The Relationship between Electron Transport Components and ...

Aust. J. Plant Physiol., 1987, 14, 157-70

The Relationship between Electron Transport Components and Photosynthetic Capacity in Pea Leaves grown at Different Irradiances John R. Evans Division of Plant Industry, CSIRO, G.P.O. Box 1600, Canberra, A.C.T. 2601, Australia.

Abstract Pea plants (Pisum sativum) were grown under different irradiances and the photosynthetic characteristics and chloroplast properties were determined on young, fully expanded leaves. The light-saturated rate of oxygen evolution for leaf discs, measured in 1% C02, correlated closely with the uncoupled wholechain electron transport activity by the thylakoid membranes. When expressed on a chlorophyll basis, the photosynthetic rate was proportional to both the cytochrome f content and the coupling factor activity. The ratio of coupling factor activity to cytochrome f content was constant across all the growth irradiance treatments. The content of photosystem I1 reaction centres, determined by [14C]atrazine binding, varied to a small extent while the content of photosystem I reaction centres was unaltered by growth irradiance. Reaction centres do not seem to limit the rate of non-cyclic electron transport. Analysis of the C02 response curves suggested that the ratio of electron transport capacity to RuP2 carboxylation capacity was greater for plants grown at higher irradiances than for plants grown at lower irradiances. Within any irradiance treatment, the ratio of the two capacities was approximately constant. The proportion of leaf nitrogen allocated to thylakoid proteins (27%) was independent of growth irradiance. Adaptation to low irradiance was associated with a reduction in the electron transport components and an increase in the light-harvesting chlorophyll a / b protein such that the amount of chlorophyll per unit of thylakoid protein nitrogen increased. By contrast, adaptation to high irradiance was associated with an increase in the electron transport capacity per unit of chlorophyll such that the electron transport rate per unit of thylakoid nitrogen was increased.

Introduction Considerable interest has focused on the comparison of the photosynthetic properties of plants adapted to high and low light environments (Boardman 1977; Bjorkman 1981). However, while changes in the relative amounts of protein and partial reaction rates have been observed, clear mechanistic relationships between the proteins and reaction rates of photosynthesis are not usually evident. Adaptation to growth irradiance in terms of both the changes in the relative abundance of the pigment-protein complexes and partial reactions have been clearly demonstrated for pea leaves (Leong and Anderson 1984a, 1984b), providing a good experimental system with which to relate changes in protein components to both the electron transport and C02 assimilation capacities. For efficient use of the proteins involved, the capacity for RuP2 consumption by the Calvin cycle enzymes, primarily RuP2 carboxylase, should be matched by the ability to regenerate RuP2. The latter requires the production of NADPH and ATP. Analysis of the response of CO2 assimilation to intercellular CO2 partial pressure, pi, (Farquhar and Caemmerer 1982), allows these two capacities to be calculated for intact leaves. The 0310-7841,87 '020157$02.00

John R. Evans

rate of oxygen evolution, or Hill activity, can be calculated from the rate of CO2 assimilation at high irradiance and high pi assuming 4 electrons per oxygen (equation A9 in Caemmerer and Farquhar 1981). Leaf age, nitrogen nutrition (Caemmerer and Farquhar 1981, 1984; Evans 1983a, 1985, 1986a) and phosphorus nutrition (Brooks 1985) do not markedly alter the ratio of Hill activity to RuP2 carboxylase activity. This supports the argument that the two processes are balanced. Adaptation to low irradiance, however, may be associated with a change in this ratio. The rate limiting steps in steady-state non-cyclic electron flow are unknown. Consequently, the reaction centre densities of both photosystem I, (PS I), and photosystem 11, (PS 11), as well as the chrochrome f content were determined. The capacities for noncyclic electron flow and ATP synthesis should also be balanced because the demand for both NADPH and ATP for C02 assimilation is relatively constant. As this has not been demonstrated, coupling factor activity was also measured.

