Analysing the responses of photosynthetic CO2 assimilation to long-term elevation of atmospheric CO2 concentration

An integrating sphere leaf chamber which allows the simultaneous measurement of light and C O 2 absorbance for the determination of the actual quantum yield of photosynthetic organs, in vivo.

Vegetatio 104/105: 33-45, 1993. J. Rozema, H. Lambers, S.C. van de Geijn and M.L. Cambridge (eds). CO 2 and Biosphere © 1993 Kluwer Academic Publishers. Printed in Belgium.

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Analysing the responses of photosynthetic CO2 assimilation to long-term elevation of atmospheric CO2 concentration

S.P. Long, N.R. Baker & C.A. Raines Dept. of Biology, University of Essex, Colchester, C04 3SQ, UK; Dept. of Applied Science, Brookhaven Natl. Lab., Upton, NY 11973, USA

Keywords: Greenhouse effect, Chlorophyll fluorescence, RubisCO, Photosystem II, Stomata, Quantum efficiency

Abstract

Understanding how photosynthetic capacity acclimatises when plants are grown in an atmosphere of rising CO2 concentrations will be vital to the development of mechanistic models of the response of plant productivity to global environmental change. A limitation to the study of acclimatisation is the small amount of material that may be destructively harvested from long-term studies of the effects of elevation of CO2 concentration. Technological developments in the measurement of gas exchange, fluorescence and absorption spectroscopy, coupled with theoretical developments in the interpretation of measured values now allow detailed analyses of limitations to photosynthesis in vivo. The use of leaf chambers with Ulbricht integrating spheres allows separation of change in the maximum efficiency of energy transduction in the assimilation of CO2 from changes in tissue absorptance. Analysis of the response of CO2 assimilation to intercellular CO2 concentration allows quantitative determination of the limitation imposed by stomata, carboxylation efficiency, and the rate of regeneration of ribulose 1:5 bisphosphate. Chlorophyll fluorescence provides a rapid method for detecting photoinhibition in heter- ogeneously illuminated leaves within canopies in the field. Modulated fluorescence and absorption spectroscopy allow parallel measurements of the efficiency of light utilisation in electron transport through photosystems I and II in situ.

Abbreviations: A, net rate of C O 2 uptake per unit leaf area (#mol m - 2 s - 1); Asat light-saturated A; AA82o, change in absorptance of PSI on removal of illumination (OD); c, CO2 concentration in air (#mol tool- 1); ca, c in the bulk air; ci, c in the intercellular spaces; ce, carboxylation efficiency (mol m- 2 s- 1); E, transpiration per unit leaf area (mol m-2 s- 1); F, fluorescence emission of PSII (relative units); Fm, maximal level of F; Fo, minimal level of F upon illumination when PSII is maximally oxidised; Fs, the steady-state F following the m peak; Fv, the difference between Fm and Fo; Fm, maximal F' generated after the m peak by addition of a saturating light pulse; F'o the minimal level of F' after the m peak determined by re-oxidising PSII by far-red light; g~, leaf conductance to CO2 diffusion in the gas phase (mol m -2 s- 1); g;, leaf conductance to water vapour diffusion in the gas phase (mol m -2 s- 1); kc and ko, the Michaelis constants for CO2 and 02, respectively, (/~mol mol-~); Jmax, the maximum rate of regeneration of rubP (/~mol m- 2 s- ~); 1, stomatal limitation to CO2 uptake (dimensionless, 0-1); LCP, light compensation point of photosynthesis (#mol m - 2 s - 1); oi, the intercellular 02 concentration (mmol mol- 1); Pi, cytosol inorganic phosphate concentration; PSI, photosystem I; PSII, photosystem II; Q, photon flux (#mol m -2 s-L); Qabs, Q absorbed by the leaf; rubisCO, ribulose 1:5 bisphosphate

34

carboxylase/oxygenase; rubP, ribulose 1:5 bisphosphate; s, projected surface area of a leaf (m2); Wc,max, is the maximum rate of carboxylation (#mol m-2 s- 1); Wc ' the rubisCO limited rate of carboxylation (#mol m - 2 s- 1); W~, the electron transport limited rate of regeneration of rubP (#mol m-2 s-1); Wp, the inorganic phosphate limited rate of regeneration of rubP (#mol m-2 s-1); ~t, absorptance of light (dimensionless, 0-1); ct a, a of standard black absorber ct~, ct of leaf;; ~s, ~t of integrating sphere walls; F, CO2 compensation point of photosynthesis (#mol mol-1); z the specificity factor for rubisCO carboxylation (dimensionless); 0, convexity of the response of A to Q (dimensionless, 0-1); 4, the quantum yield of photosynthesis on an absorbed light basis (~A/~Qabs; dimensionless); q~app, the quantum yield of photosynthesis on an incident light basis (bA/bQ; dimensionless); q~m, the maximum q~; ~bm,app, the maximum tPapp; ~bPSii, the photochemical efficiency of PSII (dimensionless, 0-1); q~PSn,m, the maximum (h, sn.

Introduction

Photosynthesis is the major physiological process by which plants sense, and by which their pro- duction can respond directly to, rising atmospheric CO2 concentrations (Mott, 1990; Long & Drake 1992). Understanding the response of photosynthesis to rising Ca is therefore fundamen- tal to understanding of the response of plant pro- duction and growth. On initial transfer to an elevated Ca the rate of photosynthetic CO2 uptake per unit leaf area (A) increases because: (i) CO2 competitively inhibits the oxygenation of ribulose 1:5 bisphosphate (rubP), and hence inhibits photorespiration; and (ii) the velocity of carboxylation increases because of the relatively low km of rubisCO for CO2 (Stitt, 1991). Since photorespiration may depress A by ca. 20-50~o in temper- ate climates (Long 1985), a substantial increase in A is predicted as Ca rises (Long 1991). However, prediction of the exact increase in A which will occur with the growth of plants in higher CO2 concentrations is complicated by acclimatisation. The term acclimatisation is used here to describe the effect of prolonged growth in elevated ca on the development and maintenance of the photosynthetic apparatus, in turn determining photosynthetic capacity. A common observation in plants grown in controlled environments was that growth in elevated Ca depressed photosynthetic capacity, in some cases to the extent that the increase resulting from inhibition of photorespiration was offset by the decrease in capacity (Sage

