PhotosynthesisResearch 43: 273-282, 1995. © 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
Occurrence of the carotenoid lactucaxanthin in higher plant LHC II D e n i s e Phillip & A n d r e w J. Y o u n g * School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AE UK; *Author for correspondence Received27 September1994; acceptedin revisedform22 March 1995 Key words: carotenoid, lactucaxanthin, light-harvesting complex, Photosystem II, xanthophyll cycle
Abstract
The pigment composition of the light-harvesting complexes of Photosystem II (LHC II) has been determined for lettuce (Lactuca sativa). In common with other members of the composite, the photosynthetic tissues of this species may contain large amounts of the carotenoid lactucaxanthin (e, e-carotene-3,3'-diol) in addition to their normal compliment of carotenoids. The occurrence and distribution of lactucaxanthin in LHC II has been examined using isoelectric focusing of BBY particles followed by reversed-phase HPLC analysis of the pigments. The major carotenoids detected in LHC IIb, LHC IIa (CP29) and LHC IIc (CP26) purified from dark-adapted lettuce were lutein, violaxanthin, neoxanthin and lactucaxanthin. Lactucaxanthin has been shown to be a major component of PS II, accounting for ,~26% of total xanthophyll in both LHC lib (,-~23% total xanthophyll) and in the minor complexes (12-16%). In this study, LHC lib was clearly resolved into four bands and their carotenoid composition determined. These four bands proved to be very similar in their pigment content and composition, although the relative amounts of neoxanthin and lutein in particular were found to increase from bands 1 to 4 (i.e. with increasing electrophoretic mobility). The operation of the xanthophyll cycle has also been examined in the LHC of L. sativa following light treatment. The conversion efficiency for violaxanthin~zeaxanthin was nearly identical for each light-harvesting complex examined at 58-61%. Nearly half of the zeaxanthin formed in PS II was associated with LHC IIb, although the molar ratio of zeaxanthin:chlorophyll a was highest in the minor LHC. Abbreviations: HPLC-high performance liquid chromatography; IEF-isoelectric focusing; LHCII-lightharvesting complex associated with Photosystem II; PS II-Photosystem II; qE-ApH-dependent nonphotochemical quenching of chlorophyll fluorescence Introduction
Compared to other photosynthetic systems such as the algae and the phototrophic bacteria, the pigment composition of the photosynthetic membranes of higher plants is highly conserved. In addition to chlorophylls a and b, the following carotenoids have been found in all higher plant species examined to date: neoxanthin (5', 6'-epoxy-6, 7-didehydro-5, 6, 5', 6~-tetrahydro /~, fl-carotene-3, 5, 3'-triol), violaxanthin (5, 6, 5', 6'-diepoxy-5, 6, 5', 6'-tetrahydro-/~,/3-carotene-3, 3'diol), antheraxanthin (5, 6-epoxy-5, 6-dihydro-/~, /~carotene-3, 3'-diol), zeaxanthin (fl,/~-carotene-3, 3'-
diol), lutein (~, e-carotene-3, 3'-diol) and ~-carotene (/3, ~-carotene). In some plant species, a-carotene (/~, e-carotene) has been found to replace some of the /3-carotene, often in significant quantities (Goodwin 1980; Young and Britton 1989; Young 1993). In members of the Compositae, such as lettuce, the carotenoid lactucaxanthin (e, e-carotene-3,3'-diol) has also been detected (Siefermann-Harms et al. 1981). (See Fig. 1 for structures). The occurrence of this carotenoid in higher plant photosynthetic membranes is therefore relatively rare but in those species where it is present it may account from only a few percent to more than one third of total thylakoid carotenoid. It has there-
274 fore been suggested that this compound has an important role in light-harvesting although little, as yet, is known concerning its organisation in the photosynthetic apparatus (Siefermann-Harms and Ninnemann 1982; Siefermann-Harms 1987). The light-harvesting complex of Photosystem II (LHC II) contains the bulk of the pigment found in higher plant photosynthetic tissues (Thornber et al. 1993; Jansson 1994). LHC II comprises at least four different chlorophyll a/b binding proteins, of which one, LHC lib, binds ,,~65% of chlorophyll associated with PS II. The minor LH(2 II complexes, namely LHC IIa, LHC IIc and LHC IId have each been shown to contain approximately 5% of total chlorophyll. A number of studies have attempted to determine the carotenoid content and composition of the pigment protein complexes of higher plants. The majority of the early studies yielded relatively high levels of free pigment and the results must therefore be viewed with some caution. This is especially true when considering the location of the xanthophyll cycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin) due to their apparent low affinity to the LHC polypeptides. More recently, the use of low levels of glycosidic surfactants has allowed the carotenoid composition of the higher plant PS II light-harvesting complexes to be more accurately determined for barley (Bassi et al. 1993), maize (Peter and Thornber 1991) and spinach (Ruban et al. 1994a). One of the main findings of these studies was that each light-harvesting complex was shown to have a unique carotenoid composition. Two of these studies, namely Bassi et al. (1993) and Ruban et al. (1994a), have paid special attention to the location and behaviour of the xanthophyll cycle carotenoids. Of these, Ruban and colleagues (1994) have reported the location of zeaxanthin in PS II as a result of light treatment of leaves prior to isolation of the complexes. Whilst both studies are in agreement about the location of lutein and neoxanthin in LHC II, there is some discrepancy concerning that of xanthophyll cycle carotenoids. Ruban et al. (1994a) showed that the bulk (,,~50%) of PS II violaxanthin was associated with LHC IIb, whereas Bassi et al. (1993) reported that the minor complexes were specifically enriched in violaxanthin and only 18% associated with LHC IIb. The data of Peter and Thornber (1991) are more difficult to interpret as their results show that the vast majority of violaxanthin was associated with a fifth light-harvesting complex, termed LHC IIe. In addition, Bassi and colleagues (1993) have reported that bulk LHC IIb from barley lacks the abili-
ty to synthesise zeaxanthin from violaxanthin and that the minor complexes are therefore the key elements in the operation of the xanthophyll cycle. In contrast, Ruban et ai. (1994a) have observed significant zeaxanthin formation in spinach LHC IIb. Gruzecki and Krupa (1993) have recently reported that LHC II has zeaxanthin epoxidase activity. In addition to the total amount of zeaxanthin formed it is also perhaps important to consider the ratio of zeaxanthin:chlorophyll a for each individual complex, especially if a direct, singlet-singlet, interaction between these molecules leads to fluorescence quenching (Owens et al. 1992; Frank et al. 1994) as opposed to an 'indirect' (amplification) action through carotenoidmediated alterations to LHC organisation (Horton et al. 1991; Ruban et al. 1993, 1994b). This ratio is consistently highest in the minor complexes (Bassi et al. 1993; Ruban et al. 1994a), suggesting an important role for these LHC in photoprotection (Bassi et al. 1993).
Materials and methods
Plant material Leaves of six week old lettuce plants (Iceberg, Webbs Wonderful) were either dark adapted or illuminated in [98% N2 2% 02]. Such light treatment has been shown to induce conversion of violaxanthin to zeaxanthin (Noctor et al. 1991). Thylakoid membranes and PS II BBY particles (Crofts and Horton 1991) were isolated from dark-adapted and light-treated leaves as described before. LHC II was prepared from triton solubilised thylakoids after the method of Burke et al. (1978), as modified by Ruban and Horton (1992).
Isoelectric focusing For separation of LHC II components by nondenaturing IEF a procedure modified from that described by Bassi et al. (1991) was used (Ruban et al. 1994a). After measurement of pH values using a flat-tip electrode, each green band was carefully collected using a spatula and eluted using columns with a minimum volume of a solution containing 100 mM Hepes (pH 7.6) and 0.06% dodecyl-/~-D-maltoside. Room temperature absorption spectra were taken in this buffer immediately after collection using a Cecil 5001 spectrophotometer.
275
0
Arttheraxanthin
HO~
OH
i,,,~l I Neoxaathin
OH
OH
OI Lactucaxanthin tt0 Violaxanthia
HO ~
'
O
H
Lutein
HO~
O
H
Zeaxanthin Fig. 1. Structuresof the xanthophylls found in the light-harvesting complexesof Lactuca sativa.
13
F
t = 30.1 min 100% A 0% B. Solvents were of HPLC grade (Fisons) and were filtered and helium-degassed during use. Spectra were recorded over the range 300600 nm on a Hewlett-Packard 1040 diode-array detector, allowing integration of each individual pigment at their Amax.Carotenoids were quantified using external standards and published extinction coefficients (Davies 1976; Young and Britton 1993).
