Hexadecenoic Acid Content of Phosphatidylglycerol: Light-harvesting ...

Plant Cell Physiol. 46(8): 1272–1282 (2005) doi:10.1093/pcp/pci136, available online at www.pcp.oupjournals.org JSPP © 2005

Temperature and Light Modulate the trans-∆3-Hexadecenoic Acid Content of Phosphatidylglycerol: Light-harvesting Complex II Organization and Nonphotochemical Quenching Gordon R. Gray 1, *, Alexander G. Ivanov 2, Marianna Król 2, John P. Williams 3, Mobashoher U. Kahn 3, Elizabeth G. Myscich 2 and Norman P. A. Huner 2 1

Department of Plant Sciences, University of Saskatchewan, Saskatoon SK S7N 5A8, Canada Department of Biology and The Biotron, University of Western Ontario, London ON N6A 5B7, Canada 3 Department of Botany, University of Toronto, Toronto ON M5S 1A1, Canada 2

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The interaction of light and temperature in the modulation of the trans-∆3-hexadecenoic acid (trans-16:1) content of phosphatidylglycerol (PG) in winter rye (Secale cereale L.) was assessed and related to the organization of light-harvesting complex II (LHCII). Increasing the growth irradiance from 50 to 800 µmol m–2 s–1 at 20°C resulted in a 1.8-fold increase in the trans-16:1 content in PG which favoured a greater preponderance of oligomeric LHCII, measured in vitro as the ratio of oligomer : monomer. Similar irradiance-dependent increases were observed during growth at 5°C; however, 1.4-fold lower trans-16:1 contents and lower LHCII oligomer : monomer ratios were observed compared with growth at 20°C and the same irradiance. These trends were also observed under natural field conditions. Thus, the accumulation of trans-16:1, as well as the organization of LHCII are modulated by both growth irradiance and growth temperature in an independent but additive manner. We also examined how changes in the supramolecular organization of LHCII affected the capacity for non-photochemical quenching (qN) and photoprotection via antenna quenching (qO). While qO was positively correlated with qN, there was no correlation with either LHCII organization or xanthophyll cycle activity under the steady-state growth conditions examined. Keywords: Antenna quenching — Light-harvesting complex II — Non-photochemical quenching — Phosphatidylglycerol — Supramolecular organization — trans-∆3-hexadecenoic acid. Abbreviations: A, antheraxanthin; EPS, epoxidation state; LHCII, light-harvesting complex II; Lhcb, Chla/b-binding light-harvesting polypeptides associated with PSII; Lut, lutein; Neo, neoxanthin; PG, phosphatidylglycerol; QA, first stable electron acceptor of PSII; qE, energy-dependent component of non-photochemical quenching; qN, coefficient of non-photochemical quenching; qO, quenching coefficient of basal fluorescence (antenna quenching); 1 – qP, relative reduction state of QA (PSII excitation pressure); trans-16:1, trans-∆3hexadecenoic acid; V, violaxanthin; Z, zeaxanthin.

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Introduction In the natural environment, plants experience and must adjust to wide daily and seasonal fluctuations in temperature and light. The ability of plants to respond to changes in their environment is crucial in determining tolerance to stress and their habitat preference. When plants are exposed to excess light, a decrease in photosynthetic capacity, known as photoinhibition, frequently occurs due to an imbalance between the absorption of light energy and utilization through reductive carbon, nitrogen and sulfur metabolism. However, plants utilize different species-specific photoprotective strategies to minimize the potential for photoinhibition and subsequent photooxidative damage, comprised of both photochemical and nonphotochemical mechanisms (Savitch et al. 2002, Öquist and Huner 2003). Growth and development of winter rye (Secale cereale L.) under conditions which result in elevated PSII excitation pressure result in a tolerance to photoinhibition (Gray et al. 1996). PSII excitation pressure is estimated by the Chl fluorescence parameter 1 – qp, and measures the relative reduction state of QA, the first stable quinone electron acceptor of PSII which, in turn, reflects the redox poise of the plastoquinone pool and intersystem electron transport chain (Dietz et al. 1985, Gray et al. 1996, Huner et al. 1998). Changes in either growth irradiance or growth temperature can modulate PSII excitation pressure in a similar manner. Lowering the temperature at constant irradiance lowers the capacity for light utilization which results in an increase in the reduction state of QA and hence an increase in 1 – qP. Alternatively, increasing the irradiance at constant temperature increases the rate of light absorption and produces an increase in reduction of QA (Maxwell et al. 1995, Gray et al. 1996, Huner et al. 1998). Furthermore, maximum photosynthetic capacity for oxygen evolution (Pmax O2), as well as non-photochemical quenching of Chl fluorescence (qN), are positively and linearly correlated with 1 – qp experienced during prevailing growth conditions in winter rye (Gray et al. 1996, Gray et al. 1997a). Non-photochemical processes, collectively known as qN, dissipate excess excitation energy in the antenna pigment bed of PSII and are considered to be the major PSII photoprotec-

