ABSTRACT ORIGINAL ARTICLE OA 220 EN E. H. MURCHIE & P. HORTON

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1 Plant, Cell and Environment (1998) 21, ORIGINAL ARTICLE OA 220 EN Contrasting patterns of photosynthetic acclimation to the light environment are dependent on the differential expression of the responses to altered irradiance and spectral quality E. H. MURCHIE & P. HORTON Robert Hill Institute, Department of Molecular Biology and Biotechnology, Western Bank, University of Sheffield S10 2TN, UK ABSTRACT Four plant species (Chamerion angustifolium, Digitalis purpurea, Brachypodium sylvaticum and Plantago lanceolata) which have previously been shown to demonstrate contrasting photosynthetic acclimatory responses to the light environment (Murchie & Horton 1997, Plant, Cell and Environment 20, pp ) were analysed at a biochemical level. Plants were grown under low irradiance with a shade-type spectrum (LFR: 50 µmol quanta m 2 s 1 ), moderately high white light (MW: 300 µmol quanta m 2 s 1 ) and low irradiance white light (LW: 50 µmol quanta m 2 s 1 ). The effects of light quality upon chlorophyll content and photosynthetic capacity were found to be species-dependent. A far-red dependent reduction in chlorophyll was found in three species, and an irradiance-dependent reduction was found in B. sylvaticum, which showed the greatest alteration in the xanthophyll cycle pool size of all species tested under these conditions. Chlorophyll a/b ratios were sensitive to both light quality and quantity in C. angustifolium and D. purpurea, being highest in MW, lowest in LFR, and intermediate in LW, whilst the other species showed no response. Ratios of photosystem II to photosystem I (PSII and PSI) demonstrated a strong irradiance-associated increase in all species except B. sylvaticum, whereas an increase in PSII/PSI in LFR compared to LW conditions was present in all species. A change in chlorophyll a/b was not always associated with a change in PSII/PSI, suggesting that the level of LHCII associated with each PSII varied in some species. Cytochrome f content showed an irradiance-dependent effect only, indicating a relationship with the capacity of electron transport. It is concluded that differing strategies of acclimation to the light environment demonstrated by these species results from differing strengths of expression of a series of independently regulated changes in the levels of photosynthetic components. Key-words: acclimation; chloroplast; irradiance; photosynthesis; photosystem; spectral quality. Abbreviations: Chl, chlorophyll; Chl a/b, ratio of chlorophyll a to chlorophyll b; Cyt f, cytochrome f; FR, far-red light; LFR, Correspondence: E. H. Murchie, Fax: , E.H.Murchie@sheffield.ac.uk low irradiance, far-red enriched growth light; LHCII, light harvesting complex associated with PSII; LW, low irradiance, white growth light; MW, moderate irradiance, white growth light; PAR, photosynthetically active radiation; P max, light and CO 2 saturated photosynthetic rate; PSI, photosystem I; PSII, photosystem II. INTRODUCTION The relative amounts of the supramolecular complexes in photosynthetic membranes are not fixed, but change according to prevailing light conditions. Such long-term modification (occurring over hours or days) involves the synthesis, assembly and insertion of new proteins and pigment-protein complexes and/or the breakdown of already existing membrane complexes (Anderson 1986; Anderson et al. 1995). Of particular interest are the changes in ratio of photosystem II to photosystem I, the functional consequences these have for photosynthetic efficiency, and the possible mechanisms by which they are controlled. The ratio of PSII to PSI can change according to both the irradiance and spectral quality of light (Chow & Anderson 1992). Studies with plants grown under relatively low light conditions have shown that altering the relative rates of excitation of PSI and PSII using light of different spectral qualities can lead to large changes in photosystem stoichiometry. Chow et al. (1990c) found that when pea plants were grown under light of a spectral quality designed to over-stimulate either PSII or PSI, changes in the ratio of PSII to PSI were observed which appeared to compensate for the deficiency in electron transport in one or other photosystem since the quantum yield of oxygen evolution was higher when leaves were probed with light of the same quality as that under which they had been grown. Similar results have been obtained with Arabidopsis thaliana (Walters & Horton 1995a). This strongly suggests that changes in photosystem stoichiometry are of importance in maintaining photosynthetic efficiency under light-limiting conditions. When spinach was switched between PSII and PSI light, concomitant changes in amounts of PSII, PSI and LHCII polypeptides occurred within 35 h suggesting a highly dynamic system constantly changing in response to incident light quality (Kim et al. 1993). There is evidence that alterations in thylakoid composition according to spectral quality are also species-dependent: pea grown in 1998 Blackwell Science Ltd 139