A
5
L 10
15
20
25
Time (rains) Typicalseparation of pigments extracted from thylakoids of dark-adapted lettuce (at 447 rim). See Materials and methods for full details of mobile and stationary phases. Peak identifications: A-neoxanthin; B-violaxanthin; C-antheraxanthin; D-lactucaxanthin; E-lutein; E Ft-chlorophyll a; G, G~-chlorophyll b; H, H~-all-trans and cis-isomers of/3-carotene. Fig. 2.
HPLC analysis Individual pigments were separated and quantified on reversed-phase HPLC (Fig. 2) using a Spherisorb ODS 2 column (5 #m particles, 25.0 c m x 4.6 ram) operating on a solvent gradient (flow rate = 1.0 ml/min) of: t = 0 min 100% solvent A (acetonitrile/water 9/1 v/v), 0% solvent B (ethyl acetate); t = 0-16 min to 40% A 60% B (linear gradient); t = 16-25 min 40% A 60% B (isocratic); t = 25.1-30.0 rain 0% A 100% B (isocratic);
Results
Thylakoids and BB Ys The pigment composition of thylakoids and BBY particles isolated from L. sativa is shown in Table 1. The major carotenoids found in these preparations were /3-carotene, lutein, violaxanthin, lactucaxanthin and neoxanthin. Other lettuce varieties and other species examined generally showed much reduced levels of lactucaxanthin, expressed as a percentage of total carotenoid (unpublished data). Lactucaxanthin represented ,,~15-16% of total carotenoid in both thylakoid and BBY preparations. The ratio of lutein:lactucaxanthin was fairly constant in these preparations at ,-~1.7:1. Following isoelectric focusing of BBY particles from L. sativa, six clearly resolved pigment-protein complexes were obtained between pI 4.05 and 4.66.
276 Table 1. Carotenoid composition of thylakoids and BBY particles isolated from dark-adapted and fight-treated lettuce (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (4- S.E.) of three replicates
Pigment
Dark adapted Thylakoids BBYs
Light-treated Thylakoids BBYs
Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin /3-Carotene
7.8 (0.2) 12.8 (0.3) N.D. N.D. 6.3 (0.3) 14.4 (0.4) 11.4 (0.5)
7.9 (0.3) 13.5 (0.4) N.D. N.D. 7.2 (0.2) 11.0 (0.4) 11.3 (0.3)
7.5 (0.2) 13.0 (0.4) 2.5 (0.1) 7.0 (0.3) 6.1 (0.2) 4.2 (0.1) 12.1 (0.4)
8.3 (0.3) 14.5 (0.5) 2.3 (0.1) 5.6 (0.2) 6.6 (0.3) 2.5 (0.1) 11.4 (0.4)
Chl a:b Car:Chl V+A+Z Z% Lut:Lact
2.9:1 0.27:1 14.4 (0.4) 1.62:1
2.6:1 0.25:1 11.0 (0.4) 1.71:1
3.1:1 0.25:1 13.7 (0.3) 51.1 (0.6) 1.72:1
2.6:1 0.23:1 10.4 (0.2) 53.8 (0.6) 1.74:1
N.D. = not detected.
A
Wavelength(rim)
4~o
s~o
6bo
Wavelength (nm) Fig. 3. Room temperature absorption spectra of eluted complexes in buffer (see Materials and methods for details): A. LHC lib (lighttreated), bands 1-4. B. LHC IIa and LHC IIc (light-treated).
No difference was observed in terms of pI values, absorption spectra or pigment composition for bands obtained from IEF using either thylakoids or BBYs. This IEF pattern was almost identical to that seen in spinach (Ruban et al. 1994a) and similar to that reported for maize (Bassi et al. 1991; Bassi and Dainese 1992), although LHC IId could not be clearly resolved for lettuce (for spinach BBYs prepared and subjected to IEF under identical conditions LHC IId could be clearly identified and excised from the gel). In the present study it was also possible to obtain sufficient resolution of IEF bands 1--4 (LHC IIb, pI 4.07--4.28) in addition to LHC IIc (pI 4.50) and LHC IIa (pI 4.68) to allow their pigment composition to be determined. Their absorption spectra (Fig. 3) were nearly identical to those reported for spinach (Ruban et al. 1994a). The ratios of chlorophyll alb for the LHC II complexes are given in Tables 2 and 3. A ratio of 1.2:1 for the LHC IIb fractions is similar to earlier published values (Bassi et al. 1993) as is the distribution of chlorophyll between these complexes: 66-67% chlorophyll in LHC IIb, ,,,4% in LHC IIa and 6-8% in LHC IIc (Peter and Thornber, 1991; Ruban et al. 1994a). The ratios of chlorophyll a:b for LHC IIa and LHC IIc were 4.2-~-4.3:1 and 2.6-2.7:1, respectively. The distribution of total carotenoid between these PS II light-harvesting complexes was very similar to that of chlorophyll. In the case of both sets of pigments,
277 Table 2. Pigment compositionof LHC lib purified from BBYs isolated from (A) dark-adapted and (B) light-treatedlettuce by IEF (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (-4-S.E.) of three replicates
Pigment Band 1
LHC lib - Dark-adapted Band 2 Band 3
Band 4
12.3 (0.2) 20.5 (0.4) N.D. N.D. 10.2 (0.2) 2. 8 (0,1)
12.8 (0.1) 21.0 (0.4) N.D. N.D. 11.4 (0.2) 2.9 (0.1)
13.1 (0.4) 22.0(0.2) N.D. N.D. 12.8 (0.1) 3.0 (0.1)
14.7 (0.2) 24.4 (0.4) N.D. N.D. 15.6 (0.3) 3.6 (0.2)
1.2:1 0.16:1 2.8 (0.1)
1.2:1 0.17:1 2.9 (0.1)
1.2:1 0.18:1 3.0 (0.1)
1.2:1 0.20:1 3.6 (0.2)
1.67:1
1.64:1
1.67:1
1.60:1
(A) Lactucaxanthin Lntein Antheraxanthin Zeaxananthin Neoxanthin Violaxanthin Chl a:b
Car:Chl V+A+Z Z% Lut:Lact (B) Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin
10.6 (0.1) 16.7 (0.3) tr 2.3 (0.1) 9.7 (0.2) 1.6 (0.1)
LHC IIb- Light-treated 10.8 (0.2) 12.2 (0.2) 18.5 (0.4) 21.4(0.5) tr tr 2.5 (0.1) 2.4 (0.1) 13.0 (0.3) 14.8 (0.3) 1.6 (0.1) 1.6 (0.1)
14.4 (0.4) 24.3 (0.4) tr 2.7 (0.1) 17.1 (0.1) 1.9 (0.1)
Chl a:b
1.2:1 1.05:1 3.9 (0.2) 58.9% 1.58:1
1.3:1 0.16:1 4.1 (0.2) 60.9% 1.71:1
1.2:1 0.20:1 4.6 (0.2) 58.7% 1.69:1
Car:Chl V+A+Z Z% Lut:Lact
1.2:1 0.19:1 4.0 (0.2) 60.0% 1.75:1
N.D. = not detected; tr = trace amounts only.
the remainder (not ascribed to any particular LH band) accounted for ,-~12% of pigment. Following light treatment, zeaxanthin was formed following de-epoxidation of violaxanthin and typically accounted for 50--60% o f the xanthophyll cycle pool, as has been typically reported for many other plant species ( D e m m i g - A d a m s 1990; D e m m i g - A d a m s and Adams 1992, 1993). Levels o f the intermediate in this reaction, antheraxanthin, were generally very small and only accounted for a few percent of total carotenoid. A high proportion o f the violaxanthin (,,~ 19%) in darkadapted spinach and ,,~24% and 10% ofzeaxanthin and antheraxanthin, respectively, formed as a result of light treatment o f leaves, was not associated with the main
light-harvesting complexes. These values are much higher than for any o f the other carotenoids present in PS II. L H C lib
The room temperature absorption spectra for the four LHC IIb fractions obtained during I E F o f BBYs all had strong characteristic peaks at 474 nm and 652 nm (Fig. 3). Little difference in their absorption spectra was observed between these separate bands for either dark-adapted or light-treated leaves. The pigment composition o f bands 1--4 is given in Tables 2A and 2B for dark-adapted and light-
278 Table 3. Pigment composition of LHC Ha and LHC IIc purified from BBYs isolated from dark-adapted and light-treated lettuce by IEF (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (~- S.E.) of three replicates Pigment
Dark-adapted LHC IIa LHC IIc
Light-treated LHC IIa LHC IIc
Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin /3-Carotene
4.9 (0.3) 12.2 (0.9) N.D. N.D. 8.4 (0.4) 16.6 (0.8) 7.5 (0.3)
7.4 (0.1) 14.8 (0.1) N.D. N.D. 7.6 (0.4) 15.4 (0.4) 11.4 (0.5)
5.1 (0.1) 13.7 (0.4) 3.0 (0.1) 9.3 (0.3) 7.5 (0.2) 4.2 (0.1) 8.2 (0.3)
7.5 (0.1) 14.1 (0.4) 3.1 (0.2) 8.7 (0.4) 6.2 (0.1) 4.0 (0.2) 12.2 (0.1)
Chl a:b
4.3:1 0.28:1 16.6 (0.8)
2.5:1 0.26:1 15.4 (0.4)
4.1:1 0.29:1 16.5 (0.3) 56.4 (0.4) 2.68:1
2.7:1 0.23:1 15.8 (0.3) 55.1 (0.3) 1.88:1
Car:Chl V+A+Z Z% Lut:Lact
2.48:1
2.00:1
N.D. = not-detected.