et al. 1989). A prominent change in the photosynthetic apparatus is a decrease in the activation or/and quantity of rubisCO protein, underlying the frequently observed decrease in carboxylation efficiency. However, this is not a universal effect. In some species no decrease in photosynthetic capacity occurs during acclimatisation. In one of the few long-term studies of perennial vegetation, an increase in photosynthetic capacity was observed after 4 years of growth at 700 #mol mol - 1 (Arp & Drake 1991). Arp (1991) has further shown that the decline in photosynthetic capacity during acclimatisation to elevated Ca observed in many of the earlier studies may have been an artifact of restriction of rooting volume and notes that this downward acclimatisation is rarely observed when plants are grown in large rooting volumes. Acclimatisation may be expected to result from a number of processes operating on different time scales. COz is known to effect the expression of at least one gene involved in photosynthesis (Coleman 1991) and CO2 may therefore directly affect the development of the photosynthetic apparatus in leaves. Increased growth with a limited supply of nitrogen will result in a dilution of N within tissues which in turn may result in decreased photosynthetic capacity. Sim- ilarly, if plants are unable to utilise the additional photosynthate resulting from increased net CO2 uptake, then carbohydrates will accumulate in the source leaves. There are a variety of mechanisms by which this increase in carbohydrate levels could produce a feed-back inhibition of photo-

synthetic rate and could also affect the expression of genes coding for photosynthetic enzymes (Stitt 1991).

Whilst the immediate effects of an increased Ca on assimilation are well known, and can be ef- fectively modelled from a mechanistic understanding, predictions are complicated by acclimatisation (Harley et al. 1991; Long 1991; McMurtrie & Wang 1992). There is evidence that acclimatisation is most marked in species where photosynthesis is limited by capacity to generate new sites of storage or utilisation, i.e. sinks, or where nitrogen is limiting (Stitt 1991; Long & Drake 1992). Development of new sinks in perennial species may require years, as the results of Arp & Drake (1991) may suggest. Since growth of plants in elevated Ca may result in pronounced changes in sink sizes, root system size, and tissue C / N ratios in the long term, acclimatisation must be expected to occur on a relatively long time scale. Indeed it is possible that loss of photosynthetic capacity in elevated Ca observed over the first weeks or months of growth may be a tem- porary phenomenon resulting from insufficient nitrogen uptake or sink size. This might later be compensated for by utilisation of the additional photosynthate in the development of a larger root system, allowing the capture of more nutrients or in the development of more and larger sinks ca- pable of fully utilising the increased photosynthesis that an elevated atmospheric CO2 concentration can support. Understanding of the mechanisms by which photosynthesis acclimatises, the diversity of these mechanisms and their kinetics will be essential to the development of effective mechanistic models of the response of ecosystem and crop primary productivity to rising %. The findings of Arp & Drake (1991) that acclimatisation of photosynthesis can occur on a time-scale of years emphasises the importance of growth of perennial vegetation at elevated Ca on a time-scale of years. However, a serious limitation here is the amount of material that can be grown in elevated ca for such extended periods. Investigation of acclimatisation over prolonged periods will require techniques which will not damage the vegetation. Statistically, random

35

sampling should not involve the destructive removal of more than 10~ of the population, for the statistical analysis to approximate to sampling with replacement (Sokal & Rohlf 1981). Disregarding statistical considerations, removal of more that 10 ~ of the population would clearly have a marked effect on the remainder, such that in a long-term experiment the manipulated areas may become unrepresentative of the surrounding vegetation. Destructive removal of 10~o of the canopy in each year is likely to have a carryover effect on the next year's vegetation. If for exam- ple we consider an open-top chamber enclosing 2 m 2 of perennial vegetation with an average leaf area index of 2, and assuming that the experiment is designed to run for 10 years, then removal of just 20 cm 2 per year would result in destructive sampling of 10~ of the leaf area over the 10 years. Whilst it might be argued that this canopy would be replaced the experimenter would be im- posing an additional artificial sink. Vegetation subjected to long-term elevation of c a provides the best available resource for the examination of change in the photosynthetic apparatus during acclimatisation, however it offers little scope for the vast array of photosynthetic research methods requiring destructive sampling of large quantities of leaves. Methods of investigation, will of necessity, be largely limited to non-invasive in vivo methods or those requiring the removal of minute quantities of vegetation.

Technological developments in the measurement of gas exchange, fluorescence and absorption spectroscopy, coupled with theoretical developments in the interpretation of measured values now allow detailed analyses of limitations to photosynthesis in vivo. This paper examines these developments in the in vivo analysis of photosynthesis and their potential in studying acclimatisation in plants growing in the field in elevated CO2 concentrations.

Measurement of photosynthetic gas exchange

Photosynthetic gas exchange by leaves, and by whole plants, is typically determined by enclosure

36

in chambers to define the area of material under analysis. Fluxes of gases between the enclosed leaf and the atmosphere are determined as the product of concentration change and air flow rate across the chamber (Long & Hallgren 1992). Car- bon dioxide concentration change is typically measured by infra-red gas analysis, and this is usually combined with measurement of water vapour concentration changes by dew point or elec- trical conductance hygrometry (Long & Hallgren 1992). A range of commercial portable gas exchange systems is now available (Bingham & Long 1992).