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Organization of LHCII in cereals

tive mechanism (Demmig-Adams et al. 1999, Horton et al. 1999, Niyogi 1999, Gilmore 2000, Gilmore and Ball 2000, Müller et al. 2001, Ort 2001, Holt et al. 2004). The generation of qN in the antenna (antenna quenching or qO) is considered to be a cooperative phenomenon which, despite intensive study, is not yet fully understood. While several processes contribute to qN, the major component is thought to be energy-dependent quenching (qE). The qE component is regulated by the ∆pH across the thylakoid membrane and also involves the PsbS protein which is essential for the formation of qE, although its precise role remains elusive (Niyogi 1999, Li et al. 2000, Ruban et al. 2002, Aspinall-O’Dea et al. 2002, Li et al. 2004). Mechanistically, qE works by switching the photosynthetic antenna from a state of energy capture to one of thermal dissipation, decreasing the efficiency of energy transfer to the PSII reaction center (Horton et al. 1996). Although it is not always the case (Hurry et al. 1997, Demmig-Adams et al. 1999, Sane et al. 2002, Ivanov et al. 2003, Sane et al. 2003, Finazzi et al. 2004), qN is thought to occur through the interconversion of the light-harvesting xanthophyll, violaxanthin (V), to the energy-quenching xanthophylls, antheraxanthin (A) and zeaxanthin (Z), otherwise known as the xanthophyll cycle (Demmig-Adams et al. 1999). In addition to its role in non-photochemical quenching, Z also appears to act as an anti-oxidant to protect against photo-oxidative stress (Havaux and Niyogi 1999, Baroli et al. 2003). Carotenoids are also important as structural components of LHCII, involved in the stabilization of the native LHCII trimer as well as folding and assembly of LHCII monomers (Plumley and Schmidt 1987, Kühlbrandt et al. 1994, Booth and Paulsen 1996, Paulsen 1999, Lokstein et al. 2002, Liu et al. 2004). Horton et al. (Horton et al. 1991, Horton et al. 1999) suggest that the xanthophyll cycle pigments, V and Z, loosely bound to the periphery of LHCII may be important in stabilizing LHCII supramolecular aggregates. Two major mechanisms have been proposed to account for antenna quenching via the xanthophyll cycle. The direct mechanism proposes that the S1 state of A and Z within LHCII is lower than that of Chl a within the antenna pigment bed. Thus, the S1 state of antenna chlorophyll is able to transfer energy to A and Z but not to V. Consequently, excited state A and Z decay to ground state with the release of heat (Frank et al. 1994). Thus, the light-dependent, reversible interconversion of V to A and Z is able to regulate the energy transfer within LHCII (Frank et al. 1994). In contrast, the indirect mechanism proposes that the trans-thylakoid ∆pH and the xanthophyll cycle pigments regulate the aggregation state of LHCII which affects the rapidly relaxing qE component of non-photochemical quenching (Horton et al. 1999, Wentworth et al. 2003). This may involve trimerization or the formation of monomeric LHCII (Elrad et al. 2002, Garab et al. 2002, Jackowski et al. 2003). Clearly, the underlying mechanism by which the xanthophyll cycle and overall LHCII organization regulate antenna quenching remains controversial (Polivka et al. 1999, Frank et