Table 4. Distribution of xanthophylls in light-harvesting complexes (LHC lib, LHC IIa and LHC IIc) of light-treated and dark-adapted lettuce. Data are expressed as a percentage of total xanthophyll applied to the gel and are the means of at least three separate analyses Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Band 7
Rem
19.4 14.4 6.9 11.8 17.2 9.0
19.4 16.4 6.9 11.8 18.7 9.0
19.9 19.2 6.9 11.6 20.8 9.4
20.4 23.3 6.9 12.8 24.9 9.6
4.3 6.5 17.2 12.0 4.8 14.8
6.5 8.9 20.7 17.2 1.8 9.9
4.8 4.5 10.3 8.6 5.4 19.8
5.4 6.8 24.1 13.8 6.4 18.7
19.7 13.4 14.8 8.6
19.8 17.4 19.7 8.8
19.8 20.1 21.3 9.0
21.7 21.4 23.0 9.3
4.0 6.7 5.3 15.2
5.7 9.4 2.3 9.4
4.5 4.8 6.1 20.4
4.8 6.4 7.5 19.3
Light-treated Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin
Dark-adapted Lactucaxanthin Lntein Neoxanthin Violaxanthin
Band identification: 1-4 LHC IIb; 5 LHC llc; 6 PS II Core; 7 LHC IIa; Rem = Remainder.
treated leaves, respectively. The ratio of chlorophyll a:b at 1.2:1 in all four LHC IIb bands is consistent with previous studies (e.g. Bassi et al. 1993). Whilst the chlorophyll composition is relatively constant, the carotenoid composition of these complexes does vary. /3-Carotene was consistently absent from all LHC IIb preparations and only xanthophylls were detect-
ed. Lutein was the single most abundant carotenoid in LHC IIb (20-24 mol/100 chlorophyll a for bands 1-4), whilst levels of neoxanthin and lactucaxanthin were lower at between 10-16 mol/100 chlorophyll a. The ratio of total carotenoid:total chlorophyll increased from 0.15:1 to 0.20:1 in bands 1-4 (i.e. with increasing migration) due to changes in the levels of all the
279 carotenoids relative to chlorophyll (Table 2). The ratio of lutein:lactucaxanthin remained fairly constant in all four bands as did the pool size of the xanthophyll cycle carotenoids (3-5 mol/100 chlorophyll a). Levels of violaxanthin and, after light treatment, zeaxanthin only ever accounted for a few percent of total carotenoid in each LHC IIb band and antheraxathin was only ever observed in trace amounts (below the levels required for integration on the HPLC). Although the xanthophyll cycle carotenoids are a relatively minor component of each of these bands, nearly half of the PS II xanthophyll cycle pool is associated with LHC IIb itself (Table 4). The data shown in Tables 2A and 2B indicate that the xanthophyll cycle pool size increased following light treatment (resulting in zeaxanthin formation). No satisfactory explanation can be given for this behaviour. The conversion efficiency for violaxanthin~zeaxanthin was similar in all four LHC IIb bands at ,-~60%.
Minor LHC H The pigment composition of the minor LHC II complexes (as defined on IEF by Bassi et al. 1991; Bassi and Dainese 1992) is shown in Table 3. Both LHC IIa and LHC IIc were each found to contain only 4 5% each of total PS II lactucaxanthin (Table 4). This compares with >80% found in LHC IIb. The ratio of lactucaxanthin:lutein was also much higher in these two minor complexes compared to LHC IIb at ,-2:1. The pigment composition of LHC IIc was, however, noticeably more variable than the other complexes which may result due to cross-contamination from LHC IIb. Analysis of the carotenoid content of the minor complexes isolated from dark-adapted leaves of L. sativa, shows that, unlike LHC IIb, violaxanthin is the single most abundant component in both LHC IIa and LHC IIc (16 mol/100 chlorophyll a, see Table 3). The distribution of violaxanthin and zeaxanthin in LHC IIa and LHC IIc is given in Table 4. LHC IIa and LHC IIc possess ,,-20% and ,,~15% of PS II violaxanthin respectively. Light treatment of leaves of induces zeaxanthin formation via violaxanthin de-epoxidation in both LHC IIa and LHC IIc. The conversion efficiency for zeaxanthin formation from violaxanthin in both of these complexes was only slightly lower than that observed in LHC lib, at 55-56%, although the deepoxidation state was higher due to the accummulation of relatively high amounts of antheraxanthin (present in only trace amounts in LHC IIb). Whilst LHC IIa only
contains 8-9% of PS II zeaxanthin and LHC IIc 12% it is clear that in these minor complexes the xanthophyll cycle carotenoids represent a much higher proportion of total carotenoid. In contrast to LHC IIb, significant amounts of/5carotene were detected in both minor LHC. This concurs with Bassi et al. (1993) who found that fl-carotene accounted for ,-5% of total carotenoid in both LHC IIa and LHC IId and concluded that ~-carotene was a genuine component of these minor PS II complexes. Data from this study for/~-carotene levels in lettuce (Table 3) were much higher than those reported by Bassi and co-workers for barley, at ,-~15% of total carotenoid for both complexes. The relative levels of ~-carotene and the xanthophylls in higher plant photosynthetic tissues are known to be affected by growth conditions (especially incident light intensity) and large inter-specific differences may be expected. No data are available for LHC IId as this complex was not observed following IEF of either thylakoids or BBYs. Under identical preparative and electrophoretic conditions for spinach BBYs, LHC IId could be clearly resolved and it's pigment composition determined by HPLC analysis.
Discussion
Lactucaxanthin in higher plant LHC Lactucaxanthin is not commonly found in the photosynthetic tissues of higher plant and levels of this carotenoid may vary substantially from one species to another. In this study on lettuce, lactucaxanthin was found to represent as much as 21% of total thylakoid carotenoid and 24% of PS II carotenoid. In some other plant species, notably Eunonymus spp. this level may rise to nearly 35% of thylakoid carotenoid so that levels may be greater than those of lutein (D. Phillip and A. Young, unpublished data). Clearly therefore this rare carotenoid may, in some species, be a very important component of the pigment bed. Lactucaxanthin was found in all the major and minor LHC II complexes of L. sativa. The ratio of lutein:lactucaxanthin was higher in both LHC IIa and LHC IIc compared to the four separate LHC IIb fractions. It is interesting to compare this data with that obtained from spinach LHC II (prepared in an identical manner; Ruban et al. 1994a; D. Phillip and A. Young, unpublished data). It is noteworthy that the molar ratio of the sum of lactucaxanthin and lutein
280 Table 5. Xanthophyll Stoichiometry in LHC II. Values for the present study have been calculated using the chlorophyll a content of these complexes determined by Bassi et al (1993): LHC l i b - 8 per monomer; LHC Ila and LHC IIc - 6 per monomer Hertrysson
Peter and
Bassi et
Ruban et
Present
et al.
Thornber
al. (1993)
al.
study
(1989)
(1991)
(1994a)
LHC lib Neoxanthin
-
1
0.5
1
1
Violaxanthin
-
0.5
tr
0.3*
0.3*
Lutein
-
2
2
1.7
Lactucaxanthin
.
2 .
.
.
1
LHC lla Neoxanthin
1
1
1
0.5
Violaxanthin
1
2
1.5
1
1
Lutein
1
2
2
1
0.7
Lactucaxanthin
.
.
.
.
0.5
0.3
LHC llc Neoxanthin
-
1
0.5
1
Violaxanthin
-
0.5
1
I
i
Lutein
-
2
2
1
1
Lactucaxanthin
.
tr = trace amounts only; *
equivalent
.
.
.