2 140 E. H. Murchie and P. Horton continuous red, white or blue light of equal irradiance had a fixed component stoichiometry, whereas Asplenium australacium or Atriplex triangularis when grown under the same set of conditions showed an increase in PSII/PSI in red compared to blue or white light (Leong & Anderson 1984a; Leong et al. 1985). Alterations in photosystem stoichiometry in response to growth irradiance have also been noted in several plant species. An increase in growth irradiance resulted in significant increases in PSII per unit chlorophyll in pea (Leong & Anderson 1984a; Chow & Anderson 1987; Evans 1987), spinach (Chow & Hope 1988; Anderson et al. 1992), mustard (Wild et al. 1986), and barley (De la Torre & Burkey 1990), whilst PSI/Chl remained relatively constant. In contrast, A. thaliana (Walters & Horton 1994), Tradescantia albiflora (Chow et al. 1991) and Alocasia macrorrhiza (Chow et al. 1987) showed no change in photosystem stoichiometry. Tradescantia albiflora and A. thaliana showed changes in photosystem stoichiometry similar to those of pea and spinach when subjected to PSII or PSI light, suggesting the existence of independent processes governing light quality-induced vs. light quantity-induced alterations. Natural shade cast by overhead vegetation is deficient in PAR due to selective filtering by photosynthetic pigments and as a result combines a low irradiance with a high farred to red ratio. Therefore natural environments are likely to expose chloroplasts to light conditions with potentially antagonistic effects: firstly, a decreased irradiance which would result in a decrease in PSII/PSI, and secondly a shade spectrum which would increase PSII/PSI. However, the exact nature of the response to shade is difficult to predict, which is at least partially due to the large temporal and spatial alterations which characterize the natural light environment. To overcome this problem, Mckiernan & Baker (1991) grew Silene dioica in open glasshouse conditions, and under shade generated by the aquatic angiosperm Lemna gibba: it was shown that the PSII/PSI ratio was lower in unshaded conditions. In contrast, Anderson & Osmond (1991) found a low PSII/PSI in leaves of most field-grown rain-forest species, implying that species respond in different ways because of inherent differences in the relative strengths of the responses to altered irradiance and spectral quality. A previous study of 22 wild-type British plant species (Murchie & Horton 1997) showed a large amount of interspecific variation in the capacity to alter the Chl a/b ratio and the maximum photosynthetic rate in response to growth under simulated natural shade (LFR) and moderate sun (MW) conditions. It was possible to link these parameters to an index of habitat preference, thereby providing a basis for understanding this variation from an ecological viewpoint. Responses in photosynthetic capacity were observed for most species, but some species did not alter Chl a/b whilst others showed large changes. Similarly, changes in chlorophyll content ranged from zero to threefold. It was suggested that this arises from the existence of at least two different mechanisms of acclimation: firstly an alteration in chlorophyll content at the leaf level, probably caused by changes in chloroplast number per unit leaf area, and secondly changes in chlorophyll content at the thylakoid level, associated with alterations in the relative proportions of electron transport components and/or pigment protein complexes. Accordingly, four broad classification groups were identified: A, species which showed only leaf level acclimation; B, species which showed acclimation at both leaf and chloroplast level; C, species which only displayed chloroplast level acclimation; D, species which showed no evidence of light acclimation. Species belonging to groups B and C showed the greatest acclimation potential and are found naturally in both shaded