Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1135–1139, February 1999 Plant Biology Unusual carotenoid composition and a new type of xanthophyll cycle in plants RALPH A. BUNGARD*†,ALEXANDER V. RUBAN‡, JULIAN M. HIBBERD*, MALCOLM C. PRESS*, PETER HORTON‡, AND JULIE D. SCHOLES* Robert Hill Institute, Departments of *Animal and Plant Sciences and ‡Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom Edited by Charles J. Arntzen, Boyce Thompson Institute for Plant Research, Ithaca, NY, and approved December 6, 1998 (received for review October 12, 1998) ABSTRACT The capture of photons by the photosynthetic regulation of stomatal function and seed dormancy. Violax- apparatus is the first step in photosynthesis in all autotrophic anthin is the biosynthetic precursor to neoxanthin but differs higher plants. This light capture is dominated by pigment- from both lutein and neoxanthin in that its concentration containing proteins known as light-harvesting complexes varies markedly between species and depending on the light (LHCs). The xanthophyll–carotenoid complement of these environment under which the plants have developed. Violax- LHCs (neoxanthin, violaxanthin, and lutein) is highly con- anthin is also the primary substrate in a reversible light-driven served, with no deletions and few, uncommon additions. We deepoxidation reaction known as the xanthophyll cycle. In this report that neoxanthin, considered an integral component of cycle, violaxanthin is converted to zeaxanthin by means of the LHCs, is stoichiometrically replaced by lutein-5,6-epoxide in intermediate antheraxanthin under conditions where light the parasitic angiosperm Cuscuta reflexa, without compromis- energy absorbed by chlorophyll is in excess of that used in ing the structural integrity of the LHCs. Lutein-5,6-epoxide photosynthetic processes, and this conversion is reversed when differs from neoxanthin in that it is involved in a light-driven light is no longer excessive. The accumulation of the deepoxi- deepoxidation cycle similar to the deepoxidation of violaxan- dation products antheraxanthin and zeaxanthin in the photo- thin in the xanthophyll cycle, which is implicated in protection synthetic apparatus is associated with harmless energy dissi- against photodamage. The absence of neoxanthin and its pation in the form of heat. The xanthophyll cycle is ubiquitous replacement by lutein-5,6-epoxide changes our understanding in higher plants and is implicated in protection against pho- of the structure-function relationship in LHCs, has implica- todamage (6, 7). tions for biosynthetic pathways involving neoxanthin (such as the plant hormone abscisic acid), and identifies one of the The Cuscutaceae is a monotypic family of ca. 150 parasitic early steps associated with the evolution of heterotrophy from plant species with low host specificity and a wide geographic autotrophy in plants. distribution. Cuscuta is at an early stage of nonphotosynthetic evolution and the genus contains both chlorophyllous and achlorophyllous species, with the latter having been derived Despite the enormous diversity in the evolution, form, and recently from the former (8–10). Thus Cuscuta provide a rare function of photosynthetically active higher plants, the basic biochemical mechanisms appear remarkably conserved. In all opportunity to observe the early evolutionary steps associated autotrophic plants studied, the first step in photosynthesis, the with the loss of photosynthesis and potential modifications to absorption of sunlight, is carried out primarily by an array of the otherwise widespread and highly conserved photosynthetic pigment-binding proteins termed the light-harvesting com- apparatus. The focus of this study is the chlorophyllous species plexes (LHCs). With no deletions and few, uncommon addi- C. reflexa (Fig. 1A), which contains just a single band of fully tions, these complexes contain the same major xanthophyll— functional photosynthetic cells located adjacent to the vascular carotenoids, namely lutein, neoxanthin, and violaxanthin. Of bundles (11). The functional significance of these cells is in the these xanthophylls, lutein appears in the highest concentration, recycling of CO2 from neighboring cells that have high respi- usually comprising between 30 and 60% of total xanthophylls ration rates. Fixation of external CO2 is unlikely because of (1, 2). Studies elucidating the structure of the major pigment- spatial isolation from the atmosphere, and the parasite obtains binding protein of the LHCs of photosystem II (LHCIIb) carbon exclusively from the host plant, mainly by means of suggest that each monomeric subunit contains two lutein phloem connections in the haustoria (Fig. 1B), which provide molecules that form a crossbrace in the center of the complex a vascular bridge between the two species (12). indicating that lutein may have a major structural role within the LHCs (3, 4). Neoxanthin comprises a smaller proportion of MATERIALS AND METHODS the total xanthophyll pool but epitomizes the