Materials and Methods For experiment 1, Pisum sativum cv. Kelvedon Wonder was grown in sterilised soil in a glasshouse with natural light supplemented by 400-W high pressure sodium lamps which extended the photoperiod t o 16 h. Light treatments were imposed by reducing the available light with shade cloth enclosures. The irradiances a t noon o n a sunny day were 1000, 130 and 40pmol PAR quanta m - 2 s - I . Net CO2 assimilation as a function of intercellular p(CO2) was measured in an open gas exchange system (Evans 1 9 8 6 ~ ) The . leaf temperature was maintained at 23°C with a leaf-to-air vapour pressure difference of 12 mbar and a n irradiance of 1100 pmol PAR quanta m - 2 s - I. At the completion of the CO2 response curve, the response to irradiance was determined with p, maintained close to 500 pbar. Rates of gas exchange and underlying biochemistry were calculated according to Caemmerer and Farquhar (1981). For experiment 2, Pisum sativum cv. Greenfeast was grown in a mixture of perlite and vermiculite in growth cabinets with VHO fluorescent tubes (Philips 140'33RS). Irradiances were varied using different numbers of tubes and shade cloth to give 540, 300, 90, 45 and 20 pmol PAR quanta m - 2 s - '. The daylength was 16 h with a constant day /night temperature of 20°C, chosen so as to match the conditions used by Leong and Anderson (1984a, 1984b). Each morning, the plants were given complete nutrient solution and were then watered each afternoon. Oxygen evolution as a function of irradiance was determined in a leaf-disc oxygen electrode (Delieu and Walker 1981) (Hansatech, King's Lynn, U.K.) at 25°C with 21% 0 2 and 1 % COz from a 1 M carbonatelbicarbonate buffer solution at p H 9. The proportion of the white light absorbed by the leaf disc was subsequently measured using an integrating sphere; it averaged 88% and was independent of growth irradiance. The irradiance response curves were fitted by the following equation:

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J~0 - J(Zo

+ Jmax) + l o J m a x = 0 ,

(1)

where J is the rate of electron transport, Jmax is the maximum rate of whole chain electron transport, 0 is a convexity term and Zo=Z(l - n / 2 is the useful light absorbed by P S I1 where Zis the light absorbed by the leaf and f is a term to correct for the spectral distribution of light (-0.15 for quartz iodide, Evans 1987). Hill activity is simply J , 4, whereas the rate of CO2 assimilation depends o n p , (for p, = 500, A=J/4~0.8). The top three unfolded leaflets from each plant were collected and homogenised at 5°C in an Omnimixer for 15 s. The isolation medium contained 50 mM MES-NaOH (pH 6.5), 5 mM MgC12, 10 mM NaC1, 2 r n M EDTA and 400 mM sorbitol. The homogenate was filtered through four layers of Miracloth and centrifuged at 1000 g for 5 min. The pellet was resuspended in the isolation medium, with 100 mM instead of 400 mM sorbitol, and centrifuged at 1000 g for 5 min. The pellet was resuspended in a small volume of the resuspension buffer and the preparation was kept on ice. Chlorophyll content was determined according to Arnon (1949). Uncoupled whole-chain electron transport from H z 0 to methyl viologen (MV) was determined in triplicate with a n oxygen electrode (Hansatech) at 2 5 T with a chlorophyll concentration of 33 p ~ The . reaction medium contained 50 mM HEPES-NaOH (pH 7.8), 5 mM MgC12, 10 mM NaC1, 1 mM EDTA, 100 mM sorbitol, 0 . 1 mM MV, 1 . 5 mM NH4C1 and 1 mM NaN3. The remaining thylakoids were frozen in liquid nitrogen for later determinations of atrazine binding, P700, cytochrome f, coupling factor activity and nitrogen content.