0.5
0.5
to 1 violaxanthin per trimer
to chlorophyll a is nearly identical to that of lutein alone in spinach. Other data obtained on other members of the compositae, including other varieties of lettuce also show this trend and although the levels of these two carotenoids are variable their sum remains relatively constant accounting for 30-35% total thylakoid carotenoid. This is similar to that observed in an early study on the pigment content of lettuce thylakoids and LHC (Siefermann-Harms and Ninnemann 1982). The calculated stoichiometry of xanthophylls in LHC II for L. sativa is shown in Table 5. Data from other recent studies are also shown for comparison. The values presented in Table 5 have been calculated using the chlorophyll a contents as used by Bassi and colleagues (1993) and adopted by many other groups: i.e. 8 chlorophyll a per LHC IIb monomer and 6 each for LHC II and LHC IIc. Pigment stoichiometry for LHC II in L. sativa is in broad agreement with those of previous studies. However, the data shown in Table 5 indicate sub-stoichiometric amounts of lutein in LHC IIb and LHC IIa and sub-stoichiometric amounts of lactucaxanthin in both minor complexes. Other studies consistently calculate there to be six lutein molecules
per trimer. Clearly in L. sativa, in which only five luteins were estimated per trimer, a proportion of lutein-binding sites must be occupied by other xanthophylls, of which lactucaxanthin is the most likely candidate. Both minor LHC have three carotenoid molecules per polypeptide. The carotenoids, lactucaxanthin (e, e-carotene-3, 3'-diol), lutein (fl, e-carotene-3, 3'-diol) and, indeed, zeaxanthin (/3, fl-carotene-3, 3'-diol) are all very similar except in degree of conjugation (n = 9, 10 and 11 conjugated double bonds, respectively). The orientation adopted by the backbone of the polyene chain with respect to an end-group is dependent on the type of end-group present, i.e. whether the conjugation extends into the ring or not, for/3- or e-end-groups, respectively. In the case of lactucaxanthin (with two e-rings) this has the effect of altering the orientation of the end-groups, rendering them perpendicular to the main polyene chain, whereas the presence of one/3end-group in lutein will cause the end-group to adopt a near-planar orientation. There is no evidence to suggest that the conformation of the carotenoid molecule may be critical in determining its protein-binding, although twisting of the polyene backbone is evident
281 from Kiahlbrandt's model. The 3.4 ,~ resolution electronic crystallography model of LHC IIb published recently by Ktihlbrandt et al. (1994) shows that lutein occupies a central position in this complex and the orientation adopted by the two end-groups types of both these luteins are clearly visible. It is quite possible that lactucaxanthin may, due to its close structural similarities to lutein, simply be able to replace a proportion of lutein molecules occupying the central position this complex. The sub-stoichiometric amounts of these carotenoids determined in this study for all three lettuce LHC II complexes examined would certainly support this. At least some, or alternatively, all of the lactucaxanthin may occupy a position peripheral to the complex similar to that suggested for neoxanthin and violaxanthin in the model proposed by Ktihlbrandt and colleagues (1994). The carotenoid content of the LHC IIb trimer (the most likely functional unit) of L. sativa would be one violaxanthin, three neoxanthin, three lactucaxanthin and five lutein. The differences in calculated stoichiometry between studies apparent in Table 5 could be due to inter-specific differences but could also arise due to the use of different growth conditions (for example, the ratio of lactucaxanthin:lutein in L. sativa is greatly affected by light intensity - probably by regulation at the level of the/3- and e-cyclases; see also Johnson et al. 1993) and the use of different isolation procedures for the complexes adopted by different laboratories.
spinach (Ruban et al. 1994a), although ,,~50% of zeaxanthin formed in L. sativa is located in LHC Ilb, rather than in the minor LHC, the molar ratio of zeaxanthin:chlorophyll a is very low. A particular feature of the minors was the relatively high ratio of zeaxanthin:chlorophyll a compared to LHC IIb. Although the molecular mechanism whereby zeaxanthin-mediated non-photochemical quenching of chlorophyll fluorescence arises is still not fully understood, this might indicate that the minor complexes have a role in this process. Unlike the study of Ruban et al. (1994a), all LHC exhibited similar conversion efficiencies for violaxanthin~zeaxanthin. In this study all the xanthophylls found in thylakoids and BBYs from L. sativa were also detected in the IEF bands corresponding to the PS II core. Whilst the core was found to contain high levels of ;3-carotene (as reported in other studies, e.g. Peter and Thornber 1991), the levels of some carotenoids (including lactucaxanthin), and neoxanthin in particular, were very low. The data shown in Table 4 also indicate that the xanthophyll cycle operates in the PS II core and that the production of zeaxanthin is very efficient when compared to LHC IIb and the minors. Clearly, the variations in the carotenoid content and composition of the PS II core (and indeed PS I as a whole; see Thayer and Bjorkman 1992) needs a more thorough study.
Acknowledgements The xanthophyll cycle Thylakoids and BBYs obtained from both darkadapted and light-treated leaves resulted in almost identical behaviour on IEE It is important to note that, in L. sativa, zeaxanthin can be formed in all LHC II complexes examined, including LHC IIb which has almost half of PS II zeaxanthin (Tables 2 and 3). This is in agreement with the observations made on spinach LHC II by Ruban et al. (1994a) but contradicts the data of Bassi et al. (1993) for barley in which LHC IIb was reported to lack the ability to deepoxidate violaxanthin. According to our data for L. sativa, violaxanthin is present in LHC IIb at ,,~0.3 molecules per monomer supporting the conclusions of Ruban et al. (1994a) that one violaxanthin molecule is present per trimer. Both minor complexes bind one violaxanthin molecule per polypeptide. The extent of de-epoxidation in bands 1-4 is similar at 58-61% of the xanthophyll cycle pool and is only slightly lower in LHC IIa and LHC IIc. As with
We thank Peter Horton, Sasha Ruban and Pam Scholes (University of Sheffield, UK) for their assistance in the preparation of complexes. This work was supported by a LJMU Research Grant.
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282 Davies BH (1976) Carotenoids. In: Goodwin TW (ed) Chemistry and Biochemistry of Plant Pigments, 2nd ed, Vol 2, pp 38-165. Academic Press, London Demmig-Adams B (1990) Carotenoids and photoprotection: A role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 124 Demmig-Adams B and Adams WW (1992) Photoprotection and other responses of plants to high light stress. Aanu Rev Plant Physiol Plant Mol Bio143:599--626 Demmig-Adams B and Adams WW (1993) The xanthophyll cycle. In: Young AJ and Britton G (eds) Carotenoids in Photosynthesis, pp 206-252. Chapman and Hall, London Frank HA, Cua A, Chynwat V, Young A J, Gosztola D and Wasielewski MR (1994) Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis. Photosynth Research, 41:389-395 Goodwin TW (1980) The Biochemistry of Carotenoids, Vol 1, Plants. Chapman and Hall, London Gruszecki WI and Krupa Z (1993) LHC II preparation exhibits properties of a zeaxanthin expoxidase. J Photochem Photobiol B Biol 17:291-292 Hen_ryssonT, Schr&ler WP, Spangfort M and/~kerlund H-E (1989) Isolation and characterisation oftbe chlorophyll a-b protein complex CP29 from spinach. Biochim Biophys Acta 977:301-308 Horton P, Ruban AV, Rees D, Pascal A, Noctor G and Young AJ (1991) Control of the light-harvesting function of chloroplast membranes by aggregation of the LHC II chlorophyll-protein complex. FEBS Lett 292:1-4 Jansson S (1994) The light-harvesting chlorophyll a/b-binding proteins. Biochim Biophys Acta 1184:1-19 Johnson GN, Young AJ, Scholes JD and Horton P (1993) The dissipation of excess excitation energy in British plant species. Plant Cell Environ 16:681-686 Kiihlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex. Nature 367:614--621 Noctor G, Rees D, Young A and Horton P (1991) The relationship between zeaxanthin, energy-dependent quenching of chlorophyll fluorescence and the transthyalkoid pH-gradient in isolated chloroplasts. Biochim Biophys Acta 1057:320-330 Owens TG, Shreve AP and Albrecht AC (1992) Dynamics and mechanism of singlet energy transfer between carotenoids and chlorophylls: Light-harvesting and non-photochemical quenching. In: Murata N (ed) Research in Photosynthesis, Vol 4, pp 179-186. Kluwer Academic Publishers, Dordrecht Peter GF and Thornber JP ( 1991) Biochemical composition and organization of higher plant Photosystem II light-harvesting pigment proteins. J Biol Chem 266:16745-16754