Since transpiration is limited by the leaf only through the conductance for water vapour diffusion from the mesophyll cell wall outer surfaces to the air surrounding the leaf (g~), this conductance may be calculated directly from the rate of water loss from the leaf (E) and from the gradient of water vapour concentration between chamber air (Wa) and the leaf intercellular air spaces (wi), which is assumed to equal the saturation humidity at the temperature of the leaf:

g; = E(wi - Wa) (1)

The conductance to CO2 (g0 is assumed to equal g[ divided by the ratio of the binary diffusivities of water vapour/air and CO2/air, with 1.6 as the accepted value for this ratio (Field et al. 1989). Where leaf boundary layer conductance consti- tutes a significant proportion of the diffusion limitation, then a separate conversion is necessary as transfer of molecules in the boundary layer will be both by diffusion and turbulent transfer (Long & Hallgren 1992). From considerations of diffusion alone it may be assumed that mean CO2 mole fraction at the mesophyll cell wall/internal air space interface (ci) will be:

ci = c, - A/g1 (2)

This calculation allows expression of A in relation to c~ and so allows separation of any stomatal effects in examining the response of photosynthesis within the mesophyll to CO 2 concentration. Experimental and theoretical considerations suggest that ci may be in close equilibrium with the concentration of CO2 within the mesophyll, i.e.

will reflect the substrate concentration available for carboxylation (reviewed: Long 1985). The use of feedback control compensating gas exchange systems also allows examination of the effects of other environmental variables while ci is maintained at a constant level by varying the CO2 concentration within the chamber air, to compen- sate for any change in A or gl.

The response of leaf gas exchange to light and to ci provide important indicators of causes underlying change in photosynthesis during acclimatisation to growth in elevated Ca. The following sections discuss these responses.

The light response of photosynthetic CO 2 assimilation

The response of A to photon flux (Q) describes a non-rectangular hyperbola of the form: A Q . ~b . . . . . + Asa t - [(~bm,ao p ' Q + Asat) 2 - 4~ . . . . . 'Asat" 0]0"5 R d. (3)

2~9

The only significant deviation from this model, seen in some leaves, is a transiently high slope (bA/bQ) below the light compensation point (LCP) termed the Kok effect. This is assumed to result from a progressive suppression of dark respiration. Beyond this point a linear response of A to Q persists between about 30 and 200 #mol m -2 s - l , which is considered to represent the maximum apparent quantum yield of CO2 uptake ((Pm,app; Bj0rkman & Demmig 1987; Long & Drake 1991). This linear region is followed by a progressive decline in the slope ( b A / b Q ) until a plateau is reached on which A is light-saturated (Asat). The convexity of the transition from light- limited to light-saturated photosynthesis is described by the coefficient 0 which may vary from 0-1. If the curve shows an ideal Blackman response, then 0 would be unity. In reality, 0 lies between these two extremes. The value of 0 is of significance in determining the extent to which changes in (~m,app may affect A at higher light levels and the extent to which Asa t may influence A below light saturation. 0 may decrease with increasing leaf thickness and may also decrease following photoinhibition of P S II (Leverenz e t al.

1990). In C3 plants, Asa t may be limited by the rate of diffusion of CO2 to the site ofrubisCO, the activity of rubisCO or/and the rate of regeneration of rubP. Light limited photosynthetic capacity, indicated by ~m,~pp is limited by the efficiency of the leaf in intercepting light and by the efficiency of transduction of the intercepted light in CO2 assimilation, this in turn is primarily limited by efficiency of rubP regeneration in low light and the proportion of that rubP utilised in CO2 assimilation. The following sections examine how these limitations to light-saturated and light- limited photosynthesis may be partitioned.

The maximum quantum yield of photosynthesis

Quantum yield (•) is the efficiency of light utilisation in photosynthesis, i.e. the number of moles of CO2 fixed per mole quanta absorbed by a leaf. Since the response of A to Q is hyperbolic, the maximum quantum yield can only be measured at low Q, when photosynthesis is strictly light- limited and proportional to Q (Fig. 1). In practice this is measured at quantum fluxes from ca. 20 to 150/~mol m -2 s-1 (Long & Drake 1991; Baker & Ort 1992).

The initial slope of the light response curve may be described as the apparent maximum quantum yield (q~m,~pp). The qualification 'apparent' is important since the estimate is based on incident, not on absorbed light; if reflected and transmitted

. ........ - 20

,~ 10

"Kok effect"

- 1 0 . . . . . , . . . . . . . . . . . . . 500 1000 1500

Q (Nmol rn z fit)

Fig. 1. An idealised response of CO 2 assimilation rate (A) to photon flux density (Q), describing a non-rectangular hyperbola (Eqn. 3)

2000

37

light are taken into account, the absolute maximum quantum yield (~Prn) may be obtained. ~Pm may be determined with a transparent leaf chamber incorporated into an Ulbricht integrating sphere (Oquist et al. 1978; Ireland et al. 1989; Long & Drake 1991). This allows the simultaneous measurement of light and CO2 absorption by the leaf.

To determine quantum yield within an integrating sphere leaf chamber the rates of CO2 and photon absorption must be determined. For each level of photon flux (Qi) the rate of CO2 uptake Jo,i may be determined as:

J c,i = (% - Co)'U. (4)

Where ce and Co are the concentrations of CO2 at the chamber inlet and outlet, respectively, (#mol tool - ~ ) and u is the flow of air through the chamber (mol s- 1).

To determine the rate of photon absorption (JQ) the absorptance (as) and the internal surface area (Ss) of the empty sphere are required. This is determined by measuring the photon flux at the wall of the sphere first when it is empty (Qempty)

and then on addition of a black absorber (Qab- sorber) of known absorptance (aa) and surface area (Sa):

as = 0~a" Sa/[ss(Oempty - Qabsorber)]" (5)

Absorptance by a leaf (al) of known surface area (Sl) is similarly determined from decrease in photon flux on the sphere walls:

a 1 = as[ss(Qempty - Qleaf)]/Sa. (6 )

The rate of photon absorption by the leaf, JQ,i ~mol s- l ) , when enclosed within the sphere is then given at any photon flux by:

JQ,i - Ss "as" (Qempty,i - Qleaf, i) • (7)

~Pabs is obtained by determination of the slope of the response of the rate of CO2 uptake by the leaf Jo,i (#tool s- 1) to JQ,i by finding the best-fitting straight line with the least-squares method and the linear regression model:

Jc,i = Jc,o q" (Pm,abs "JQ, i . (8)

Where Jc,o is the rate of uptake at Q = 0.