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al. 2000, Polivka et al. 2002, Dreuw et al. 2003a, Dreuw et al. 2003b, Ma et al. 2003, Holt et al. 2004). The seminal work of Trémolières and co-workers showed that phosphatidylglycerol (PG), the major phospholipid present in chloroplast thylakoid membranes, and its fatty acid composition play a crucial role in stabilizing the oligomeric state of LHCII (Dubacq and Trémolières 1983). A positively charged trimerization motif has been identified as a site on Lhcb which interacts with the negatively charged head group of PG (Hobe et al. 1994, Hobe et al. 1995, Trémolières and Siegenthaler 1998). Removal of this motif prevents the in vitro trimerization of Lhcb. In addition, Nußberger et al. (1993) showed that crystallization of trimeric LHCII was dependent upon the presence of both PG and digalactosyldiacylglycerol. Furthermore, in vitro studies by Trémolières and co-workers (Trémolières and Siegenthaler 1998) showed that the apparent stability of oligomeric LHCII is dependent not on PG content, but rather on the molecular species composition of PG such that oligomeric LHCII was stabilized when thylakoids exhibit high levels of PG 16:0/trans-∆3-hexadecenoic acid (trans-16:1) relative to PG 16:0/16:0 (Dubertret et al. 1994, Selstam 1998). Trans16:1 of PG is a unique fatty acid for several reasons: (i) this fatty acid is only found in thylakoid membranes and exhibits a double bond in the trans configuration whereas all other fatty acids of the highly unsaturated chloroplast thylakoid membrane are in the cis configuration (Selstam 1998); (ii) trans-16: 1 is always esterified specifically to the sn-2 position of the glycerol backbone of PG; (iii) trans-16:1 is the only chloroplast fatty acid in angiosperms whose biosynthesis is strictly light dependent (Gray et al. 1997b, Trémolières and Siegenthaler 1998); and (iv) trans-16:1 is found exclusively in eukaryotic, Chl a/b-containing photoautotrophs (Selstam 1998). Huner and co-workers showed that acclimation of rye to low temperature resulted in a specific decrease in the content of PG containing trans-16:1 with a concomitant shift in the supramolecular organization of LHCII from its oligomeric to monomeric state (Huner et al. 1987, Huner et al. 1989). Furthermore, purification of rye LHC showed that PG is specifically bound to LHCII, and in vitro reconstitution experiments of delipidated rye LHCII showed that the conversion of monomeric LHCII to oligomeric LHCII in rye was strictly dependent upon the presence of PG containing trans-16:1 (Krupa et al. 1987, Krupa et al. 1992). Chloroplast biogenesis showed that LHCII is inserted into the thylakoid membrane in its monomeric form which subsequently is stabilized in its oligomeric form (Krol et al. 1988, Krol et al. 1989, Dreyfuss and Thornber 1994). Monitoring the kinetics of the light-dependent accumulation of trans-16:1 indicated that maximum trans-16:1 accumulation preceded the conversion of monomeric LHCII to oligomeric LHCII (Krol et al. 1988, Krol et al. 1989). However, it is clear that trans-16:1 in PG is not required to stabilize oligomeric LHCII in all plant species. Although orchids do not synthesize trans-16:1, LHCII is stabilized in its oligomeric form (Huner et al. 1989, Selstam 1998). Similarly, a fatty acid

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Fig. 1 The relative reduction state of QA estimated as 1 – qP in leaves of winter rye (S. cereale L. cv Musketeer). (A) and (B) Light–response curves. Development occurred at a temperature of 20°C with an irradiance of 800 (filled diamonds), 250 (filled circles) or 50 (filled squares) µmol m–2 s–1 or 5°C with an irradiance of 250 (open circles) or 50 (open squares) µmol m–2 s–1. Response was measured at the respective growth temperature of 20°C (A) or 5°C (B). C, Determinations under steady-state growth conditions. Measurements in (C) were performed at the growth temperature (20°C, filled bars; 5°C, open bars) and growth irradiance as indicated. All values represent means ± SE; n = 3. When not present, error bars are smaller than symbol size.