Electron Transport Components and Photosynthesis

Atrazine binding was assayed according to Tischer and Strotmann (1977). Thylakoids (55 nmol Chl) were mixed with atrazine in 1 ml of 25 mM Tricine-NaOH (pH 8.0), 50 mM NaCl and 5 mM MgC12. The concentration of atrazine was varied from 50 to 500 nM by adding different amounts of 10 p~ (in 10% ~ Bq mol-'). ethanol, water, v , v) [14c]atrazine obtained from Amersham (specific activity 5 . 9 2 10" The mixtures were centrifuged (Eppendorf 5414) for 2 min to pellet the membranes and 900 p1 of the supernatant liquid were removed for counting. Assays were run in duplicate and membrane-bound counts were assumed to be equal to the difference between the counts in the supernatant with and without thylakoid membranes. In this laboratory, atrazine binding has been found to give the same PS I1 content as that determined by oxygen yield per flash for both spinach and pea (Chow and Hope 1987). P ~ Ocontent O was calculated from the absorbance changes at 702 nm induced by flashes of blue-green light (Corning 4-72, 450pnol quanta m - 2 s - ' ) after correcting for the fluorescence, according to Haehnel et a/. (1980). Thylakoids (33 p~ Chl) were suspended in 50 mM TES-NaOH (pH 7 . 9 , 10 mM NaCI, 5 mM MgC12, 400 mM sucrose, 1 mM sodium ascorbate, 0.1 mM MV, 5 p~ DCPIP, 2 p~ DCMU and 0.05% nonidet. The signal from 10 flashes were summed to improve the signal-to-noise ratio and was used (Hiyama and Ke 1972). Cytochrome f was estian extinction coefficient of 64 m ~ - cm' mated from hydroquinol-reduced minus ferricyanide-oxidised difference spectra according to Bendall et al. (1971). For both P ~ Oand O cytochrome f , assays were conducted in triplicate. The ~g~ -specific ATPase activity of the thylakoids (1 1 p~ Chl) was assayed in the presence of octyl glucopyranoside according to Pick and Bassilian (1981). The reaction medium contained 5 0 m ~ Tricine-NaOH (pH 8.0), 40 mM octyl glucoside, 2 mM MgClz and 0.1 mM EDTA. After preincubation of the samples for 5 min at 3 7 T , the reaction was started by the addition of ATP (final concentration 4 mM) and stopped after 60 s with 10% trichloroacetic acid (TCA). Samples were run in triplicate and included a blank without thylakoids to determine the phosphate released from the ATP by the TCA. Inorganic phosphate was assayed according to Ames (1966). The nitrogen contents of the leaves and thylakoid preparations were determined by Kjeldahl digestion.

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Results The photosynthetic capacity of pea leaves declined when adapted to low irradiance. The C02 response curves (expt 1, Fig. 1A) show that both the mesophyll conductance (the slope of the A :pi response curve near the CO2-compensation point) and the Hill activity (proportional to the rate of C 0 2 assimilation at high pi) were reduced. On average, for plants adapted to high irradiance the ratio of intercellular to ambient p(C02), pilp,, at the standard initial conditions was 0 . 7 1 5 0.01 compared with 0.82+0.01 for the two low irradiance treatments. Within any irradiance treatment, the ratio of Hill activity to mesophyll conductance was approximately constant, but the ratio was considerably higher for plants grown at 1000 pmol quanta m - 2 s - (Fig. 1B). The latter is readily apparent when the CO2 response curves for leaves from the 1000 and 130 pmol quanta m - 2 s - ' treatments with the same mesophyll conductance are compared (Fig. 2). It suggests that the capacity for electron transport is reduced more than that of the Calvin cycle during adaptation to low irradiance. The irradiance response curves were similar whether determined by 0 2 evolution with growth-cabinet material (expt 2, Fig. 3A) or by C02 assimilation with glasshouse-grown material (expt 1, Fig. 38). The scale in Fig. 3B has been increased to make the two figures equivalent since the rate of CO2 assimilation with pi = 500 pbar represents 80% of the oxygen evolution rate required to sustain it (i.e. 20% of the RuP2 is consumed by photorespiration). The convexity term 0 was independent of both growth irradiance and method of measurement. A maximum photosynthetic capacity of -35 pmol 0 2 m - 2 s - ' was apparent for pea leaves. Increasing the growth irradiance from 285 to 500 pmol quanta m - 2 s - ' in the cabinet made no difference to the maximum rate per unit leaf area, although it was associated with considerable starch accumulation, and the rate was similar to that observed for glasshouse material grown at high irradiance. For expt 2, the constant irradiance for 16 h in the growth cabinet meant that plants

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John R. Evans

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200

400

lntercellular p(C0,) ( p b a r )

0

0.05

0.10

0.1 5

Mesophyll conductance ( m o l CO,rn-Z s-' bar-')

Fig. 1. A . Rate of CO2 assimilation versus intercellular p(CO2), pi for glasshousegrown peas. Data from two replicate leaves are shown for each growth irradiance. A A , 1000. 0 H , 130. 0 .,43 (pnol quanta m - s - noon irradiance). Arrows indicate points measured at the initial standard conditions with ambient l m-2 s p(CO2) = 340 pbar, 21 % O r , 1100 ~ m o quanta B. Calculated Hill activity versus mesophyll conductance from the A :pi curves. H = 7 . 8 1 + 276 g,, r 2 = 0.967. -----------Symbols as in 1A. H=272g,-0.7, r2=0.968.