38

Most gas exchange studies have been con- cerned with light- saturated rates of CO2 assimilation (Asat ) , perhaps because in most C3 crops assimilation is saturated by light levels well below full sunlight. However, in the field situation and particularly in canopies with a large leaf area index (LAI), many leaves may experience light-limiting conditions, since even in full sunlight some leaves will be shaded by others. The significance of this may be seen in measurements of CO2 assimilation by whole stands. Unlike single leaves, stands of plants show a much longer phase in which CO2 assimilation responds linearly to increase in Q (Long 1985). In mature canopies, assimilation may be influenced as much by q~m,app as by Asa t

(Long 1985; 1989). Thus understanding acclimatisation in tpm,app is also important.

Known mechanisms of acclimatisation of photosynthesis to elevated CO2 concentration, primarily changed activity of rubisCO, should not affect ~bm, since photosynthesis during the initial linear phase of the light response curve is limited by the rate of regeneration of rubP and not by rubisCO activity. Increase in Ca should increase ~m since decreased oxygenation ofrubP will mean that an increased proportion of rubP will be available for carboxylation (Stitt 1991).

Following the parameters and equations of the model of Farquhar et al. (1980), a doubling of Ca

0.10

0.08

0.08

0.04

0.02

0.00 10

; . . ' 7 . . . ' 7 . . . - - - - - - _

700 ~ ~ ' ~ " . . . 500 . . . . . ~ .

20 30 40

Leaf t e m p e r a t u r e (*C)

Fig. 2. The predicted response of the maximum quantum yield of CO 2 uptake (~b) to leaf temperature at three atmospheric CO 2 concentrations (Ca; after Long, 1991). Points indicate mean ~p ( + 1 se) for plants of Scirpus olneyi grown for 3 years at a c a of 350 #mol mol-1 (closed symbol) and 680 /~mol mol- 1 (open symbol), data from Long & Drake (1991).

from 350 #mol mol- ~ to 700/~mol mol- ~ should result in a ca. 25 % increase in tp. Long & Drake (1991) observed that q~m increased by ca. 22~o, in plants of Scirpus olneyi grown in 680/~mol mol- 1 for 3 years and measured in 680 #mol mol- 1, in comparison to controls grown and measured in 350 #mol mol - 1 (Fig. 2). Measurement of q~ in an integrating sphere chamber allows separation of any effect of acclimatisation on the maximum efficiency of energy transduction in CO2 assimilation from change in the absorptance of the tissue (~tL), since the apparent maximum quantum yield (tPm,app) will be given by:

~m,app = (Prn ' (~s" (9)

Analysis of light-saturated photosynthesis

Above the initial slope of the A/Q response, photosynthesis may be co-limited by three processes: (i) the delivery of CO2 to the site of carboxylation; (ii) carboxylation efficiency; and (iii) regeneration of rubP. The response of A to ci provides an in vivo method of separating the quantitative contri- bution of these three processes to any change in msat.

Separation of stomatal from mesophyll limitations

Leaf conductance (gi) will typically decrease in plants on exposure to elevated Ca, due to partial closure of the stomata (Eamus 1991). This does not necessarily mean that stomata will be an increased limitation on photosynthesis. Farquhar & Sharkey (1982) developed a simple method of separating stomatal and mesophyll limitations using the A/c i response. Assimilation rate (A), measured at the normal atmospheric COz concentration (ca = 354/~mol mol- 1), is subtracted from A o, the rate which would occur if there were no stomatal or other gas phase diffusive limitation interpolated from the A/c i response curve at ci = 350 #mol mol- 1)(see Fig. 3). The relative limitation (1) which the stomata impose, may then be calculated:

l= (Ao - A)/Ao. (10)

39

rubP rubP 40 [ saturat,d. I . . . . . . . l i m i t e d . . . . .

7~ o. 30 - / .

/ oo ' ~ "" t i o n

<

0 250 500 7 5 0 1000

ci (/~mol tool -I)

Fig. 3. Agcncralised response ofAto ci, indicating the points used in the calculation of stomatal limitation (l, eqn. 10).

Where A indicates the photosynthetic rate with a c a of 354

/~mol tool- i and A o the rate with a c i of 354/~mol tool- 1

This method (Eqn. 10) has the advantage, when calculated graphically, that it makes no assump- tions regarding the shape of the response of A to ci. If an increase in stomatal limitations is the cause of a reduction in A, then 1 (Eqn. 10) must increase accordingly; if on the other hand an increase in limitations within the mesophyll domi- nates then a smaller or zero change in 1 might be expected. If the shape of the A/ci response curve is unchanged by the treatment, then increased stomatal limitation is also indicated by a decrease in ci. Examination of many A/c~ response curves shows that the operating c~ (i.e. the value of ci obtained when Ca is 354/~mo1 mol- 1, the current atmospheric average) is often maintained at a point close to the inflection in the response (Fig- ure 3; e.g. Stitt 1991). At a vapour pressure def- icit of 2 kPa, the ratio of Ci/C a in C3 species has been observed to be close to 0.7 (Wong et al. 1979). Because A describes a hyperbolic response to Ca, if Ca is increased from 350 #mol tool- 1 to 700 #mol mol- 1 gl must decrease to maintain a Ci/C a a t 0.7. Thus, elevation of ci by increase in Ca will move the operating point along the A / c i re-

s p o n s e away from the point of inflection so that 1 will decrease. Thus a common effect of rising Ca is a decrease in stomatal conductance coupled with a decrease in the limitation imposed on photosynthesis by stomata.

