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Abstract
The parasitic angiosperm Cuscuta reflexa has a highly unusual carotenoid composition in that it does not contain neoxanthin, an otherwise ubiquitous component of the major light-harvesting complex protein (LHCIIb) in all other higher plant species studied to date. Combined HPLC and mass spectrometric analysis has enabled us to detect in tissues of C. reflexa two new types of xanthophylls: lutein-5,6-epoxide and 9-cis-violaxanthin. We have isolated the LHCIIb complex from thylakoids and analyzed chlorophyll and carotenoid composition. The data show that the 9-cis-violaxanthin is present in amounts similar to that of neoxanthin in most plants. On the other hand, lutein-5,6-epoxide was found to be in substoichiometric quantities, suggesting a peripheral location similar to the loosely-associated all-trans-violaxanthin and also enabling suitable accessibility for the de-epoxidase (VDE). Absorption spectroscopy revealed close similarities of the excited state energies of neoxanthin and 9-cis-violaxanthin in vitro and in intact LHCIIb complex. Resonance Raman analysis clearly indicates a cis conformation of violaxanthin in the complex, confirming the pigment analysis data and proving that not only does violaxanthin replace neoxanthin as an intrinsic component of LHCIIb in C. reflexa but it also adopts the same 9-cis conformation of neoxanthin. These results suggest that the N1 binding site of LHCIIb preferentially binds 9-cis-5,6-epoxy carotenoids, which has implications for the features of this binding site and its role in the photosystem II antenna assembly and stability.
Highlights
The parasitic angiosperm Cuscuta reflexa has a highly unusual carotenoid composition in that it does not contain neoxanthin, an otherwise ubiquitous component of the major light-harvesting complex protein (LHCIIb) in all other higher plant species studied to date [1]
HPLC/MS Analysis of Pigments—The carotenoids and chlorophyll composition of intact tissue and LHCIIb were analyzed by HPLC/MS with HPLC coupled to positive-ion, atmospheric pressure chemical ionisation mass spectrometry (APCI-MS)
C. reflexa, did not contain neoxanthin and instead contained relatively high concentrations of lutein-5,6-epoxide (Lx) and another xanthophylls that we identify as 9-cis-violaxanthin (9cV, Fig. 2). 9-cis-violaxanthin was identified from its mass (Fig. 1), by furanoid rearrangement to confirm the existence of two epoxy functional groups,2 by comparison to isomers produced from iodine-catalyzed photoisomerization of all-trans-violaxanthin (Fig. 2), and through comparison with petal tissue from Viola tricolor, which has been shown to be enriched in 9-cis-violaxanthin [25]
Summary
Plant Material and Experimental Conditions—Plant material from C. reflexa Roxb. (dodder) used for HPLC/MS pigment analysis and thylakoid preparations from which LHCIIb protein was subsequently isolated was grown on Coleus blumei L. as the host species. HPLC/MS Analysis of Pigments—The carotenoids and chlorophyll composition of intact tissue and LHCIIb were analyzed by HPLC/MS with HPLC coupled to positive-ion, atmospheric pressure chemical ionisation mass spectrometry (APCI-MS). For this analysis, a Waters 2790 HPLC system (Waters Corporation, Milford, MA) was coupled to a Micromass LCT Time-of-Flight mass spectrometer (Waters Corporation). Carotenoids were identified and quantified by comparing absorption spectra and retention times with published data and purified standards, and by analysis of furanoid re-arrangements and iodine-catalyzed isomerization products together with mass data obtained through APCI-MS analysis. Excitation was provided by Coherent Argon (Innova 100) laser lines at 457.9 and 488.0 nm (Coherent)
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