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Fig. 2. Rate of CO2 assimilation versus intercellularp(C02) for two replicates from high and intermediate growth irradiance treatments with similar mesophyll conductances. Symbols as in Fig. 1A.

lntercellular p(C0,) ( p b a r )

grown at 43 pmol quanta m - 2 s - ' had higher photosynthetic capacities than the equivalent glasshouse-grown material. The capacity for oxygen evolution per unit of chlorophyll measured with the intact leaf closely matched the uncoupled rate of whole-chain electron transport (Fig. 4A). It is assumed that, in 1% CO;? and saturating irradiance, the rate of oxygen evolution reflects the electron transport capacity and can be related to the components of the electron transport chain. Both cytochrome f content and coupling factor (CF) activity (Figs 4B, 4C) were proportional to the capacity for oxygen evolution. This indicates that the capacities for both non-cyclic electron transport and ATP regeneration are well coordinated. The two capacities must be balanced because the turnover times appeared to be constant and independent of photosynthetic capacity and the ratio of CF activity to cytochrome f content was constant. By contrast, the content of the photosystem


Absorbed irradiance ( p rnol PAR quanta m-2 s-')

Fig. 3. A . Rate of 0 2 evolution from leaf discs versus absorbed irradiance measured at 21% 02, 1% CO2. Plants were grown in cabinets. Curves are fitted by equation (1) as follows: 0 500 (growth irradiance, pmol quanta m - s - '), 180 (Jmax,pmol electrons m - 2 S-'1, 0.77 (8); A 285, 184, 0.77; W 80, 115, 0.77; 43, 87, 0.71, respectively. Each curve is the mean of 14 leaves. B. Rate of CO2 assimilation versus absorbed irradiance measured at 21% 02, 500 pbar p,. Plants were grown in the glasshouse. Curves fitted as for Fig. 3A as follows: A 1000, 182, 0.74; W 130, 71, 0.74; 0 40, 45, 0.74. Each curve is the mean of six leaves. Arrows indicate growth irradiance at noon.

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Fig. 4. Rate of oxygen evolution from leaf discs versus: A. Whole-chain electron transport, H z 0 + MV ( + NHdC1) (mmol02 mol- Chl s - '). A , 500 (growth irradiance, pmol quanta m - 2 S - '1. 0 , 2 8 5 ; 0, 80; 0 , 43; 0, 20. A , glasshouse, full sunlight. y=3.09+1.03x, r2=0.77. B. Cytochrome f content (mmol f molChl). y = 1.89+34.3x, r 2 = 0 . 7 1 . C. Coupling factor activity (mmol Pi mol-' ~ h l s - ' ) y=0.174x-5.08, . r2=0.89. D. Atrazine binding sites, Q (mmol Q mol- Chl). y=29.Ox- 15.2, r2=0.446. E. P700 content (mmol P700 moI- chi). F. Total leaf nitrogen (mol N mol- ' Chl). y=0.516x-44.8, r 2 = 0 . 6 4 .

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John R. Evans

reaction centres per unit of chlorophyll was poorly correlated with photosynthetic capacity (Figs 4 0 , 4E). While P700 content was insensitive to growth irradiance, the content of atrazine binding sites varied by about 40Yo compared to a 70% change in photosynthetic capacity. The content of atrazine binding sites was proportional to the chlorophyll a / b ratio (data not shown) such that low contents were associated with low chlorophyll a / b ratios. The latter reflects an increase in the relative amount of lightharvesting chlorophyll a / b protein. An analysis of the nitrogen cost of the thylakoid membranes is presented in Table 1 . The nitrogen costs have been revised from the initial estimates by Evans (1983b), but still contain a considerable element of uncertainty. It is assumed that protein is 16% nitrogen by weight. While the molecular weights of the proteins are becoming accurately known from the DNA sequences, the amount of chlorophyll and carotenoids bound to each is still uncertain. Table 1. The nitrogen cost calculated for components in the chloroplast thylakoid membranes Component

1A

2B 1x 2 Low irradianceD

3' 1x3 High irradianceD

Light-harvesting Chl a/b complex Photosystem I1 Photosystem I Chlorophyll N-cost for light-harvesting and reaction centres (mol N mol- Chl) Electron transport and coupling factor N-cost per unit of light-harvesting (mol N mol - Chl) N-cost per unit of electron transport (mol N mmol- c y t f )

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Calculated proportion of chlorophyll in each A N-cost (mol N mol-' Chl) derived in the text. Proportion of chlorophyll in each complex (Leong and Anderson complex, see text. Fmmol cyt 1984b). Chlorophyll a/b ratio 2.5 (low), 2.9 (high). mol N mmol-I cyt f. f mol-' Chl.