and unshaded habitats. In order to explore these ideas further four species were selected which showed different responses to LFR and MW conditions, and analysed in more detail for their thylakoid membrane composition. The objectives were, firstly, to discover whether changes in the relative amounts of PSII, PSI and/or light harvesting complexes would account for acclimation at the chloroplast level. The second objective was to establish the extent to which the responses to the LFR shade condition were due to the enrichment in far-red light or to the reduced irradiance. MATERIALS AND METHODS Selection of species The species selected were: Digitalis purpurea L. (Foxglove) (shade association index for a full description of this parameter see Murchie & Horton (1997) group C/D above), a species which is mostly restricted to shaded habitats (Grime et al. 1988); Brachypodium sylvaticum (Hudson) Beauv. (Slender False Brome) (shade association index 0 752, group D) which is mainly found in shaded habitats although it can be widespread in open areas, where signs of photobleaching have been observed (Grime et al. 1988); Chamerion angustifolium (L.) J. Holub (Rosebay Willow-Herb) (shade association index 0 526, group B), found in marginally shaded areas as well as open ones (Grime et al. 1988); and Plantago lanceolata L. (Ribwort), (shade association index 0 1, group A/B) which is mostly absent from woodland. The four species were grown under the conditions described in Murchie & Horton (1997) supplying MW (moderate, white: 300 (± 40) µmol quanta m 2 s 1 ) and LFR (low, far-red enriched shade: 50 (± 15) µmol quanta m 2 s 1 ) light. To investigate the effects of growth under light of the same quality as MW but of reduced irradiance, plants were also grown at a distance from the light sources approximately twice that of the MW-grown plants, such that the irradiance was then 50 (± 15) µmol quanta m 2 s 1. This growth light condition is termed low, white (LW). The growth period depended on species and light conditions, but varied between three weeks and two months. Mature, exposed, healthy leaves were used throughout. Measurements of leaf photosynthetic oxygen evolution rates and leaf chlorophyll content were carried out as

3 Patterns of photosynthetic acclimation 141 described in Murchie & Horton (1997). Photosynthetic capacity was achieved at 1000 µmol quanta m 2 s 1 (see Murchie & Horton 1997). Assay of photosystem II, photosystem I and cytochrome f Leaves were selected for thylakoid isolation from the exposed upper part of the plant. Care was taken during the growth of plants that the leaves used for a single thylakoid preparation had received as nearly as possible the same irradiance. Thylakoids were isolated by the method of Walker (1980), except that the grinding medium was supplemented with 10 mol m 3 PVP and 0 5% w/v BSA. The first pellet, which contained a mixture of intact chloroplasts and thylakoids, was washed twice using a soft paint brush in chilled grinding medium, and once in a solution containing 20 mol m 3 KCl; 5 mol m 3 MgCl 2 ; 10 mol m 3 MES-KOH (ph 7 6) to shock the intact chloroplasts osmotically. Thylakoids were resuspended in a solution containing 330 mol m 3 sorbitol; 100 mol m 3 HEPES- KOH (ph 7 6); 20 mol m 3 KCl; 2 5 mol m 3 MgCl 2 and 1 mol m 3 Na 2 EDTA, and stored on ice, and were used within one hour for the PSII atrazine-binding assays, and within five hours for measurements of Cyt f and PSI. The content of PSII was estimated using [ 14 C] atrazine, which binds reversibly to the D1 polypeptide at appropriate herbicide concentrations (McCarthy et al. 1988; Chow et al. 1990b). [ 14 C] atrazine was obtained from Sigma (specific activity 4 5 mci mmol 1 ). Thylakoids were mixed with an appropriate volume of buffer (400 mol m 3 sucrose, 50 mol m 3 TES-NaOH (ph 7 5), 10 mol m 3 NaCl, 5 mol m 3 MgCl 2 ) The concentration of [ 14 C] atrazine was varied from 0 05 to 0 50 µm, with a chlorophyll concentration of 50 µm. An additional series of tubes was run with the omission of thylakoids. Tubes were left at room temperature (25 ± 2 C) for three minutes in the dark, and then centrifuged for one minute at r.p.m. to pellet the membranes. The difference between the counts in the supernatant with and without thylakoid membranes was assumed to be equal to the membrane-bound counts. The content of PSI was assayed by monitoring the chemically induced redox changes of P 700 (Whitmarsh & Ort 1984). The content of Cyt f was assayed by monitoring chemically induced absorbance