Separation of limitations due to carboxylation and to capacity for regeneration of rubP

Acclimation to elevated ca has been commonly, but not invariably, shown to involve" a decrease in rubisCO activity (reviewed: Bowes 1991; Long & Drake 1992). Another factor leading to acclimatisation may be an increase in leaf carbohydrate levels leading to an end-product inhibition of A (Stitt 1991). Analysis of the A/ci response and coupled with measurement of Asa t in varying 02 partial pressures allows separation of these effects. From a steady-state model of photosynthetic carbon metabolism, it is suggested that the A/ci response consists of two phases, an initial linear response where the efficiency of carboxylation (i.e. amount of active rubisCO) determines the slope bA/bci, followed by an inflection to a slower rise where A is limited by the supply of rubP for carboxylation (Farquhar et al. 1980; Fig. 3). Following the model of Farquhar et al. (1980) and subsequent modifications (Harley et al. 1992) it may be stated that A is related to ci in the following manner:

A = [1 0.5_o!l.min {Wc,Wj,Wp}- Rd. z'ci A

(11)

Where, Wc is assumed to obey Michaelis- Menten kinetics:

V c , m a x " Ci Wc = (12)

ci + kc (1 + oi/ko) "

At low values of ci, A will be limited by Wc (Eqn. 11; Fig. 3), since there will be an excess of capacity for regeneration of rubP. Since, the affin- ities of rubisCO for CO2 and 02 are considered to vary little between terrestrial C3 species, the main determinant of A at low ci will be Vc,ma x which will be directly dependent on the quantity of active rubisCO in vivo. The initial slope of the A/ci response, or carboxylation efficiency (ce), therefore provides an in vivo measure of the activity of rubisCO (mol m -2 s - t ) . Studies have shown a close agreement between the initial slope of the A / c i curve, or ce, predicted by the method

40

of Farquhar et al. (1980) and the extractable activity of rubisCO (Long 1985).

Beyond the inflection of the A / c i response (Fig. 3) A is limited by the capacity of the leaf to regenerate rubP for carboxylation. Two factors are thought to limit A at these higher ci values. 1. Wj, the potential rate of non-cyclic electron transport, which is a function of the photon flux, size of the apparatus for light capture, efficiency of energy transduction on the photosynthetic mem- brane; 2. Wp, the rate of regeneration of rubP that may be supported by the available inorganic phosphate. Wp may be of importance under conditions which lead to an accumulation of soluble carbohydrates in the leaf(Harley et aL 1991). The upper portion of the A/ci response therefore provides an in vivo measure of the maximum capacity of the leaf to regenerate rubP. Beyond the inflection of the A/ci response an increase in A with further increase in ci continues, but on a lower gradient. This continued increase occurs, because the proportion of carbon lost in photorespiration will continue to decline, as predicted by eqn. 11. Thus even though limited by the supply of rubP, an increasing proportion of this rubP will be used in carboxylation rather than oxygenation. If A is limited by Wp, rather than Wj, this will be apparent in two ways: (i) bA/bci will be zero beyond the inflection of the curve and (ii) A will not increase with inhibition of photorespiration on decreasing the atmospheric O2 concentration to 10 mmol mol-1 (Sharkey & Vander- veer 1989).

Changes in the A/ci curve with rising atmospheric C02 concentrations

Decline in carboxylation efficiency, i.e. the initial slope of the A/ci response represents a common, but not a universal, feature of acclimatisation of the photosynthetic apparatus to elevated CO2 concentration (Stitt 1991; Long & Drake 1992). Why should such a decrease occur? For a wide range of C3 species it has been found that the ci which they attain under current atmospheric CO2 concentrations is close to the transition between

Wo and Wj/Wp limited photosynthesis, i.e. at the point where the quantity of active rubisCO and the capacity for regeneration of rubP are co- limiting (reviewed: Long 1985; Fig. 3). An envi- ronmentally induced change in capacity for carboxylation of rubP appears often to be matched by change in the capacity for rubP regeneration, and vice versa, such that the two processes re- main co-limiting (reviewed Long & Drake 1992). This suggests an optimisation of the distribution of resources within the chloroplast so that neither active rubisCO nor the apparatus for regeneration of rubP are in excess. If optimisation of the distribution of resources between components of the photosynthetic apparatus is a ubiquitous phenomenon, then adjustment must also be expected in plants grown in elevated Ca which will increase carboxylation efficiency. Given the large invest- ment of energy and nitrogen in rubisCO, up to 25~ of leaf nitrogen, strong selective pressures for adjustment of carboxylase levels are likely.

5 0 . "'..." i'.. e - ' - V e , . . ~ - 8 0 Z

i ~ .. / / , . . . . ....... _ _ ,i.ma x + 66z~ I 4 0

• .. / ' . , ' .

3 0 : ~

.,¢ tO /" ': """ / ' ":, , . . . '"i]..:...

~ 0 0 4 0 0 8 0 0 8 0 0 1 0 0 0

c I (/J, m o l t oo l -1 )

Fig. 4. The simulated response of light-saturated rates of leaf CO 2 uptake (msat) with intercellular CO2 concentration (ci) calculated from the equations and parameters of Long (1991). The solid line indicates the curve based on the parameters of a plant grown at 350 #tool mol- 1 and is typical of the response observed in many C 3 plants grown at current CO2 concentrations. Arrows indicate the operating points, i.e. the ci obtained for a given c a. The dotted lines joining c i and c a on the three curves, indicate the supply function. The broken lines illustrate two potential patterns of acclimatisation to growth in elevated CO 2. The lower line indicates the result of a 30% decrease in V . . . . a n d Womax, simulating a loss of rubisCO activity and the upper line a 65% increase in J . . . . simulating an increase in the maximum capacity for regeneration of rubP.