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For the light-harvesting chlorophyll a / b protein, 11 chlorophylls are bound (Mullet 1983) per 24.5 kDa polypeptide (Coruzzi et al. 1983), which corresponds to 25.5 mol N mol-' Chl. For the PS I1 complex, the total molecular weight obtained using the DNA sequences where available (Murphy 1986; Oh-Oka et a[. 1986) is 3 17.8 kDa. From discontinuous SDS-PAGE, the PS 11 complex contains about 126 Chl per 1000 Chl (Leong and Anderson 1 9 8 4 ~ ) .As these leaves contain 2.44 PS I1 reaction centres per 1000 Chl (Fig. 4D), this represents 52 Chl per PS I1 complex and 69.8 mol N molChl. For PS I, there are two core polypeptides, 83.2 and 82.5 kDa, each binding about 20 Chl and an 18 and 15 kDa polypeptide (Murphy 1986). Again from gel electrophoresis, the PS I complexes CPI and CPIa contain about 308 Chl per 1000 Chl (Leong and Anderson 1984a). There are 1.84 PS I reaction centres per 1000 Chl (Fig. 4 E ) , giving 167 Chl per PS I complex. Given the 40 Chl bound to the core polypeptides, 127 Chl are bound by the light-harvesting chlorophyll a / b protein of photosystem I (LHCI). If we assume that there are 18 LHCI monomers per photosystem I reaction centre, this means that each 22 kDa monomer binds 7 Chl. In all, 167 Chl are bound per 594.7 kDa, giving 40.9 mol N mol- Chl. For simplicity it will be assumed that the cytochrome b / f complex, plastocyanin, ferredoxin-NADP-reductase (FNR) and coupling factor all vary in parallel (see Fig. 4 ) .

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The nitrogen cost for the electron transport components and coupling factor can then be expressed per mmol of cytochrome f . The approximate molecular weights for the cytochrome b / f complex, FNR and the coupling factor are 137, 37 and 500 kDa (Anderson 1986; Murphy 1986) and their relative abundances are approximately 1 : 1.05 : 0.71 (Strotmann et al. 1974; Bohme 1977; Berzborn et al. 1981; Hurt and Hauska 1981). This gives 6.07 mol N mmol- cyt f , to which must be added 0 . 2 mol N for plastocyanin and ferredoxin. Having established the approximate nitrogen costs for the chlorophyll-protein complexes, electron transport chain and coupling factor, it is necessary to calculate the distribution of chlorophyll between the complexes. From Leong and Anderson (1984a), the proportions of chlorophyll in the light-harvesting complex and the P S I1 and I complexes for a plant grown under high irradiance with a chlorophyll a / b ratio of 2.9 were 0.566, 0.126 and 0.308, respectively. At low irradiance, there was a 20% reduction in the number of atrazine binding sites (Fig. 4 0 ) so that the proportion of chlorophyll associated with the reaction centre of PS I1 declines to 0.101. This chlorophyll a is reallocated to the light-harvesting complex and, assuming a chlorophyll a / b ratio of 1 12 (Anderson et al. 1978), means that the proportion of chlorophyll in the light-harvesting complex increases to 0.614 and the chlorophyll a / b ratio declines to 2.45. This is close to that actually observed (2.5, Table 1 ) . By difference, the proportion of chlorophyll in PS I declines slightly to 0.285. The calculated distribution of chlorophyll between the light-harvesting complex, and the PS I1 and I complexes of 0.614, 0.101 and 0.285, compare favourably with that observed by Leong and Anderson (1984a) of 0.62, 0.11 and 0.27, respectively. The lower chlorophyll a / b ratio associated with adaptation to low irradiance reduced the light-harvesting nitrogen cost by - 4% (Table 1). Including the electron transport components and coupling factor meant a reduction of 11%. For adaptation to high irradiance, increasing the coupling factor and the cytochrome b / f complex reduces the nitrogen cost in terms of electron transport capacity by 30%. At higher growth irradiances, increased electron transport capacity was associated with an increased nitrogen content (Fig. 4F). This reflects not only the extra protein invested at high irradiance into electron transport components such as the cytochrome b/f complex and coupling factor, but the Calvin cycle enzymes as well. The amount of leaf nitrogen partitioned into the thylakoid membranes remained surprisingly constant at 27% while the capacity for electron transport varied over 3-fold. The nitrogen cost for electron transport is analysed in Fig. 5. Since electron transport capacity was directly proportional t o the cytochrome f content (Fig. 4 B ) , the number of moles of thylakoid nitrogen per cytochrome f represent the nitrogen cost for electron transport. Each point was the measured nitrogen content for the thylakoid preparation from plants grown at different irradiances. Also included are some data for spinach grown in a glasshouse in full sunlight. The solid curve is not a fit of the data but represents the nitrogen cost predicted from the dashed lines derived in Table 1 .