change at 554 nm, also as described by Whitmarsh & Ort (1984). Carotenoid content Plants were dark-adapted for 24 h before leaf samples were taken. Leaf material was cut (one to two cm 2 ) from mature, healthy leaves and immediately frozen to 196 C. Samples were stored at 70 C until extraction and analysis, which was carried out as in Johnson et al. (1993) RESULTS Photosynthetic activity and leaf chlorophyll content Table 1 shows the basic features of photosynthetic acclimation from the previous paper (Murchie & Horton 1997) for the four selected species, and compares them against the new growth condition utilized in this study, LW (low irradiance white light). Comparing MW and LFR, the different categories of response are seen for the species shown: in C. angustifolium and D. purpurea, chlorophyll per unit leaf area was the same for MW and LFR conditions, whereas for P. lanceolata the chlorophyll content was 100% greater in MW than LFR. The Chl a/b ratio was unchanged in the latter species but lower in LFR than MW for both D. purpurea and C. angustifolium. These three Table 1. Chl per unit leaf area, Chl a/b, light and CO 2 saturated rates of photosynthesis (P max ) per unit leaf area and per unit Chl for the four species grown under: moderate, white light (MW: 300 µmol m 2 s 1 ); low, white light (LW: 50 µmol m 2 s 1 ) and low, far-red enriched light (LFR: 50 µmol m 2 s 1 ). Means of at least six replicates ± SEM. Values for a given parameter and a given species are significantly different statistically (one way analysis of variance) from others for that species at the 5% or 10% level except where shown (* = no difference at the 10% level between condition marked and MW, = no difference at the 10% level between LFR and LW). Groups A D refer to habitat preferences as described in Murchie & Horton (1997) Growth C. angustifolium P. lanceolata B. sylvaticum D. purpurea conditions (group B) (group A/B) (group D) (group C/D) mg Chl m 2 MW 127 ± ± ± ± 10 LW 165 ± 7 236* ± ± ± 8 LFR 130* ± ± * ± * ± 14 Chl a/b MW 4 21 ± ± ± ± 0 06 LW 3 73 ± * ± * ± ± 0 08 LFR 3 00 ± * ± * ± ± 0 05 P max /area MW 8 70 ± ± ± ± 1 3 LW 6 04* ± ± ± ± 0 6 LFR 2 20 ± ± * ± ± 1 0 P max/ Chl MW 67 5 ± ± ± ± 6 LW 37 1 ± ± ± ± 3 LFR 18 0 ± ± * ± ± 3

4 142 E. H. Murchie and P. Horton species all showed substantial changes in P max per unit chlorophyll. The differences in P max per unit area were greatest for P. lanceolata. Rather different behaviour was shown by B. sylvaticum, since this species exhibited no changes in chlorophyll content, Chl a/b ratio, P max /Chl or P max /area when comparing MW and LFR. Also shown in Table 1 are the effects of growth in low irradiance white light (LW), and the data clearly show how some aspects of the interspecies variation can be explained by the differential response to irradiance and spectral quality. In P. lanceolata the large increase of chlorophyll content in MW, symptomatic of photosynthetic acclimation at the leaf level, was mostly due to the altered spectral quality from LFR rather than the increased irradiance. This effect can account for the 40% increase in P max /area in P. lanceolata in LW compared to LFR. The change in P max /Chl between LW and LFR in this species and in D. purpurea was minimal, but differences were greater for C. angustifolium and B. sylvaticum. Increased irradiance gave rise to a strong effect on P max /Chl in all species, so that the increase in this parameter in MW compared to LFR was predominantly an effect of irradiance which was therefore exerting control of acclimation at the chloroplast level. The increase in P max /area in MW compared to LFR originated from the irradiance-associated behaviour of P max /Chl in P. lanceolata and D. purpurea, but originated from the spectral quality-associated effect in C. angustifolium, where a decrease in chlorophyll content in MW antagonized the irradiance-dependent increase in P max /Chl. In B. sylvaticum which showed only a limited degree of acclimation for most parameters, the possibility of an increase in P max /area from LFR to MW was negated by a reduction in P max /Chl in LW compared to LFR, and an irradiancedependent reduction in chlorophyll, which offset the increase in P max /Chl (in MW compared to LFR). P max /Chl may be affected either by changes in Chl a/b which reflects the proportion of chlorophyll in LHCII relative to other components, and/or by other factors (e.g. the Rubisco content). In fact, changes in Chl a/b did not consistently relate to an equivalent change in P max /Chl. For example, Chl a/b was lower in LFR-grown C. angustifolium and D. purpurea than in LW-grown plants. The P max /Chl also declined, but Chl a/b in LFR and LW-grown B. sylvaticum were unchanged whilst P max /Chl was substantially lower in LW-grown plants. Clearly a number of factors influence P max /Chl. Changes in Chl a/b arise from an change in the antenna size of PSII or from an alteration in PSII content. Since Chl a/b responds to both irradiance and spectral quality in two species but to neither of these parameters in the other two, it was important to determine the reaction centre contents in the three different light environments. Chloroplast composition PSII per unit chlorophyll for all species and growth conditions is shown in Table 2. PSII/Chl showed a strong irradiance-dependent effect, being twice as high in MWgrown D. purpurea and C. angustifolium than in LWgrown plants, and 1 5 times as great in P. lanceolata with the exception of B. sylvaticum, however, where PSII/Chl remained constant at 2 15 mmol mol 1 Chl. For LFR conditions PSII/Chl was higher than in LW-grown plants in all species except B. sylvaticum. Additionally no species showed a difference in PSII/Chl in LFR compared to MW, with the exception of C. angustifolium. There were species-dependent differences both in the amounts of PSII for a given growth condition, and in the differences observed between the three growth lights for a given plant species. For example in MW conditions, PSII/Chl was the same for both C. angustifolium and P. lanceolata (around 3 00 mmol mol 1 Chl), and slightly lower in D. purpurea (2 69 mmol mol 1 Chl) and lowest in B. sylvaticum (2 00 mmol mol 1 Chl). Reducing the irradiance did not change PSII/Chl by a proportionally similar amount in each species: B. sylvaticum had the highest value for LW-grown plants, and D. purpurea the lowest. The numbers of atrazine-binding sites per unit chlorophyll given here are within the range quoted for pea, barley, spinach and mustard grown under a similar range of irradiances, i.e. between 1 75 and 4 00 mmol mol 1 Chl (Leong & Anderson 1984b; Wild et al. 1986; Evans 1987; Chow & Hope 1988; De la Torre & Burkey 1990). There have been few assays of atrazine-binding sites for wild Table 2. PSII, PSI, and Cyt f content per unit total Chl (all values expressed as mmol mol 1 Chl) for the four species grown under MW, LW and LFR light (see text and Table 1 for details). Means of at least three independent experiments ± SEM. For details of symbols see Table 1 Growth conditions C. angustifolium P. lanceolata B. sylvaticum D. purpurea PSII/Chl MW 3 08 ± ± ± ± 0 03 LW 1 49 ± ± * ± ± 0 10 LFR 1 99 ± * ± * ± * ± 0 20 PSI/Chl MW 1 09 ± ± ± ± 0 04 LW 1 09* ± * ± ± * ± 0 06 LFR 1 16* ± * ± ± * ± 0 02 Cyt f/chl MW 1 05 ± ± ± ± 0 15 LW 0 69 ± ± ± ± 0 07 LFR 0 65 ± ± * ± ± 0 19

5 Patterns of photosynthetic acclimation 143 species: between 2 0 and 3 0 mmol mol 1 Chl for Silene dioica (Mckiernan & Baker 1990), A. macrorrhiza (Chow et al. 1987) and A. thaliana (Walters & Horton 1994). Table 2 shows that there were species-specific differences in the values for PSI/Chl for a given growth light: in C. angustifolium this parameter was lower than for the other species under all growth conditions, at around 1 10 mmol PSI mol 1 chlorophyll and this was not affected by light quantity or quality. Similarly, PSI/Chl for D. purpurea did not change ( 1 5 mmol mol 1 Chl). In P. lanceolata, PSI/Chl in LW was lower than for MW or LFR-grown plants. PSI/Chl in B. sylvaticum appeared to be the most sensitive to growth light and was lowest in LFR and highest in MW. However, PSI expressed on a chlorophyll basis showed changes which were generally smaller than those for PSII. There was apparently no consistent trend in the effect of spectral quality on PSI content. There are many reports of an invariant PSI/Chl over a range of growth irradiances in a number of species (Chow et al. 1987; Evans 1987; Lee & Whitmarsh 1989; Walters & Horton 1994). However, PSI/Chl has been found to vary with spectral quality in pea (Glick et al. 1985), spinach (Chow & Anderson 1992) and T. albiflora (Chow et al. 1991) with a reduction in PSI/Chl in response to a low red:far-red ratio, or growth in red as compared to blue light. Cyt f/chl was higher in MW-grown plants than in either LW- or LFR-grown plants, again with the exception of B. sylvaticum for which values did not change (Table 2). For all species there was no difference in Cyt f/chl between LW and LFR-grown plants. Therefore amounts of Cyt