Ruban AV and Horton P (1992) Mechanism of ApH-dependent dissipation of absorbed excitation energy of photosynthetic membranes. I. Spectroscopic analysis of isolated light-harvestingcomplexes. Biochim Biophys Acta 1102:30-38 Ruban AV, Young, AJ and Horton P (1993) Induction of nonphotochemical energy dissipation and absorbance changes in leaves. Evidence for changes in the state of the light-harvesting system of Photosystem II in vivo. Plant Physiol 102:741-750 Ruhan AV, Young A J, Pascal AA and Horton P (1994a) The effects of illumination on the xanthophyll composition of the Photosystem II light-harvesting complexes of spinach thylakoid membranes. Plant Physiol 104:227-234 Ruban AV, Young AJ and Horton P (1994b) Modulation of chlorophyll fluorescence quenching in isolated light-harvesting complex of Photosystem II. Biochim Biophys Acta 1186: 123-127 Siefermann-Harms D (1987) The light-harvesting and protective functions of carotenoids in photosynthetic membranes. Physiol Plantarum 69:561-568 Siefermann-Harrns D and Ninnemann H (1982) Pigment organization in the light-harvesting chlorophyll-a/b-protein complex of lettuce chloroplasts. Evidence obtained from protection against proton attack and from energy transfer. Photochem Photobio135: 719-731 Siefermann-Harms D, Hertzberg S, Borch G and Liaaen-Jensen S (1981) Lactucaxanthin, an e, e-carotene-3,3t-diol from Lactuca sativa. Phytocbemistry 20:85-88 Thayer SS and Bj0rkman O (1990) Leaf xanthophyli content and composition in sun and shade determined by HPLC. Photosynth Res 33:213-226 Thayer SS and Bjtrkman 0 (1992) Carotenoid distribution and deepoxidation in thylakoid pigment protein complexes from cotton leaves and bundle sheath cells of maize. Photosynth Res 33:213226 Thomber JE Peter GF, Morishige DT, Gomez S, Anandan S, Welty BA, Lee A, Kerfeld C, Takeuchi T and Preiss S (1993) Lightharvesting systems I and II. Biochem Soc Trans 21:15-18 Young AJ (1993) Occtm~nce and distribution of carotenoids in photosynthetic systems. In: Young AJ and Britton G (eds) Carotenoids in Photosynthesis, pp 16-71. Chapman and Hall, London Young AJ and Britton G (1989) The distribution of a-carotene in the photosynthetic pigment-protein complexes of higher plants. Plant Sci 64:179-183 Young AJ and Britton G (eds) (1993) Carotenoids in Photosynthesis. Chapman and Hall, London
Occurrence of the carotenoid lactucaxanthin in higher plant LHC II D e n i s e Phillip & A n d r e w J. Y o u n g * School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AE UK; *Author for correspondence Received27 September1994; acceptedin revisedform22 March 1995 Key words: carotenoid, lactucaxanthin, light-harvesting complex, Photosystem II, xanthophyll cycle
Abstract
The pigment composition of the light-harvesting complexes of Photosystem II (LHC II) has been determined for lettuce (Lactuca sativa). In common with other members of the composite, the photosynthetic tissues of this species may contain large amounts of the carotenoid lactucaxanthin (e, e-carotene-3,3'-diol) in addition to their normal compliment of carotenoids. The occurrence and distribution of lactucaxanthin in LHC II has been examined using isoelectric focusing of BBY particles followed by reversed-phase HPLC analysis of the pigments. The major carotenoids detected in LHC IIb, LHC IIa (CP29) and LHC IIc (CP26) purified from dark-adapted lettuce were lutein, violaxanthin, neoxanthin and lactucaxanthin. Lactucaxanthin has been shown to be a major component of PS II, accounting for ,~26% of total xanthophyll in both LHC lib (,-~23% total xanthophyll) and in the minor complexes (12-16%). In this study, LHC lib was clearly resolved into four bands and their carotenoid composition determined. These four bands proved to be very similar in their pigment content and composition, although the relative amounts of neoxanthin and lutein in particular were found to increase from bands 1 to 4 (i.e. with increasing electrophoretic mobility). The operation of the xanthophyll cycle has also been examined in the LHC of L. sativa following light treatment. The conversion efficiency for violaxanthin~zeaxanthin was nearly identical for each light-harvesting complex examined at 58-61%. Nearly half of the zeaxanthin formed in PS II was associated with LHC IIb, although the molar ratio of zeaxanthin:chlorophyll a was highest in the minor LHC. Abbreviations: HPLC-high performance liquid chromatography; IEF-isoelectric focusing; LHCII-lightharvesting complex associated with Photosystem II; PS II-Photosystem II; qE-ApH-dependent nonphotochemical quenching of chlorophyll fluorescence Introduction
Compared to other photosynthetic systems such as the algae and the phototrophic bacteria, the pigment composition of the photosynthetic membranes of higher plants is highly conserved. In addition to chlorophylls a and b, the following carotenoids have been found in all higher plant species examined to date: neoxanthin (5', 6'-epoxy-6, 7-didehydro-5, 6, 5', 6~-tetrahydro /~, fl-carotene-3, 5, 3'-triol), violaxanthin (5, 6, 5', 6'-diepoxy-5, 6, 5', 6'-tetrahydro-/~,/3-carotene-3, 3'diol), antheraxanthin (5, 6-epoxy-5, 6-dihydro-/~, /~carotene-3, 3'-diol), zeaxanthin (fl,/~-carotene-3, 3'-
diol), lutein (~, e-carotene-3, 3'-diol) and ~-carotene (/3, ~-carotene). In some plant species, a-carotene (/~, e-carotene) has been found to replace some of the /3-carotene, often in significant quantities (Goodwin 1980; Young and Britton 1989; Young 1993). In members of the Compositae, such as lettuce, the carotenoid lactucaxanthin (e, e-carotene-3,3'-diol) has also been detected (Siefermann-Harms et al. 1981). (See Fig. 1 for structures). The occurrence of this carotenoid in higher plant photosynthetic membranes is therefore relatively rare but in those species where it is present it may account from only a few percent to more than one third of total thylakoid carotenoid. It has there-
274 fore been suggested that this compound has an important role in light-harvesting although little, as yet, is known concerning its organisation in the photosynthetic apparatus (Siefermann-Harms and Ninnemann 1982; Siefermann-Harms 1987). The light-harvesting complex of Photosystem II (LHC II) contains the bulk of the pigment found in higher plant photosynthetic tissues (Thornber et al. 1993; Jansson 1994). LHC II comprises at least four different chlorophyll a/b binding proteins, of which one, LHC lib, binds ,,~65% of chlorophyll associated with PS II. The minor LH(2 II complexes, namely LHC IIa, LHC IIc and LHC IId have each been shown to contain approximately 5% of total chlorophyll. A number of studies have attempted to determine the carotenoid content and composition of the pigment protein complexes of higher plants. The majority of the early studies yielded relatively high levels of free pigment and the results must therefore be viewed with some caution. This is especially true when considering the location of the xanthophyll cycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin) due to their apparent low affinity to the LHC polypeptides. More recently, the use of low levels of glycosidic surfactants has allowed the carotenoid composition of the higher plant PS II light-harvesting complexes to be more accurately determined for barley (Bassi et al. 1993), maize (Peter and Thornber 1991) and spinach (Ruban et al. 1994a). One of the main findings of these studies was that each light-harvesting complex was shown to have a unique carotenoid composition. Two of these studies, namely Bassi et al. (1993) and Ruban et al. (1994a), have paid special attention to the location and behaviour of the xanthophyll cycle carotenoids. Of these, Ruban and colleagues (1994) have reported the location of zeaxanthin in PS II as a result of light treatment of leaves prior to isolation of the complexes. Whilst both studies are in agreement about the location of lutein and neoxanthin in LHC II, there is some discrepancy concerning that of xanthophyll cycle carotenoids. Ruban et al. (1994a) showed that the bulk (,,~50%) of PS II violaxanthin was associated with LHC IIb, whereas Bassi et al. (1993) reported that the minor complexes were specifically enriched in violaxanthin and only 18% associated with LHC IIb. The data of Peter and Thornber (1991) are more difficult to interpret as their results show that the vast majority of violaxanthin was associated with a fifth light-harvesting complex, termed LHC IIe. In addition, Bassi and colleagues (1993) have reported that bulk LHC IIb from barley lacks the abili-
ty to synthesise zeaxanthin from violaxanthin and that the minor complexes are therefore the key elements in the operation of the xanthophyll cycle. In contrast, Ruban et ai. (1994a) have observed significant zeaxanthin formation in spinach LHC IIb. Gruzecki and Krupa (1993) have recently reported that LHC II has zeaxanthin epoxidase activity. In addition to the total amount of zeaxanthin formed it is also perhaps important to consider the ratio of zeaxanthin:chlorophyll a for each individual complex, especially if a direct, singlet-singlet, interaction between these molecules leads to fluorescence quenching (Owens et al. 1992; Frank et al. 1994) as opposed to an 'indirect' (amplification) action through carotenoidmediated alterations to LHC organisation (Horton et al. 1991; Ruban et al. 1993, 1994b). This ratio is consistently highest in the minor complexes (Bassi et al. 1993; Ruban et al. 1994a), suggesting an important role for these LHC in photoprotection (Bassi et al. 1993).