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Discussion RuP2 Regeneration Relative to RuP2 Carboxylation Capacity Using the analysis of Farquhar and Caemmerer (1982) to interpret CO2 response curves, it has been apparent that peas adapting to their growth irradiance alter the balance between the capacity for RuP2 regeneration and RuP2 carboxylation (Figs 1 , 2). Plants adapted to high irradiance had more Hill activity relative to RuP2 carboxylation capacity. Within any treatment, however, the ratio of the two capacities remained nearly constant. A similar response can be inferred for the rainforest tree, Agathis, where plants grown in full sun had relatively greater Hill activity (Langenheim et a/. 1984). For shade-grown clones of Solanurn dulcarnara, one out of three also showed

John R. Evans

mmol f mol-' chi Fig. 5. Nitrogen cost for electron transport (mol thylakoid N mmol-I cytochrome f ) versus cytochrome f content. Pea. A Spinach. Lines are derived in Table 1. (1) Electron transport components and coupling factor. (2) Light-harvesting and reaction centres. (3) = (1) (2). The solid line is (3) divided by the cytochrome f content.

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a similar response when transferred to high irradiance, the other clones showing no change in the ratios of the two capacities (Ferrar and Osmond 1986). Wheat plants subjected to similar treatments as the peas in this experiment had reduced photosynthetic capacity, but the ratio of the Hill activity to mesophyll conductance remained constant (Evans, unpublished data). For beans, the results are conflicting. Caemmerer and Farquhar (1981, 1984) observed that the ratio of Hill activity to mesophyll conductance increased during adaptation to reduced irradiance whereas Seemann, Sharkey and Osmond (unpublished data) found little change. One possible cause for these species differences is that photoinhibition may have taken place as at least 1 h at half of full svnlight was required in order to measure the C02 response curve for the plants adapted to low irradiance. The adaptation by peas is therefore not a general phenomenon with some species showing the reverse while others show no change in the ratio of Hill activity to mesophyll conductance. By contrast, leaf age (Evans 1983a, 1986a), nitrogen stress (Caemmerer and Farquhar 1981; Evans 1983a, 1985) and phosphorus stress (Brooks 1985), do not alter this ratio. The increase in mesophyll conductance relative to Hill activity during adaptation to low irradiance was accompanied in peas by an increased stomata1 conductance such that the intercellular CO2 partial pressure, pi, was 40 pbar higher than for plants adapted to high irradiance. These two factors result in maximising the rate of CO2 assimilation at ambient conditions. Firstly, operating with a higher pi increases the rate of COz assimilation. Secondly, increasing the mesophyll conductance could increase the rate at ambient conditions until the regeneration of RuP2 became limiting. Generally leaves measured under standard conditions and high irradiance seem to operate close to the region where the two capacities are in balance (Caemmerer and Farquhar 1981; Evans 1986a). This seems to be the case for the low and intermediate irradiance treatments but, for the plants grown at high irradiance, the operating point was consistently below the transition to an RuP2 regeneration limitation (Fig. 1).


