetic rates (AL) were calculated from Eq. (2) and daily total net photosynthesis was then computed by integrating over the day.
Results
Leaf area expansion The crop sown on 23 April was exposed to unusually mild temperatures at the beginning of the growing season. For example, during the first 20 days after sowing, air temperature fell below 5 °C on only one occasion,
220 40
(a)
6O
g-.
30
(9 v
40
o
20
-