Materials and methods
Plant material Leaves of six week old lettuce plants (Iceberg, Webbs Wonderful) were either dark adapted or illuminated in [98% N2 2% 02]. Such light treatment has been shown to induce conversion of violaxanthin to zeaxanthin (Noctor et al. 1991). Thylakoid membranes and PS II BBY particles (Crofts and Horton 1991) were isolated from dark-adapted and light-treated leaves as described before. LHC II was prepared from triton solubilised thylakoids after the method of Burke et al. (1978), as modified by Ruban and Horton (1992).
Isoelectric focusing For separation of LHC II components by nondenaturing IEF a procedure modified from that described by Bassi et al. (1991) was used (Ruban et al. 1994a). After measurement of pH values using a flat-tip electrode, each green band was carefully collected using a spatula and eluted using columns with a minimum volume of a solution containing 100 mM Hepes (pH 7.6) and 0.06% dodecyl-/~-D-maltoside. Room temperature absorption spectra were taken in this buffer immediately after collection using a Cecil 5001 spectrophotometer.
275
0
Arttheraxanthin
HO~
OH
i,,,~l I Neoxaathin
OH
OH
OI Lactucaxanthin tt0 Violaxanthia
HO ~
'
O
H
Lutein
HO~
O
H
Zeaxanthin Fig. 1. Structuresof the xanthophylls found in the light-harvesting complexesof Lactuca sativa.
13
F
t = 30.1 min 100% A 0% B. Solvents were of HPLC grade (Fisons) and were filtered and helium-degassed during use. Spectra were recorded over the range 300600 nm on a Hewlett-Packard 1040 diode-array detector, allowing integration of each individual pigment at their Amax.Carotenoids were quantified using external standards and published extinction coefficients (Davies 1976; Young and Britton 1993).
A
5
L 10
15
20
25
Time (rains) Typicalseparation of pigments extracted from thylakoids of dark-adapted lettuce (at 447 rim). See Materials and methods for full details of mobile and stationary phases. Peak identifications: A-neoxanthin; B-violaxanthin; C-antheraxanthin; D-lactucaxanthin; E-lutein; E Ft-chlorophyll a; G, G~-chlorophyll b; H, H~-all-trans and cis-isomers of/3-carotene. Fig. 2.
HPLC analysis Individual pigments were separated and quantified on reversed-phase HPLC (Fig. 2) using a Spherisorb ODS 2 column (5 #m particles, 25.0 c m x 4.6 ram) operating on a solvent gradient (flow rate = 1.0 ml/min) of: t = 0 min 100% solvent A (acetonitrile/water 9/1 v/v), 0% solvent B (ethyl acetate); t = 0-16 min to 40% A 60% B (linear gradient); t = 16-25 min 40% A 60% B (isocratic); t = 25.1-30.0 rain 0% A 100% B (isocratic);
Results
Thylakoids and BB Ys The pigment composition of thylakoids and BBY particles isolated from L. sativa is shown in Table 1. The major carotenoids found in these preparations were /3-carotene, lutein, violaxanthin, lactucaxanthin and neoxanthin. Other lettuce varieties and other species examined generally showed much reduced levels of lactucaxanthin, expressed as a percentage of total carotenoid (unpublished data). Lactucaxanthin represented ,,~15-16% of total carotenoid in both thylakoid and BBY preparations. The ratio of lutein:lactucaxanthin was fairly constant in these preparations at ,-~1.7:1. Following isoelectric focusing of BBY particles from L. sativa, six clearly resolved pigment-protein complexes were obtained between pI 4.05 and 4.66.
276 Table 1. Carotenoid composition of thylakoids and BBY particles isolated from dark-adapted and fight-treated lettuce (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (4- S.E.) of three replicates
Pigment
Dark adapted Thylakoids BBYs
Light-treated Thylakoids BBYs
Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin /3-Carotene
7.8 (0.2) 12.8 (0.3) N.D. N.D. 6.3 (0.3) 14.4 (0.4) 11.4 (0.5)
7.9 (0.3) 13.5 (0.4) N.D. N.D. 7.2 (0.2) 11.0 (0.4) 11.3 (0.3)
7.5 (0.2) 13.0 (0.4) 2.5 (0.1) 7.0 (0.3) 6.1 (0.2) 4.2 (0.1) 12.1 (0.4)
8.3 (0.3) 14.5 (0.5) 2.3 (0.1) 5.6 (0.2) 6.6 (0.3) 2.5 (0.1) 11.4 (0.4)
Chl a:b Car:Chl V+A+Z Z% Lut:Lact
2.9:1 0.27:1 14.4 (0.4) 1.62:1
2.6:1 0.25:1 11.0 (0.4) 1.71:1
3.1:1 0.25:1 13.7 (0.3) 51.1 (0.6) 1.72:1
2.6:1 0.23:1 10.4 (0.2) 53.8 (0.6) 1.74:1
N.D. = not detected.
A
Wavelength(rim)
4~o
s~o
6bo
Wavelength (nm) Fig. 3. Room temperature absorption spectra of eluted complexes in buffer (see Materials and methods for details): A. LHC lib (lighttreated), bands 1-4. B. LHC IIa and LHC IIc (light-treated).
No difference was observed in terms of pI values, absorption spectra or pigment composition for bands obtained from IEF using either thylakoids or BBYs. This IEF pattern was almost identical to that seen in spinach (Ruban et al. 1994a) and similar to that reported for maize (Bassi et al. 1991; Bassi and Dainese 1992), although LHC IId could not be clearly resolved for lettuce (for spinach BBYs prepared and subjected to IEF under identical conditions LHC IId could be clearly identified and excised from the gel). In the present study it was also possible to obtain sufficient resolution of IEF bands 1--4 (LHC IIb, pI 4.07--4.28) in addition to LHC IIc (pI 4.50) and LHC IIa (pI 4.68) to allow their pigment composition to be determined. Their absorption spectra (Fig. 3) were nearly identical to those reported for spinach (Ruban et al. 1994a). The ratios of chlorophyll alb for the LHC II complexes are given in Tables 2 and 3. A ratio of 1.2:1 for the LHC IIb fractions is similar to earlier published values (Bassi et al. 1993) as is the distribution of chlorophyll between these complexes: 66-67% chlorophyll in LHC IIb, ,,,4% in LHC IIa and 6-8% in LHC IIc (Peter and Thornber, 1991; Ruban et al. 1994a). The ratios of chlorophyll a:b for LHC IIa and LHC IIc were 4.2-~-4.3:1 and 2.6-2.7:1, respectively. The distribution of total carotenoid between these PS II light-harvesting complexes was very similar to that of chlorophyll. In the case of both sets of pigments,
277 Table 2. Pigment compositionof LHC lib purified from BBYs isolated from (A) dark-adapted and (B) light-treatedlettuce by IEF (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (-4-S.E.) of three replicates
Pigment Band 1
LHC lib - Dark-adapted Band 2 Band 3
Band 4
12.3 (0.2) 20.5 (0.4) N.D. N.D. 10.2 (0.2) 2. 8 (0,1)
12.8 (0.1) 21.0 (0.4) N.D. N.D. 11.4 (0.2) 2.9 (0.1)
13.1 (0.4) 22.0(0.2) N.D. N.D. 12.8 (0.1) 3.0 (0.1)
14.7 (0.2) 24.4 (0.4) N.D. N.D. 15.6 (0.3) 3.6 (0.2)
1.2:1 0.16:1 2.8 (0.1)
1.2:1 0.17:1 2.9 (0.1)
1.2:1 0.18:1 3.0 (0.1)
1.2:1 0.20:1 3.6 (0.2)
1.67:1
1.64:1
1.67:1
1.60:1
(A) Lactucaxanthin Lntein Antheraxanthin Zeaxananthin Neoxanthin Violaxanthin Chl a:b
Car:Chl V+A+Z Z% Lut:Lact (B) Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin
10.6 (0.1) 16.7 (0.3) tr 2.3 (0.1) 9.7 (0.2) 1.6 (0.1)
LHC IIb- Light-treated 10.8 (0.2) 12.2 (0.2) 18.5 (0.4) 21.4(0.5) tr tr 2.5 (0.1) 2.4 (0.1) 13.0 (0.3) 14.8 (0.3) 1.6 (0.1) 1.6 (0.1)
14.4 (0.4) 24.3 (0.4) tr 2.7 (0.1) 17.1 (0.1) 1.9 (0.1)
Chl a:b
1.2:1 1.05:1 3.9 (0.2) 58.9% 1.58:1
1.3:1 0.16:1 4.1 (0.2) 60.9% 1.71:1
1.2:1 0.20:1 4.6 (0.2) 58.7% 1.69:1
Car:Chl V+A+Z Z% Lut:Lact
1.2:1 0.19:1 4.0 (0.2) 60.0% 1.75:1
N.D. = not detected; tr = trace amounts only.