f are sensitive to growth irradiance but not quality. Cyt f/chl is plotted against P max /Chl in Fig. 1. There was a weak correlation (previously shown by Evans 1987) and this would appear to hold for three of the species in this study, the exception being D. purpurea which showed a particularly high Cyt f/chl when MW-grown. In order to determine changes in carotenoid amounts during photosynthetic acclimation in the four selected species, dark-adapted leaf carotenoid levels were determined by HPLC. Results are expressed as a percentage of total carotenoids (Fig. 2). There was interspecific variation in the total carotenoid/chl, with P. lanceolata showing the highest values. The total carotenoid content differed only slightly between MW and LFR conditions and the differences between levels of neoxanthin, lutein and β-carotene were small. Previous studies have shown large increases (up to four-fold) in the xanthophyll cycle pool size in plants grown in high irradiance (Thayer & Björkman 1990; Demmig-Adams & Adams 1992), although higher irradiances (up to 2000 µmol quanta m 2 s 1 ) were used. In Fig. 2 it is seen that the expected increase in xanthophyll pool size (% violaxanthin) occurred, although only to a relatively small extent. The largest changes were shown by B. sylvaticum and C. angustifolium. DISCUSSION Murchie & Horton (1997) defined two processes contributing to the changes in leaf photosynthesis that occur during growth in sun or shade conditions: chloroplast level acclimation and/or leaf level acclimation. Varying combinations of responses at the chloroplast and leaf levels were found to combine to define the acclimation potential, that in turn was related to the shade association index which defines habitat preference of the species. In this paper we have shown that both levels of acclimation are in turn the sum of irradiance-dependent responses and the effects of light quality. The data allow us to consider how differences in the relative strengths of these responses can account for the different acclimation effects in individual species. Further, it can be seen that the irradiance response is itself the result of two conflicting influences, photosynthesis and photoprotection. Figure 1. Relationship between light and CO 2 -saturated rate of photosynthesis (P max ) per unit Chl and Cyt f content per unit Chl for the four species grown under MW, LFR and LW conditions (see text and Table 1 for details)., D. purpurea; s, B. sylvaticum;, P. lanceolata; t, C. angustifolium. Variation in the relative strengths of responses to spectral quality and to irradiance In natural shade, simulated by our LFR conditions, the rates of excitation of PSI relative to PSII are markedly increased. It has been suggested that an increase in the number of PSII units in FR-enriched conditions might play a vital role in the maintenance of optimal quantum yield in low-irradiance, shaded conditions (Chow et al. 1990c; Walters & Horton 1995a). The lower PSII/PSI ratios for LW than for LFR for all four species shown in Table 3 strengthens the suggestion that this is a fundamental process in chloroplast regulation. Table 3 also clearly shows that the ratio PSII/PSI is dependent on irradiance. Therefore, the adjustment of photosystem content to natural shade conditions is a sum of the responses to a decrease in irradiance and an enrichment in

6 144 E. H. Murchie and P. Horton Figure 2. Dark-adapted leaf carotenoid content and carotenoid per unit Chl of the four species grown under MW (open bars) and LFR (hatched bars) conditions. Percentages are fractions of total carotenoid. Zeaxanthin and antheraxanthin were undetectable in these extracts. Means of five leaves ± SEM. Table 3. Stoichiometries of thylakoid membrane components for the four species grown under MW, LW and LFR light (see text and Table 1 for details). Values shown were calculated directly from Table 2 Growth conditions C. angustifolium P. lanceolata B. sylvaticum D. purpurea PSII/PSI MW LW LFR FR light. A drop in irradiance resulted in a lower PSII/PSI (with the exception of B. sylvaticum) whereas an increase in FR resulted in a higher ratio. The two antagonistic effects can be of equal strength so that no net effect is observed (e.g. in D. purpurea). In other species the spectral quality effect was weak and a significant difference between LFR and MW was observed (e.g. C. angustifolium). In B. sylvaticum, the irradiance actually reduced the PSII/PSI ratio slightly so that a large overall decrease was found in MW compared to LFR.