Fig. 4 illustrates how this might occur. Without any adjustment of the photosynthetic apparatus the A/ci curve would be unchanged. Considering a typical A/ci curve for a leaf developed at current CO2 concentrations (based on the parameters of Long 1991), the operating ci when Ca = 350/~mol mol-~ will be at the point of inflection of the curve (point 'a' on Fig. 3), assuming Ci/Ca = 0.7. If Ca is nOW doubled, ci moves to 490/~mol mol - ~, and is now on the upper portion of the A/ci response (point 'b' on Fig. 3). Here Asa t would be limited by regeneration ofrubP (Wj) and rubisCO activity will be in considerable excess. Acclima- tisation of the A/C i c u r v e so that the inflection of the curve moves to 490 /~mol mol-1 could be achieved in two ways (Fig. 4). Either the quantity of active rubisCO is decreased (Vcmax$), SO that Wc is decreased to equal Wj at ci = 490/~mol mol - ~, or the capacity for regeneration of rubP is increased (Jma×l"), SO that Wj is increased to equal W~ at ci = 490/~mol mol - 1 (point 'c', Fig. 3).

L e a f f luorescence and absorpt ion s p e c t r o s c o p y

Chlorophyll fluorescence and absorption spectroscopy have two applications in the in vivo analysis of acclimatisation of photosynthesis: (i)in the measurement of change in PSII efficiency, in par- ticular resulting from photoinhibition; and (ii) for the measurement of the oxidation states of the reaction centres ofphotosystems I and II, and the flow of electrons through these reaction centres.

Detecting photoinhibition

If the light absorbed by the photosynthetic pigments of a leaf exceeds that which may be dissipated through the normal channels of photochemistry (including photosynthesis), thermal deactivation and fluorescence, then an apparent impairment of photosynthetic efficiency, termed photoinhibition ensues. Photoinhibition is defined here as a light-dependent depression of photosynthetic capacity irrespective of the molecular mechanism• Photoinhibition is promoted when

41

other environmental factors are sub-optimal and inhibitory to CO2 assimilation and hence photochemical dissipation of absorbed light energy, e.g. low temperature, water stress and nitrogen supply (Powles 1984). However, transient photoinhibition may also be observed on days with clear sunshine when conditions are otherwise appar- ently optimal (Ogren & Sj6str6m 1990). Photo- inhibition, commonly characterised by a decrease in q~m has been shown to correlate with a decrease in the efficiency with which crops convert intercepted light into biomass (Farage & Long 1991).

Demonstrating a decrease in ~m in the field during photoinhibition is complicated by the light environment of most canopies, where duration and level of exposure to full sunlight will vary dynamically between leaves. A large number of measurements are therefore necessary to detect any statistically significant change. However, measurement of q~m with an integrating sphere will typically require ca. 15 min, severely limiting the number of samples which may be taken. Pho- toinhibitory decrease in q~m is often closely cor- related with a decrease in the maximum efficiency of photosystem II (q~sn,m) which may be measured rapidly in situ with a simple chlorophyll fluorimeter (Bolhfir-Nordenkampf et al. 1989).

q~PSII,m is calculated from the induction of chlorophyll fluorescence (Fig. 5). If leaves are placed

f a s t s l o w t.o / • / . _ _ F m

0.8

& o.e

O

0.4 o

0.2 M o

o.o , , / . . . . . . 010 .2 0.4 0.6 10 20 30 40 50 60

I Time (s)

Fig. 5. A typical induction curve of PSH chlorophyll fluorescence. F, fluorescence emission of PSII (relative units). Fo, minimal level of F upon illumination when PSII is maximally oxidised. Fn, indicates the maximal level (m peak) of fluorescence. F v is the difference between F m and F o.

42

in darkness the PSII reaction centres will be maximally oxidised, this is termed dark adaptation. The initial level of fluorescence is that which will occur when PSII centres are maximally oxidised; the Fo level. Beyond this point the absorbed energy closes PSII centres decreasing the possible routes of dissipation of excitation energy and increasing the level of fluorescence until a peak is reached (Fm), at which point all PSII centres are assumed to be closed. The difference between F o and Fm, is termed variable fluorescence (Fv). By consideration of dissipation pathways it may be shown (Butler & Kitijima 1975) that:

(])PSII,max = Fv/Fm • (13)

Fully portable fluorimeters are available commer- cially for this measurement in the field. These instruments include clips which may be used to dark-adapt tissue in the field prior to measurement (Bolhfir-Nordenkampfet al. 1989; Bingham & Long 1992). Once leaves have been dark- adapted the measurement of Fv/Fm requires only ca. 2s, with such instruments allowing examination of a large number of samples at regular in- tervals through a day. Two sources of error though need careful attention. Interpretation assumes that all active PSII centres are open at Fo and closed at F m. If the dark adaptation period is insufficient, F o will be overestimated. F m will be underestimated if the excitation photon flux is insufficient to close all centres before the flow of electrons from PSII to PSI exceeds the rate of PSII photochemistry. Leaves will vary in the periods of dark adaptation and excitation photon fluxes required. If photoinhibition occurs th, si ~- ,max will decline. It is unclear how rising Ca may affect the occurrence of photoinhibition. In- creased photosynthesis may increase the dissipation of absorbed energy possibly averting photoinhibition. Similarly, decreased transpiration may improve plant water status, avoiding inhibition of photosynthesis which may in turn facilitate photoinhibition (Ludlow & Powles 1989). However, end-product inhibition of photosynthesis, due to an accumulation of carbohydrates in the leaf, could favour photoinhibition.

Determining electron flux through PSII and PSI

By combining in vivo measurements of gas exchange with measurements of chlorophyll fluorescence and absorption spectroscopy, changes in the relationship of efficiency of light use in CO2 assimilation (~) and in electron transport (q~sii) may be determined (Genty et al. 1989; 1990). At current atmospheric CO2 concentrations significant quantities of energy are directed via electron transport into other photosynthetic pathways, primarily the photosynthetic carbon oxidation pathway (leading to photorespiration), nitrite reduction, glutamate synthesis, and superoxide for- mation. Rising Ca decreases the proportion of NADPH utilised in photorespiration, and possibly into other competing processes. This change in the ratio of CO 2 uptake to whole chain electron transport can be detected by concurrent in vivo measurement of fluorescence and CO2 uptake.