conservative nature of the photosynthetic apparatus in that its concentra- Plant Material and Experimental Conditions. Cuscuta re- tion is remarkably consistent across a wide range of contrasting flexa Roxb. were grown with either Coleus blumei L. or Ricinus species and growth environments, where it accounts for be- communis L. as the host species. Host plants and Spinacia tween 9 and 14% of total xanthophylls (1, 2). This apparent oleracea (spinach) were grown in peat-based potting mix in a stoichiometry has led to the suggestion that neoxanthin, like glasshouse with temperature in the range 15°C to 35°C under lutein, is also an integral structural component of LHCs. natural light conditions between April and September. Neoxanthin is also considered to be a precursor of abscisic acid (ABA) (5), a plant hormone thought to have multiple func- This paper was submitted directly (Track II) to the Proceedings office. tions in higher plants including, among others, roles in the Abbreviations: LHCs, light-harvesting complexes; LHCIIb, the major pigment-binding protein of the LHCs of photosystem II; ABA, abscisic The publication costs of this article were defrayed in part by page charge acid. †To whom reprint requests should be addressed at Robert Hill payment. This article must therefore be hereby marked ‘‘advertisement’’ in Institute, Department of Animal and Plant Sciences, University of accordance with 18 U.S.C. §1734 solely to indicate this fact. Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. PNAS is available online at www.pnas.org. e-mail: [email protected]. 1135 Downloaded by guest on October 2, 2021 1136 Plant Biology: Bungard et al. Proc. Natl. Acad. Sci. USA 96 (1999) FIG. 2. Carotenoid composition of higher plants. (A) HPLC separation of pigments from dark-adapted samples from S. oleracea (dark) and C. reflexa (dark), C. reflexa exposed to light at 1,200 mmol photons m22 s21 for 2–4 h [C. reflexa (light)]. The identification of the lutein-5,6-epoxide in C. reflexa was achieved by comparing its elution times and absorption spectrum (inset) with a pure sample of lutein- 5,6-epoxide (standard); they were identical. N, neoxanthin; V, violax- anthin; Lx, lutein-5,6-epoxide; A, antheraxanthin; L, lutein; Z, zeax- anthin; a, chlorophyll a; b, chlorophyll b;C,b-carotene. (B) Biosyn- thetic pathway of carotenoids showing that neoxanthin is synthesized later than b-carotene-derived xanthophyll cycle carotenoids and dis- tinct from a-carotene-derived lutein. light by grinding in liquid N2 followed by grinding in degassed 100% acetone at 0–4°C. On occasion a small amount of z 21 FIG. 1. Cuscuta species are parasitic on the above-ground parts of NaHCO3 (around 0.1 g g plant material) was added to plant other plants. The parasites consist of a twining stem that lacks leaves material before extraction to ensure no acidification of the and has no roots. They are dependent on the host plant for organic and extract occurred; however, this had no influence on carotenoid inorganic solutes that they extract by means of haustoria, primarily composition. Pigments were extracted from thylakoids and from the host phloem (12). (A) C. reflexa growing on a Coleus host and isoelectric focusing protein bands by adding 100% acetone to (B) a close view of the host–parasite attachment with C. reflexa samples to give a final concentration of 90% acetone. Lutein- attached by haustoria to the stem of Coleus. 5,6-epoxide ([3S, 5R, 6S, 39R, 69R]-5, 6-epoxy-5, 6-dihydro- b « 9 The pigment composition data presented in Fig. 2 and Table , -carotene-3, 3 -diol; taraxanthin) in C. reflexa was identified 1 were obtained from either plant material that had been by comparing its elution times and absorption spectrum with maintained in the dark for a total of 12 h before sampling a pure sample. All other pigments were identified as (dark-adapted) or plant material that had been dark-adapted described (2). and subsequently exposed to irradiance at 1,200 mmol m22 s21 Separation and Purification of Thylakoids and LHCs. Thy- for 2 to 4 h (light). The results in Fig. 3A are from a time-course lakoids from C. reflexa were prepared by grinding plant analysis of the pigment composition after exposure of C. material in a polytron tissue homogenizer in pH 6.5 buffer (100 21 y 21 y reflexa to high irradiance. In this experiment, C. reflexa were mmol l Hepes 0.3 mol l sorbitol) containing 2% (wt vol) y y y 21 dark-adapted for 12 h before an initial time-zero sample was polyvinylpyrrolidone 0.5% (wt vol) BSA 0.5 mmol l taken. Detached filaments were then exposed to irradiance of EDTA. Homogenate was then filtered twice through four 1,200 mmol m22 s21 and light-exposed samples taken accord- layers of cheesecloth. The filtered solution was centrifuged ingly. At each sampling time, samples were plunged into liquid (5,000 g for 10 min), and the resulting pellet was resuspended, first in pH 7.3 buffer, then pH 7.6 buffer containing 1.0 mmol nitrogen where they were stored until HPLC analysis of 2 pigments was carried out.