the remainder (not ascribed to any particular LH band) accounted for ,-~12% of pigment. Following light treatment, zeaxanthin was formed following de-epoxidation of violaxanthin and typically accounted for 50--60% o f the xanthophyll cycle pool, as has been typically reported for many other plant species ( D e m m i g - A d a m s 1990; D e m m i g - A d a m s and Adams 1992, 1993). Levels o f the intermediate in this reaction, antheraxanthin, were generally very small and only accounted for a few percent of total carotenoid. A high proportion o f the violaxanthin (,,~ 19%) in darkadapted spinach and ,,~24% and 10% ofzeaxanthin and antheraxanthin, respectively, formed as a result of light treatment o f leaves, was not associated with the main
light-harvesting complexes. These values are much higher than for any o f the other carotenoids present in PS II. L H C lib
The room temperature absorption spectra for the four LHC IIb fractions obtained during I E F o f BBYs all had strong characteristic peaks at 474 nm and 652 nm (Fig. 3). Little difference in their absorption spectra was observed between these separate bands for either dark-adapted or light-treated leaves. The pigment composition o f bands 1--4 is given in Tables 2A and 2B for dark-adapted and light-
278 Table 3. Pigment composition of LHC Ha and LHC IIc purified from BBYs isolated from dark-adapted and light-treated lettuce by IEF (see Materials and methods for details). The data are expressed as moles pigment per 100 chlorophyll a and are the means (~- S.E.) of three replicates Pigment
Dark-adapted LHC IIa LHC IIc
Light-treated LHC IIa LHC IIc
Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin /3-Carotene
4.9 (0.3) 12.2 (0.9) N.D. N.D. 8.4 (0.4) 16.6 (0.8) 7.5 (0.3)
7.4 (0.1) 14.8 (0.1) N.D. N.D. 7.6 (0.4) 15.4 (0.4) 11.4 (0.5)
5.1 (0.1) 13.7 (0.4) 3.0 (0.1) 9.3 (0.3) 7.5 (0.2) 4.2 (0.1) 8.2 (0.3)
7.5 (0.1) 14.1 (0.4) 3.1 (0.2) 8.7 (0.4) 6.2 (0.1) 4.0 (0.2) 12.2 (0.1)
Chl a:b
4.3:1 0.28:1 16.6 (0.8)
2.5:1 0.26:1 15.4 (0.4)
4.1:1 0.29:1 16.5 (0.3) 56.4 (0.4) 2.68:1
2.7:1 0.23:1 15.8 (0.3) 55.1 (0.3) 1.88:1
Car:Chl V+A+Z Z% Lut:Lact
2.48:1
2.00:1
N.D. = not-detected.
Table 4. Distribution of xanthophylls in light-harvesting complexes (LHC lib, LHC IIa and LHC IIc) of light-treated and dark-adapted lettuce. Data are expressed as a percentage of total xanthophyll applied to the gel and are the means of at least three separate analyses Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Band 7
Rem
19.4 14.4 6.9 11.8 17.2 9.0
19.4 16.4 6.9 11.8 18.7 9.0
19.9 19.2 6.9 11.6 20.8 9.4
20.4 23.3 6.9 12.8 24.9 9.6
4.3 6.5 17.2 12.0 4.8 14.8
6.5 8.9 20.7 17.2 1.8 9.9
4.8 4.5 10.3 8.6 5.4 19.8
5.4 6.8 24.1 13.8 6.4 18.7
19.7 13.4 14.8 8.6
19.8 17.4 19.7 8.8
19.8 20.1 21.3 9.0
21.7 21.4 23.0 9.3
4.0 6.7 5.3 15.2
5.7 9.4 2.3 9.4
4.5 4.8 6.1 20.4
4.8 6.4 7.5 19.3
Light-treated Lactucaxanthin Lutein Antheraxanthin Zeaxanthin Neoxanthin Violaxanthin
Dark-adapted Lactucaxanthin Lntein Neoxanthin Violaxanthin
Band identification: 1-4 LHC IIb; 5 LHC llc; 6 PS II Core; 7 LHC IIa; Rem = Remainder.
treated leaves, respectively. The ratio of chlorophyll a:b at 1.2:1 in all four LHC IIb bands is consistent with previous studies (e.g. Bassi et al. 1993). Whilst the chlorophyll composition is relatively constant, the carotenoid composition of these complexes does vary. /3-Carotene was consistently absent from all LHC IIb preparations and only xanthophylls were detect-
ed. Lutein was the single most abundant carotenoid in LHC IIb (20-24 mol/100 chlorophyll a for bands 1-4), whilst levels of neoxanthin and lactucaxanthin were lower at between 10-16 mol/100 chlorophyll a. The ratio of total carotenoid:total chlorophyll increased from 0.15:1 to 0.20:1 in bands 1-4 (i.e. with increasing migration) due to changes in the levels of all the
279 carotenoids relative to chlorophyll (Table 2). The ratio of lutein:lactucaxanthin remained fairly constant in all four bands as did the pool size of the xanthophyll cycle carotenoids (3-5 mol/100 chlorophyll a). Levels of violaxanthin and, after light treatment, zeaxanthin only ever accounted for a few percent of total carotenoid in each LHC IIb band and antheraxathin was only ever observed in trace amounts (below the levels required for integration on the HPLC). Although the xanthophyll cycle carotenoids are a relatively minor component of each of these bands, nearly half of the PS II xanthophyll cycle pool is associated with LHC IIb itself (Table 4). The data shown in Tables 2A and 2B indicate that the xanthophyll cycle pool size increased following light treatment (resulting in zeaxanthin formation). No satisfactory explanation can be given for this behaviour. The conversion efficiency for violaxanthin~zeaxanthin was similar in all four LHC IIb bands at ,-~60%.
Minor LHC H The pigment composition of the minor LHC II complexes (as defined on IEF by Bassi et al. 1991; Bassi and Dainese 1992) is shown in Table 3. Both LHC IIa and LHC IIc were each found to contain only 4 5% each of total PS II lactucaxanthin (Table 4). This compares with >80% found in LHC IIb. The ratio of lactucaxanthin:lutein was also much higher in these two minor complexes compared to LHC IIb at ,-2:1. The pigment composition of LHC IIc was, however, noticeably more variable than the other complexes which may result due to cross-contamination from LHC IIb. Analysis of the carotenoid content of the minor complexes isolated from dark-adapted leaves of L. sativa, shows that, unlike LHC IIb, violaxanthin is the single most abundant component in both LHC IIa and LHC IIc (16 mol/100 chlorophyll a, see Table 3). The distribution of violaxanthin and zeaxanthin in LHC IIa and LHC IIc is given in Table 4. LHC IIa and LHC IIc possess ,,-20% and ,,~15% of PS II violaxanthin respectively. Light treatment of leaves of induces zeaxanthin formation via violaxanthin de-epoxidation in both LHC IIa and LHC IIc. The conversion efficiency for zeaxanthin formation from violaxanthin in both of these complexes was only slightly lower than that observed in LHC lib, at 55-56%, although the deepoxidation state was higher due to the accummulation of relatively high amounts of antheraxanthin (present in only trace amounts in LHC IIb). Whilst LHC IIa only
contains 8-9% of PS II zeaxanthin and LHC IIc 12% it is clear that in these minor complexes the xanthophyll cycle carotenoids represent a much higher proportion of total carotenoid. In contrast to LHC IIb, significant amounts of/5carotene were detected in both minor LHC. This concurs with Bassi et al. (1993) who found that fl-carotene accounted for ,-5% of total carotenoid in both LHC IIa and LHC IId and concluded that ~-carotene was a genuine component of these minor PS II complexes. Data from this study for/~-carotene levels in lettuce (Table 3) were much higher than those reported by Bassi and co-workers for barley, at ,-~15% of total carotenoid for both complexes. The relative levels of ~-carotene and the xanthophylls in higher plant photosynthetic tissues are known to be affected by growth conditions (especially incident light intensity) and large inter-specific differences may be expected. No data are available for LHC IId as this complex was not observed following IEF of either thylakoids or BBYs. Under identical preparative and electrophoretic conditions for spinach BBYs, LHC IId could be clearly resolved and it's pigment composition determined by HPLC analysis.