For this measurement a modulated fluorimeter is required. Here a weak modulated light is used to excite fluorescence and the amplifier is syn- chronised to discriminate the additional fluorescence resulting from the modulated light, from fluorescence generated by the flux of continuous light onto the leaf. In this way the measured fluorescence signal is unaffected by changes in the quantity of non-modulated light received by the leaf (t3gren & Baker 1985; Schreiber & Bilger 1987). As stated earlier, (]~PSII,max for dark- adapted tissue is given by the ratio Fv/Fm. Fol- lowing Fro, fluorescence is quenched by the induction of photosynthesis which increases dissipation of energy, and hence quenching of fluorescence; this is termed photochemical quenching (qQ). In addition, a second process, non- photochemical quenching (qNP), quenches the fluorescence signal further. Fluorescence typically declines to a steady-state level Fs which corre- sponds to the development of a steady-state rate of CO2 assimilation (Fig. 6). If a saturating pulse of light (10000 #mol m - 2 s - 1, 0.5s) is now added (Fig. 6), a second induction is seen, with fluorescence rising to a second and lower m peak (Fm). The height of Fm above Fs reflects the number of PSII centres that were open at the addition of the

i

l

Fluorescence emission

F V F ° s~AL(off) ~ iFR

Fig. 6. Experimental protocol for determination of qQ and ~t~sn, indicating fluorescence emission kinetics with the addition and subtraction of four light sources. ML weak modulated light, AL actinic light SP saturating light pulse, and FR far red light.

saturating light pulse, i.e. the quenching due to photochemical processes. F" may be determined once Fs is obtained, by removal of actinic light and addition of a far-red light to re-oxidise PSII by selectively exciting PSI (Fig. 6). qQ is determined by:

¢

Fm - Fs qQ = (14a)

F m - F~

= ( F m - F ~ ) / F , ) . (14b)

The efficiency of excitation energy capture by open PSII reaction centres is defined as F;/Fm, if it is assumed that non-photochemical quenching is unchanged by the saturating pulse. The efficiency with which excitation energy is used to drive PSII photochemistry is therefore given by the product of qQ and Fv/Fm:

(PPSII = (F~ - Fs)/F,~ x F'./Fm (15a)

= (Fro - Fs)/F~ (15b)

Application of this model has shown a close relationship of tpPSII, calculated in this manner, with the measured tp. If elevation of ca results in

43

an increased partitioning of NADPH into CO2 assimilation then ~ppsn/tp will decrease.

Effective utilisation of light in CO2 assimilation requires co-ordination of PSI and PSII activity. P700, the PSI reaction centre, in its oxidised state is a strong absorber of far red radiation at 820 nm. Changes in PSI absorption (A82o) may be measured by modulated absorption spectroscopy (Harbinson & Woodward 1987; Schreiber et al.

1988). On removal of white light from a leaf, A820 declines rapidly, reflecting the rapid re-oxidation of P700 in the dark. The relative proportion of non-oxidised P700 within the leaf at any point in time is given by 1-AA82o/AA82omax which defines the photochemical efficiency of PSI (tPPSi). The decrease in Aszo observed after 0.5-1s (AA82o) estimates the proportion of PT0o that was non- oxidised at steady state under the actinic light. The absorptance change corresponding to the maximal oxidation state of P700 may be estimated from extrapolation of AA82 o values, generated over a range of photon fluxes (Q), to infinite photon flux by plotting AA82o against the reciprocal of Q. Alternatively AA82omax may be estimated by addition of a strong far-red light to preferentially excite P700. Genty et al. (1990) have shown a linear relationship of q~PSii with q~PSi over a range of photon fluxes and oxygen partial pressures, suggesting close co-ordination of the flow of electrons through the two reaction centres. Such simultaneous measurements of tPpsi and tppsn will allow changes in the balance between non-cyclic and cyclic electron transport to be determined, assuming cyclic electron transport does occur in vivo (Baker 1991; Baker & Ort 1992). Such changes may occur in response to environmental changes as the metabolic requirements of leaves for ATP and reductants change (Baker & Ort 1992).

C o n c l u s i o n

Although the study of acclimatisation of photosynthesis to growth in elevated C O 2 c o n c e n t r a -

t i o n s will be limited by the availability of material for destructive analyses, much can now be de-

44

duced on the bases of acclimatisation of photosynthesis to elevated c a ill $itu. These methods provide the opportunity to study the bases of changes in both light-saturated and light-limited photosynthesis in the same leaves throughout their lives and without destroying the tissue. These in vivo measurements will also provide important guides of the key questions that will need to be addressed by in vitro methods and allow effective use of the limited quantities of material that may be destructively harvested from long-term studies of growth in elevated CO2 concentrations.

References

Arp W.J. & Drake B.G. 1991. Increased photosynthetic capacity of Scirpus olneyi after 4 years of exposure to elevated CO2. Plant Cell Environ. 14: 869-875.

Baker N.R. 1991. A possible role for photosystem II in environmental perturbation of photosynthesis. Physiol. Plant. 81: 563-570.

Baker N.R. & Ort D.R. 1992. Light and crop photosynthetic performance. In: N.R. Baker & H. Thomas (eds.), Crop Photosynthesis, Elsevier, Amsterdam (in press).

Bingham M.J. & Long S.P. 1992. Equipment for crop and environmental plant physiology research. In: Hall D.O., Scurlock J.M.O., Bolhfir-Nordenkampf H.R., Leegood R.C. & Long S.P. (eds.), Techniques in Photosynthesis and Bioproductivity, Chapman & Hall, London (in press).

Bj6rkman O. & Demmig B. 1987. Photon yield of 02 evolu- tion and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170: 489- 504.

Bowes G. 1991. Growth at elevated CO2: photosynthetic responses mediated through rubisCO. Plant Cell Environ. 14: 795-806.