Irradiance Response Curves The response of photosynthetic rate t o irradiance was measured in two ways, by oxygen evolution with 1Yo C 0 2 and by C 0 2 assimilation with p, maintained at 500 pbar (Fig. 2). Equation (1) was fitted to both sets of data and yielded the same curvature factor 0 in all cases, of about 0.74. A similar maximum photosynthetic capacity was evident for the highest irradiance in the glasshouse and for the two highest irradiances in the growth cabinet. Since the curves fitted for photosynthesis measured by either oxygen evolution or C02 assimilation were virtually identical, this demonstrates that the two methods are comparable. The irradiance within the leaf declines with depth. An irradiance response curve for a n intact leaf is therefore the sum of many individual curves. The equation used to fit the irradiance response curves has the property that, if the maximum Hill activity of each chloroplast or cell layer is proportional to the relative irradiance at that layer, and 0 is constant, then the single equation (1) can be used for the whole leaf (Evans1983b). A n identical conclusion for this optimal distribution of Hill activity was reached by Oya and Laisk (1976) and Terashima and Saeki (1985). Biochemical evidence in support of a gradient in the photosynthetic capacity of chloroplasts has been demonstrated firstly between palisade and spongy mesophyll tissue (Terashima and Inoue 1985a) and subsequently between 10 paradermal sections (Terashima and Inoue 1985b). Terashima and Saeki (1985) argue that the shape of the curve can be influenced by the pattern of light absorption through the leaf. Both Oya and Laisk (1976) and Terashima (1986) have demonstrated that the irradiance response curve is altered when the abaxial surface of the leaf is illuminated. If the adaxial surface was illuminated, 0 was greater, implying that Hill activity was affected by the intraleaf gradient in irradiance. The value of 0 for the intact leaf can reflect both the property of the chloroplast and the distribution of photosynthetic capacity through the leaf. If the leaf is measured in a light environment similar to that it is grown in, then the 8 value of the curve may reflect that of the chloroplast. Marshall and Biscoe (1980) derived a similar equation to describe irradiance response curves and found values of 0 close t o 1. The value 0 is dependent on the fit in the quantum yield region. The lower the quantum yield that is used, the more 0 tends towards 1 and the higher the value for Jma,. The quantum yields measured for this material in white light were 0.089 mol 0 2 mol- photons absorbed, which is close to a quantum requirement of 10 at the red maximum (Evans 1987). These high quantum yields result in the value of 0 obtained here being considerably less than 1, suggesting that the curve for a single chloroplast would not be a Blackman-type of curve (Blackman 1905). A value for 0 considerably less than 1 is also observed for dilute (4 PM Chl) thylakoid suspensions (Terashima and Evans, unpublished data). The value of 0 was independent of growth irradiance. Since the measurement conditions should mean that the values of 0 obtained here reflect mainly the chloroplast property, it appears as though 0 is not affected by changes in the stoichiometry of the various electron transport components or the chlorophyll a/b ratio. For plants adapted to low irradiance, the earlier saturation of the irradiance response curve gives the false impression that 0 should be larger. O n fitting equation (1) to curves of Solanum dulcamara (Osmond 1983), values of 0 r 0 . 7 5 were obtained for plants adapted to high or low irradiances.

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Determinants of Electron Transport Capacity It is well recognised that cytochrome f content is one of the most responsive of the electron transport components during adaptation to growth irradiance (Boardman 1977; Bjtirkman 1981). More recently, cytochrome f content has been found to vary in response to growth irradiance in Betula (Oquist et al. 1982), Sinapis (Wild er al. 1984,

John R. Evans

1986), Pisum (Leong and Anderson 1984b; Shmeleva and Ivanov 1985) and Phaseolus (Sampath and Kulandaivelu 1983). This would be expected given that electron transport capacity correlates best with cytochrome f content during senescence of barley leaves (Holloway et al. 1983). The results presented in Fig. 4 also show a clear linear correlation between cytochrome f content and Hill activity. This relationship was also apparent in the coupling factor activity. Other workers have also noted that coupling factor activity varies with growth irradiance. Berzborn et al. (1981) observed a 70% increase in the coupling factor content of spinach when grown at 270 rather than 100 pmol quanta m - 2 S - l , while a 300% increase was observed for peas and lettuce (Leong and Anderson 1984b; Davies et al. 1986). The coupling factor content has also been shown to vary as a function of irradiance within the leaf (Terashima and Inoue 1985a). Although Haslett and Cammack (1976) found no change in the Fd-NADP-reductase (FNR) content of bean leaves grown at different irradiances, this seems unusual. Nikolaeva and Osipova (1983) observed an increase in FNR activity of 230% and Terashima and Inoue (1985a) observed a doubling of FNR between chloroplasts isolated from spongy mesophyll and palisade tissue. FNR binds to the 17.5 kDa polypeptide of the cytochrome b/f complex in the presence of cations (Clarke and Hind 1983; Vallejos et al. 1984). However, since FNR is present only on stroma-exposed thylakoids (Jennings et al. 1979), while the cytochrome b/f complex is present in both stroma-exposed and appressed thylakoids (Anderson 1986), for FNR to remain in fixed ratio to cytochrome f requires that the FNR complex also interacts with another complex such as P S I . Given that the ratio of coupling factor to cytochrome f was unchanged by adaptation to different growth irradiances, the ratio between FNR and cytochrome f may also be fairly constant. The electron carrier pools of plastoquinone, plastocyanin and ferredoxin would also be expected to change, although they may not a11 vary in strict proportion with, for example, cytochrome f . While a clear relationship exists between maximum Hill activity and both cytochrome f and coupling factor activity, none is apparent with the reaction centre content of PS I or 11. This suggests that PS I turnover never limits the maximum rate of whole-chain electron transport. The slight dependence of PS I1 content on growth irradiance may not be related to the electron transport capacity. For example, wheat plants grown under low irradiance had greatly reduced electron transport capacities although PS I1 content was unchanged (Evans, unpublished data). For peas, the reduction in the PS I1 content with declining growth irradiance is associated with a reduction in the chlorophyll a/b ratio and may relate to the pigment organisation within the membrane and the protein cost as discussed below. The constancy of the term 8 in the irradiance response curve also suggests that the altered density of the reaction centre of PS I1 does not affect the electron transport kinetics. Adaptation of the Thylakoid Membranes The adaptation of pea leaves to lower irradiances during growth is characterised by two features. Firstly, there is a dramatic reduction in components of electron transport and photophosphorylation and, secondly, there is a reduction in the chlorophyll a/b ratio (Leong and Anderson 1984a, 1984b). The first adaptation results in a large reduction in the protein cost associated with the formation of thylakoid membranes. At lower irradiances, the plant needs to maximise light absorption and can reduce the maximum Hill activity. Calvin cycle enzymes are also reduced relative to chlorophyll (Boardman 1977; Bjorkman 1981). More protein can thus be allocated towards lightharvesting. Lowering the chlorophyll a/b ratio achieves a further saving of protein since the light-harvesting chlorophyll a/b complex holds more pigment molecules per unit of nitrogen than the core P S I and I1 complexes. The correlation between P S I1 reaction centre content and chlorophyll a/b ratio is expected because, while PS I content remains