7 Patterns of photosynthetic acclimation 145 The ratio Chl a/b, which indicates an alteration in the relative amounts of LHCII, also responds to both light intensity and quality and again gives rise to particular patterns of net change. Chl a/b has previously been shown to increase in response to far-red enriched light (Glick et al. 1985; Anderson et al. 1992) and results from co-ordinated changes in the amounts of proteins of PSII, PSI and LHCII (Kim et al. 1993). D. purpurea and C. angustifolium showed similar responses i.e. an increase in PSII/PSI in LFR compared to LW was concurrent with a decrease in Chl a/b, implying an increase in LHCII content. P. lanceolata and B. sylvaticum showed no change in Chl a/b in LFR compared to LW yet had the same trends in photosystem content as D. purpurea and C. angustifolium. Thus in P. lanceolata there was evidence of a decrease in the ratio of LHCII to PSII in LFR. Finally, in B. sylvaticum there was no change in Chl a/b despite an increase in the content of PSII relative to PSI, implying a reduction in LHCII relative to PSII. Thus, the changes in photosystem content in farred-enriched light may not necessarily be concomitant with those of antenna size and it must be noted that the particular species under study is of great importance. As emphasized by Mckiernan & Baker (1991), under light limiting conditions it is the total amount of chlorophyll associated with PSII as opposed to PSI that determines the quantum efficiency, rather than the number of reaction centres. A higher irradiance also resulted in an increased Chl a/b, again only in C. angustifolium and D. purpurea, whereas in P. lanceolata there was no change. Because in these three species there were significant increases in PSII content in MW compared to LW, then in each case there was a decrease in the amount of LHCII associated with PSII with increasing irradiance. Such reductions in LHCII arise from the selective loss of the peripheral Lhcb2 protein (Anderson 1986; Melis 1991). We have shown that the photosynthetic capacity of a leaf (P max /area) is determined by rather complex interplay between the effects of spectral quality and irradiance on the chlorophyll content and P max /Chl. Spectral quality exerted an effect on the chlorophyll content of the leaf in three of the four species examined, suggesting that the enrichment of far-red light in the shade environment was having a strong influence on acclimation at the leaf level. A similar effect of far-red light on chlorophyll content has been observed in pea (Chow et al. 1990a). Increased irradiance resulted in a very variable effect on chlorophyll content: either a decrease, an increase, or no effect. Obviously such responses altered the net difference between MW and LFR. P max /Chl is used here as an indicator of acclimation at the chloroplast level: there was an irradiance-associated increase in all species tested and hence any difference between MW and LFR in this parameter was also predominantly an irradiance effect. We conclude that irradiance was exerting chloroplast-level acclimation throughout. Altered spectral quality exerted an extremely variable effect on P max /Chl, and such effects either added to, subtracted from, or had no effect on, the net change in this parameter. Of the parameters tested, it was only the level of Cyt f which showed exclusively an irradiance-associated response and no effect of spectral quality. This irradiance response was observed for all species, again with the exception of B. sylvaticum. The changes in Cyt f and photosystem content observed in C. angustifolium, D. purpurea and P. lanceolata are comparable to those previously observed (Leong & Anderson 1984a,b; Evans 1987; De la Torre & Burkey 1990). Levels of Cyt f have frequently been correlated with the capacity for photosynthetic electron transport (Anderson et al. 1995). Variation in the strengths of the irradiance response Two strategies govern the responses of plants to increased irradiance: firstly, an increase in photosynthetic capacity to utilize the extra excitation energy for assimilatory processes and secondly, photoprotective mechanisms. The latter includes the dissipation of energy in excess of that which can be used in photosynthesis, a process in which the xanthophyll cycle has an essential role (Demmig-Adams & Adams 1992; Horton et al. 1996). The xanthophyll cycle pool size can vary several-fold with growth irradiance (Thayer & Björkman 1990; Demmig-Adams & Adams 1992), and its increase is a measure of the extent to which the irradiance level is in excess of photosynthetic capacity. When the data shown in Fig. 2 are expressed on a chlorophyll basis the greatest percentage increase in xanthophyll cycle pool size when comparing MW to LFR plants was seen in B. sylvaticum (98%), whereas for other species the changes were much smaller (48%, 32% and 18% in C. angustifolium, P. lanceolata and D. purpurea, respectively). The increase shown by B. sylvaticum is to a large extent predicted because this species had a very low value for P max in MW, and no difference between MW and LFR, and therefore even moderate light levels were sufficient to induce a photoprotective strategy. The chlorophyll content also declined in MW in this species. A loss of chlorophyll accompanied by an increase in xanthophyll cycle pool size is a strategy most clearly expressed in extremely slow-growing stress-tolerant species such as Guzmania monostachia (Maxwell et al. 1994). Consistent with this notion, it has been found that when B. sylvaticum is found in open conditions, such as in woodland gaps, there is a yellowing of the leaves (Grime et al. 1988). The lack of a significantly large difference in xanthophyll cycle content between MW and LFR in the other species strongly suggests that enhanced photoprotection was unnecessary because the increased excitation energy was being utilized via an increased capacity for electron transport and carbon fixation. Where such large increases in photosynthetic capacity occurred, an increase in the content of Cyt f and PSII was also observed. B. sylvaticum had a limited ability to