Butler W.L. & Kitijima M. 1975. A tripartite model for chloroplast fluorescence, in Avron, M. (ed.) Proceedings of the 3rd International Congress on Photosynthesis, pp. 13-24, Elsevier, Amsterdam.

Coleman J.R. 1991. The molecular and biochemical analyses of CO2 concentrating mechanisms in cyanobacteria and microalgae. Plant Cell Environ. 14: 861-867.

Eamus D. 1991. The interaction of rising CO2 and temperature with water use efficiency. Plant Cell Environ. 14: 843- 852.

Farage P.K. & Long S.P. 1991. The occurrence of photoinhibition in an over-wintering crop of oil-seed rape (Brassi- ca napus L.) and its correlation with changes in crop growth. Planta 185: 279-286.

Farage P.K., Long S.P., Lechner E.G. & Baker N.R. 1991. The sequence of change within the photosynthetic appara-

tus of wheat following short-term exposure to ozone. Plant Physiol. 95: 529-535.

Farquhar G.D. & Sharkey T.D. 1982. Stomatal conductance and photosynthesis. Ann. Rev. Plant Physiol. 33: 317-345.

Farquhar G.D., Von Caemmerer S. & Berry J.A. 1980. A biochemical model of photosynthetic (CO2) assimilation in leaves of C3 species. Planta 149: 78-90.

Field C.B., Ball J.T. & Berry J.A. 1989. Photosynthesis: prin- ciples and field techniques. In: Pearcy J.W., Ehleringer J., Mooney H.A. & Rundel P.W. (eds), Plant Physiological Ecology: Field Methods and Instrumentation, pp. 208-253, Chapman & Hall, London.

Genty B., Briantais J.-M. & Baker N.R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Bio- chim. Biophys. Acta 990: 87-92.

Genty B., Harbinson J. & Baker N.R. 1990. Relative quantum efficiencies of the two photosystems of leaves in photorespiratory and non-photorespiratory conditions. Plant Physiol. Biochem. 28: 1-10.

Harbinson J. & Headley C.L. 1988. The kinetics of P700 reduction in leaves: a novel in situ probe of thylakoid func- tioning. Plant Cell Environ. 12: 357-369.

Harbinson J. & Woodward F.I. 1987. The use of light-induced absorbance changes at 820nm to monitor the oxidation state of P700. Plant Cell Env. 10: 131-140.

Harley P.C., Thomas R.B., Reynolds J.F. & Strain B.R. 1991. Modelling photosynthesis of cotton grown in elevated CO z. Plant Cell Environ. (in press).

Ireland C.R., Long S.P. & Baker N.R. 1989. An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO 2 and water vapour exchange, light adsorption and chlorophyll fluorescence of attached leaves. Plant Cell Environ. 12: 947-958.

Long S.P. 1985. Leaf gas exchange. In: Barber J. & Baker N.R. (eds), Mechanisms and the Environment, pp. 453- 500, Elsevier, Amsterdam.

Long S.P. 1989. Gas exchange of plants in the field. In: Grubb P.J. & Whittaker J.B. (eds), Toward a More Exact Ecol- ogy pp. 33-62, 30th Symposium of the British Ecological Society, Blackwell, Oxford.

Long S.P. 1991. Modification of the response of photosynthetic productivity to rising temperature by atmospheric CO2 concentrations: Has its importance been underestimated? Plant Cell Environ. 14: 729-739.

Long S.P. & Drake B.G. 1991. Effect of the long-term elevation of CO2 concentration in the field on the quantum yield of photosynthesis of the C 3 sedge, Scirpus olneyi. Plant Physiol. 96: 221-226.

Long S.P. & Drake B.G. 1992. Photosynthetic COa assimilation and rising atmospheric CO 2 concentrations. In: Baker N.R. & Thomas H. (eds.), Topics in Photosynthe- sis vol. 11, Elsevier, Amsterdam (in press).

Long S.P. & Hallgren J.-E. 1992. Photosynthetic gas exchange. In: Hall D.O, Scurlock J.M.O, BolhM-Norden- kampf S.R., Leegood R.C. & Long S.P. (eds.), Techniques

in Photosynthesis and Bioproductivity, Chapman & Hall, London (in press).

McMurtrie R.E. & Wang Y.-P. 1992. Mathematical models of the photosynthetic response of plant stands to rising CO2 levels and temperatures. Plant Cell Environ. (in press).

Mott, K.A. 1990. Sensing of atmospheric CO 2 by plants. Plant Cell Environ. 13: 731-737.

Ogren E. & Sj6str0m M. 1990. Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy. Planta 181: 560-567.

Ogren E. & Baker N.R. 1985. Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light. Plant Cell Environ. 8: 539-547.

Oquist G., Hallgren J.-E. & Brunes L. 1978. An apparatus for measuring photosynthetic quantum yields and quanta absorption spectra of intact plants. Plant Cell Environ. 1: 21-27.

Sage R.F., Sharkey T.D. & Seemann J.R. 1989. Acclimation

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of photosynthesis to elevated CO2 in five C 3 species. Plant Physiol. 89: 590-596.

Schreiber U. & B ilger W. 1987. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorimeter. Photosyn. Res. 10: 51-62.

Schreiber U., Klughammer C. & Neubauer C. 1988. Measur- ing P700 absorbance changes around 830 nm with a new type of pulse modulation system. Z. Naturforsch. 43c: 686- 698.

Sharkey T.D. & Vanderveer P.J. 1989. Stromal phosphate concentration is low during feedback-limited photosynthesis. Plant Physiol. 91: 679-684.

Stitt M. 1991. Rising CO2 levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ. 14: 741-762.

Wong S.C., Cowan I.R. & Farquhar G.D. 1979. Stomatal conductance correlates with photosynthetic capacity. Na- ture 282: 424-426.

Analysing the responses of photosynthetic CO2 assimilation to long-term elevation of atmospheric CO2 concentration

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