the same (Leong and Anderson 19846; Shmeleva and Ivanov 1985; Chow and Hope 1987; Fig. 4E), to increase PS I1 content requires that the proportion of the chlorophyll in the light-harvesting chlorophyll a / b complex decline. Line 3 (Fig. 5 ) demonstrates the saving in terms of nitrogen per unit of light absorbed associated with adaptation to low irradiance. Associated with the lower chlorophyll a / b ratios is an increase in the amount of grana stacking (Anderson et al. 1973; Terashima and Inoue 19856; Aro et al. 1986), but little change in the leaf absorptance. Curiously, increased grana stacking does not affect the quantum yield of photosynthesis and the consequences of altering the ratio of appressed to non-appressed membranes are still unclear (Evans 1986b). The lower chlorophyll a / b ratio also results in a larger antenna for PS 11. For plants grown at high irradiance, there were 566 Chl per 1000 Chl associated with the lightharvesting complex, 85% of which is located in regions of membrane appression (Anderson and Anderson 1980). Given that there were 2.44 PS I1 reaction centres per 1000 Chl and 11 Chl per monomer of the light-harvesting complex, this gives 18 monomers per PS 11. At low irradiance, there were 614 Chl associated with the lightharvesting complex per 1-95 PS I1 reaction centres, giving 24 monomers per PS 11. The antenna size increased by nearly 30% from 250 to 320 Chl per PS I1 reaction centre during the adaptation by pea leaves to low irradiance. A similar change has been observed for tomato leaves adapted to low irradiance by measuring the initial rate constant of fluorescence induction curves (Hodges 1984). The increase in the number of light-harvesting complexes for PS I1 is also consistent with freeze-fracture evidence. The ratio of small particles on the PF, face to large particles on the EF, face increases for Atriplex plants adapted to low irradiance (Bjorkman et al. 1972) and is also higher for shade species relative to Atriplex grown at high irradiance (Goodchild et al. 1972). In adapting to high irradiance, the plant should maximise the electron transport capacity to utilise the expensive light-harvesting pigment array to best advantage. The solid line (Fig. 5) represents the nitrogen cost for electron transport. Plants adapted to high irradiances clearly have the lowest nitrogen cost for electron transport. The distribution of nitrogen between the thylakoid proteins should therefore be unaffected by nitrogen stress. For spinach grown in full sunlight with various amounts of nitrate nitrogen, a 3-fold change in the nitrogen content per unit of leaf area was not accompanied by any change in the relative amounts of the thylakoid components examined (Evans and Terashima 1987).

-

Acknowledgments This work was carried out during the tenure of a Royal Commission for the Exhibition of 1851 scholarship at the Plant Breeding Institute, Cambridge, U.K., and later a CSIRO post-doctoral fellowship. I thank Drs Jan M. Anderson, W. S. Chow, G. D. Farquhar and I. Terashima for comments on the manuscript.

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