Discussion
Lactucaxanthin in higher plant LHC Lactucaxanthin is not commonly found in the photosynthetic tissues of higher plant and levels of this carotenoid may vary substantially from one species to another. In this study on lettuce, lactucaxanthin was found to represent as much as 21% of total thylakoid carotenoid and 24% of PS II carotenoid. In some other plant species, notably Eunonymus spp. this level may rise to nearly 35% of thylakoid carotenoid so that levels may be greater than those of lutein (D. Phillip and A. Young, unpublished data). Clearly therefore this rare carotenoid may, in some species, be a very important component of the pigment bed. Lactucaxanthin was found in all the major and minor LHC II complexes of L. sativa. The ratio of lutein:lactucaxanthin was higher in both LHC IIa and LHC IIc compared to the four separate LHC IIb fractions. It is interesting to compare this data with that obtained from spinach LHC II (prepared in an identical manner; Ruban et al. 1994a; D. Phillip and A. Young, unpublished data). It is noteworthy that the molar ratio of the sum of lactucaxanthin and lutein
280 Table 5. Xanthophyll Stoichiometry in LHC II. Values for the present study have been calculated using the chlorophyll a content of these complexes determined by Bassi et al (1993): LHC l i b - 8 per monomer; LHC Ila and LHC IIc - 6 per monomer Hertrysson
Peter and
Bassi et
Ruban et
Present
et al.
Thornber
al. (1993)
al.
study
(1989)
(1991)
(1994a)
LHC lib Neoxanthin
-
1
0.5
1
1
Violaxanthin
-
0.5
tr
0.3*
0.3*
Lutein
-
2
2
1.7
Lactucaxanthin
.
2 .
.
.
1
LHC lla Neoxanthin
1
1
1
0.5
Violaxanthin
1
2
1.5
1
1
Lutein
1
2
2
1
0.7
Lactucaxanthin
.
.
.
.
0.5
0.3
LHC llc Neoxanthin
-
1
0.5
1
Violaxanthin
-
0.5
1
I
i
Lutein
-
2
2
1
1
Lactucaxanthin
.
tr = trace amounts only; *
equivalent
.
.
.
0.5
0.5
to 1 violaxanthin per trimer
to chlorophyll a is nearly identical to that of lutein alone in spinach. Other data obtained on other members of the compositae, including other varieties of lettuce also show this trend and although the levels of these two carotenoids are variable their sum remains relatively constant accounting for 30-35% total thylakoid carotenoid. This is similar to that observed in an early study on the pigment content of lettuce thylakoids and LHC (Siefermann-Harms and Ninnemann 1982). The calculated stoichiometry of xanthophylls in LHC II for L. sativa is shown in Table 5. Data from other recent studies are also shown for comparison. The values presented in Table 5 have been calculated using the chlorophyll a contents as used by Bassi and colleagues (1993) and adopted by many other groups: i.e. 8 chlorophyll a per LHC IIb monomer and 6 each for LHC II and LHC IIc. Pigment stoichiometry for LHC II in L. sativa is in broad agreement with those of previous studies. However, the data shown in Table 5 indicate sub-stoichiometric amounts of lutein in LHC IIb and LHC IIa and sub-stoichiometric amounts of lactucaxanthin in both minor complexes. Other studies consistently calculate there to be six lutein molecules
per trimer. Clearly in L. sativa, in which only five luteins were estimated per trimer, a proportion of lutein-binding sites must be occupied by other xanthophylls, of which lactucaxanthin is the most likely candidate. Both minor LHC have three carotenoid molecules per polypeptide. The carotenoids, lactucaxanthin (e, e-carotene-3, 3'-diol), lutein (fl, e-carotene-3, 3'-diol) and, indeed, zeaxanthin (/3, fl-carotene-3, 3'-diol) are all very similar except in degree of conjugation (n = 9, 10 and 11 conjugated double bonds, respectively). The orientation adopted by the backbone of the polyene chain with respect to an end-group is dependent on the type of end-group present, i.e. whether the conjugation extends into the ring or not, for/3- or e-end-groups, respectively. In the case of lactucaxanthin (with two e-rings) this has the effect of altering the orientation of the end-groups, rendering them perpendicular to the main polyene chain, whereas the presence of one/3end-group in lutein will cause the end-group to adopt a near-planar orientation. There is no evidence to suggest that the conformation of the carotenoid molecule may be critical in determining its protein-binding, although twisting of the polyene backbone is evident
281 from Kiahlbrandt's model. The 3.4 ,~ resolution electronic crystallography model of LHC IIb published recently by Ktihlbrandt et al. (1994) shows that lutein occupies a central position in this complex and the orientation adopted by the two end-groups types of both these luteins are clearly visible. It is quite possible that lactucaxanthin may, due to its close structural similarities to lutein, simply be able to replace a proportion of lutein molecules occupying the central position this complex. The sub-stoichiometric amounts of these carotenoids determined in this study for all three lettuce LHC II complexes examined would certainly support this. At least some, or alternatively, all of the lactucaxanthin may occupy a position peripheral to the complex similar to that suggested for neoxanthin and violaxanthin in the model proposed by Ktihlbrandt and colleagues (1994). The carotenoid content of the LHC IIb trimer (the most likely functional unit) of L. sativa would be one violaxanthin, three neoxanthin, three lactucaxanthin and five lutein. The differences in calculated stoichiometry between studies apparent in Table 5 could be due to inter-specific differences but could also arise due to the use of different growth conditions (for example, the ratio of lactucaxanthin:lutein in L. sativa is greatly affected by light intensity - probably by regulation at the level of the/3- and e-cyclases; see also Johnson et al. 1993) and the use of different isolation procedures for the complexes adopted by different laboratories.
spinach (Ruban et al. 1994a), although ,,~50% of zeaxanthin formed in L. sativa is located in LHC Ilb, rather than in the minor LHC, the molar ratio of zeaxanthin:chlorophyll a is very low. A particular feature of the minors was the relatively high ratio of zeaxanthin:chlorophyll a compared to LHC IIb. Although the molecular mechanism whereby zeaxanthin-mediated non-photochemical quenching of chlorophyll fluorescence arises is still not fully understood, this might indicate that the minor complexes have a role in this process. Unlike the study of Ruban et al. (1994a), all LHC exhibited similar conversion efficiencies for violaxanthin~zeaxanthin. In this study all the xanthophylls found in thylakoids and BBYs from L. sativa were also detected in the IEF bands corresponding to the PS II core. Whilst the core was found to contain high levels of ;3-carotene (as reported in other studies, e.g. Peter and Thornber 1991), the levels of some carotenoids (including lactucaxanthin), and neoxanthin in particular, were very low. The data shown in Table 4 also indicate that the xanthophyll cycle operates in the PS II core and that the production of zeaxanthin is very efficient when compared to LHC IIb and the minors. Clearly, the variations in the carotenoid content and composition of the PS II core (and indeed PS I as a whole; see Thayer and Bjorkman 1992) needs a more thorough study.
Acknowledgements The xanthophyll cycle Thylakoids and BBYs obtained from both darkadapted and light-treated leaves resulted in almost identical behaviour on IEE It is important to note that, in L. sativa, zeaxanthin can be formed in all LHC II complexes examined, including LHC IIb which has almost half of PS II zeaxanthin (Tables 2 and 3). This is in agreement with the observations made on spinach LHC II by Ruban et al. (1994a) but contradicts the data of Bassi et al. (1993) for barley in which LHC IIb was reported to lack the ability to deepoxidate violaxanthin. According to our data for L. sativa, violaxanthin is present in LHC IIb at ,,~0.3 molecules per monomer supporting the conclusions of Ruban et al. (1994a) that one violaxanthin molecule is present per trimer. Both minor complexes bind one violaxanthin molecule per polypeptide. The extent of de-epoxidation in bands 1-4 is similar at 58-61% of the xanthophyll cycle pool and is only slightly lower in LHC IIa and LHC IIc. As with
We thank Peter Horton, Sasha Ruban and Pam Scholes (University of Sheffield, UK) for their assistance in the preparation of complexes. This work was supported by a LJMU Research Grant.
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