8 146 E. H. Murchie and P. Horton augment photosynthetic capacity, and this was linked to the lack of a significantly large change in the amount of Cyt f. It is important to note that growth of D. purpurea at irradiances higher than those used here results in a significant increase in the xanthophyll cycle pool size (Webster 1996). Therefore we conclude that it is possible to distinguish between irradiances over which significant thylakoid acclimation occurs to enhance photosynthetic capacity, and irradiances over which xanthophyll-mediated photoprotective responses are induced. These ranges of irradiance are not absolute, but differ for each species so that each has a defined window for the photosynthetic acclimation and photoprotective responses. Conclusion: different strategies for photosynthetic acclimation to light environment Table 4 rationalizes the differing effects of light quality and quantity upon acclimation of photosynthesis given by the data presented in this paper and summarizes the possible combinations of response that can be found amongst plant species. By examining photosynthetic characteristics at LW, LFR and MW, it was possible to understand both the strategy behind the effects induced purely by the spectral quality of the shade-light environment, and the responses to MW which arise from the tendency towards a greater photosynthetic capacity at higher irradiance. This increase is predominantly an increase in P max /Chl and indicates acclimation at the chloroplast level, associated with increases in the content of Cyt f and PSII and a decrease in LHCII. These changes were particularly strong in C. angustifolium and D. purpurea, species known to frequent both shaded and unshaded habitats. P. lanceolata showed weaker irradiance-dependent changes in PSII and Chl a/b but the largest change in Cyt f. This species shows not only the largest change in P max but also the highest value for this parameter, and it is clearly adapted for maximum exploitation of sunlight in open environments, consistent with its low shade association index. This is to be contrasted with B. sylvaticum, a species found predominantly in shade conditions, which showed no response to an increased growth irradiance in terms of amounts of LHCII and Cyt f, photosystem stoichiometry, or P max. We extend the work of Chow et al. (1991) on the shade plant T. albiflora to suggest that a total loss of the ability to adjust light-harvesting components to high light may be a common feature of shadeassociated plants. This fixed mode of light-harvesting probably resulted from adaptation to an understorey environment, and may be considered to be an extreme example of the generally dampened acclimatory capacity of such species (Murchie & Horton 1997). Specific responses to altered spectral quality form an important part of the acclimation response. Changes in chlorophyll content in response to light quality appear to be common and may be associated with significant changes in photosynthesis. Principally however, photosystem stoichiometry changes occurred in all species thus far examined and therefore they appear to be a universal response to the shade environment, including species such as B. sylvaticum. As discussed by Anderson et al. (1995), photosynthetic acclimation appears to be controlled by a number of receptors. Although there is evidence that phytochrome does not control responses at the chloroplast level (Chow et al. 1990a; Walters & Horton 1995b) the far-red dependent reductions in chlorophyll observed here could be phytochrome responses. Increases in Rubisco and Cyt f have been linked to the action of a blue light receptor (Lopez-Juez & Hughes 1995). There is increasing evidence that photosynthesis itself controls the acclimation response via one or more redox sensing mechanisms (Allen 1993, 1995). In theory such mechanisms could provide the sensor for altered irradiance and spectral quality. The term excitation pressure has been introduced to describe how such a process can explain the response to irradiance (Huner et al. 1996); the changes in content of thylakoid constituents observed here can clearly be explained by this type of mechanism, including not only the adjustments associated with increased P max but also those associated with increased photoprotection. However, questions concerning the way in which such an array of responses can be differentially controlled, so that different irradiances and spectral qualities are integrated, giving rise to the ecologically important features of different species will remain unanswered until the molecular mechanisms of signal transduction are discovered. Leaf level acclimation Chloroplast level acclimation Increased FR: R reduced Chl increased PSII/PSI Increased irradiance increased Chl increased P max /Chl (photosynthesis) (Cyt f, PSII, Rubisco); reduced LHCII Increased irradiance reduced Chl increased xanthophyll cycle; (photoprotection) reduced Chl per chloroplast Table 4. Effects of altered irradiance and spectral quality on photosynthetic acclimation. The existence of these responses arises from this work and also that previously published. The changes in chlorophyll at the leaf level are inferred to result from altered chloroplast number arising from increases in leaf thickness. This matrix of stimuli and responses can rationalize the diversity of acclimation responses found in plant species. It must be noted that the magnitudes of response can vary (see text for details)

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