mutant of Arabidopsis thaliana which specifically lacks trans16:1 still exhibits oligomeric LHCII (McCourt et al. 1985). Clearly, factors other than molecular species of PG are also involved in the stabilization of oligomeric LHCII. We are unaware of any study which attempts to integrate data on the accumulation of this unique fatty acid, trans-16:1, in thyakoid PG and the supramolecular organization of LHCII with non-photochemical quenching through the antenna. In the present study, we assessed how light, temperature and excitation pressure modulate the trans-16:1 content of PG and relate this to the organization of LHCII both under controlled environment and under natural field conditions. We conclude that photoprotection through antenna quenching is not regulated by the supramolecular organization of LHCII in winter rye.

Fig. 2 Prevailing maximum and minimum mean daily temperatures (A) and precipitation (B) for the duration of the field experiments presented in Fig. 3.

800 µmol m–2 s–1) or 5°C at an irradiance of 50 and 150 µmol m–2 s–1 are illustrated in Fig. 1. Increasing the growth irradiance at either 20 (Fig. 1A) or 5°C (Fig. 1B) caused a significant decrease in 1 – qP at all light intensities measured. Furthermore, the value for 1 – qP at a measured irradiance of 800 µmol m–2 s–1 for rye leaves developed at 20/800 [growth temperature (°C)/growth irradiance (µmol m–2 s–1)] was comparable with 1 – qP measured at 250 µmol m–2 s–1 for rye leaves developed at 5/250 (Fig. 1C). Similarly, 1 – qP at a measured irradiance of 250 µmol m–2 s–1 for leaves developed at 20°C was similar to that of rye leaves developed at 5°C but measured at an irradiance of 50 µmol m–2 s–1 (Fig. 1C). Plants grown at 20/50 exhibited the lowest 1 – qP when measured at its respective growth irradiance (Fig. 1C). These results are consistent with previous reports for rye (Gray et al. 1997a, Gray et al. 1998) and confirm that rye leaves developed at 20/800 experience an excitation pressure comparable with those plants grown at 5/250, and those developed at 20/250 experience an excitation pressure comparable with those leaves developed at 5/50.

Results Relative reduction state of QA The 1 – qP light response curves for winter rye leaves developed at either 20°C and increasing irradiance (50, 250 and

Thylakoid lipid and fatty acid composition It is well established that low growth temperatures induce an increase in the extent of unsaturation of membrane lipids (Nishida and Murata 1996, Los and Murata 2002). The growth


Table 1

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Lipid composition from total leaf extracts of winter rye (S. cereale L. cv Musketeer)

Growth regime (°C/µmol m–2 s–1) 20/800 20/250 20/50 5/250 5/50

Lipid composition (mol %) PC MGDG DGDG

PG

PE

11 ± 1 9±3 9±2 10 ± 1 8±2

5±1 5±1 7±1 6±1 6±2

13 ± 3 14 ± 1 15 ± 2 14 ± 1 15 ± 1

36 ± 0 37 ± 2 37 ± 4 39 ± 3 38 ± 15

28 ± 2 27 ± 5 24 ± 2 25 ± 0 26 ± 13

SQDG 7±3 8±2 8±2 6±1 7±2

Lipid content was calculated as the molar ratio (mol%) of the total. All values represent means ± SD; n = 3. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol.

regime had minimal effects on the lipid composition (Table 1) and the unsaturation levels of monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol (data not shown), which is consistent with previous reports for rye (Huner et al. 1987). Huner and co-workers previously reported that low temperature induces a decrease in trans-16:1 levels of PG in various cold-tolerant plants (Huner et al. 1987, Huner et al. 1989). The data presented in Table 2 confirm that growth of rye at 5°C resulted in a lower ratio of trans-16:1/16: 0 than rye grown at 20°C and the same irradiance. Furthermore, there is a clear irradiance dependence on this ratio since the trans-16:1/16:0 ratio increased from 0.52 to 1.54 with increasing growth irradiance at 20°C and similarly increased from 0.29 to 0.70 with increasing growth irradiance at 5°C (Table 2). However, the trans-16:1/16:0 ratio was not correlated with 1 – qP since this ratio at 5/250 is 55% lower than that observed at 20/800 even though both growth conditions induce comparable values of 1 – qP (Fig. 1C). Similar trends were observed for the trans-16:1/16:0 ratio for rye grown at 5/50 and that grown at 20/250 (Table 2). The lipid and fatty acid analyses presented in Tables 1 and 2 represent experiments performed under controlled environment conditions. We examined the modulation of the trans-16: 1 content of PG in 10 cultivars of S. cereale L., four cultivars of Triticum aestivum L. and six cultivars of Hordeum vulgare L. under natural field conditions. Fig. 2 illustrates the prevailing environmental conditions for that year, including maxi-

Table 2

mum and minimum mean daily temperatures (Fig. 2A) and precipitation (Fig. 2B). Fig. 3A illustrates that, generally, the trans-16:1 content of PG in the 10 rye cultivars tested was highest in late June and lower in leaves sampled between November and March when the air temperature was the lowest (Fig. 2A). The lower trans-16:1 was associated with a concomitant increase in the content of 16:0 in PG (data not shown). Similar trends were observed for the wheat and barley cultivars, although the absolute amounts of trans-16:1 varied from that observed for rye under the field conditions (data not shown). Thus the changes in trans-16:1 content of PG from field samples are consistent with the data obtained under controlled growth conditions (Table 2). However, we note that the absolute levels of trans-16:1 in PG for samples collected in late June (40 mol%) were consistently higher than those observed for samples collected during growth under any regime in the growth cabinet. In fact, the highest level of trans-16:1 in PG (29 mol%) that we observed under controlled growth conditions was for rye grown at 20/800 (Table 2). Supramolecular organization of LHCII The effects of the growth regime on the supramolecular organization of LHCII was examined by using a non-denaturing electrophoretic gel system that allows for the separation of CP1, the Chl a protein complex containing P700; CP1a, the light-harvesting Chl–protein complex of PSI; CPa, the Chl a protein complex containing P680 and the associated core

Fatty acid profile of PG in total leaf extracts of winter rye (S. cereale L. cv Musketeer)

Growth regime (°C/µmol m–2 s–1) 20/800 20/250 20/50 5/250 5/50

16:0

trans-16:1

19 ± 1 25 ± 4 32 ± 2 27 ± 0 38 ± 2

29 ± 1 27 ± 2 16 ± 1 19 ± 0 11 ± 2

Fatty acid (mol %) trans-16:1/16:0 18:0 1.54 ± 0.09 1.07 ± 0.19 0.52 ± 0.02 0.70 ± 0.01 0.29 ± 0.06

1±0 2±1 1±0 1±0 1±1

18:1

18:2

18:3

1±0 2±1 2±0 1±0 1±1

9±1 8±1 10 ± 1 7±1 8±0

41 ± 1 36 ± 2 39 ± 2 45 ± 0 41 ± 1

Fatty acids were calculated as the molar ratio (mol%) of the total. All values represent means ± SD; n = 3. PG, phosphatidylglycerol; trans-16:1, trans-∆3-hexadecenoic acid.

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Fig. 3 Field data for 10 different cultivars of rye (S. cereale L.). (A) Content of trans-16:1 in PG. (B) Organization of LHCII. Plants were sampled in the summer (June), late fall (November), spring (March) and late spring (May) as indicated in the legend presented in (B). Values represent means ± SD; n = 3. PG, phosphatidylglycerol; trans-16: 1, trans-∆3-hexadecenoic acid.

antenna of PSII; as well as the monomeric and oligomeric forms of LHCII, the major Chl a/b LHC complex associated with PSII (Huner et al. 1987, Krol et al. 1988). The deoxycholic acid : SDS : Chl ratio utilized in our experiments minimized the amount of free pigment observed (