The xanthophyll cycle, its enzymes, pigments and regulation

Marie Eskling Carlsson

Research output: ThesisDoctoral Thesis (compilation)


The xanthophyll cycle involves the light-dependent and reversible conversion of violaxanthin to zeaxanthin. Zeaxanthin has been implicated in the protection of the photosynthetic machinery from over-excitation. The enzyme violaxanthin de-epoxidase catalyses the conversion of violaxanthin to zeaxanthin. The enzyme is located in the thylakoid lumen and its activity is strictly pH controlled. Apart from violaxanthin, ascorbate and monogalactosyldiacylglycerol are required for activity. The Km for ascorbate showed a pH dependence from which we conclude that the acid form of ascorbate is the substrate for the reaction. Release of the enzyme from the thylakoid membrane is strongly pH dependent, with a cooperativity of 4 with respect to protons. A new method for the irreversible inhibition of violaxanthin de-epoxidase was developed. From the pH dependence of this inhibition, we suggest that violaxanthin de-epoxidase undergoes a conformational change upon membrane binding. Purification of violaxanthin de-epoxidase by gel filtration, hydrophobic interaction chromatography and DEAE anion exchange chromatography resulted in a 5 000-fold enrichment and identification of a 43 kDa protein as violaxanthin de-epoxidase. The sequence of the enzyme is unique and show no apparent homology with other known proteins.

Pigment analysis of sub-thylakoid membrane domains shows that violaxanthin is evenly distributed in the membrane. Addition of purified violaxanthin de-epoxidase to these domains converted violaxanthin to zeaxanthin from both sides of the membrane to the same maximum degree, independent of pigment-protein complex composition. It is therefore concluded that this conversion takes place in the lipid matrix and not in the pigment-protein complexes. When spinach plants are shifted from low to high light the quantities of the xanthophyll cycle pigments and ascorbate are increased, but the amount and activity of violaxanthin de-epoxidase are decreased. The increase in xanthophyll cycle pigments and ascorbate only partly explains the increased rate of conversion of violaxanthin to zeaxanthin. The most probable explanation of a faster conversion is an increased accessibility of violaxanthin in the membrane. Temperature has a strong influence on both the rate and degree of maximum conversion. It is proposed that diffusion of violaxanthin between pigment protein complexes and the lipid matrix regulates the degree of conversion.
Original languageEnglish
Awarding Institution
  • Biochemistry and Structural Biology
  • [unknown], [unknown], Supervisor, External person
Award date1998 May 20
ISBN (Print)91-628-2988-2
Publication statusPublished - 1998

Bibliographical note

Defence details

Date: 1998-05-20
Time: 13:15
Place: Lund University, Solvegatan 35A, Lund, Sweden

External reviewer(s)

Name: Aro, Eva-Mari
Title: Prof
Affiliation: Dept. of Biology, University of Turku, FIN-20014 Turku, Finland


Subject classification (UKÄ)

  • Biological Sciences

Free keywords

  • zeaxanthin
  • xanthophyll cycle
  • violaxanthin de-epoxidase
  • thylakoid
  • spinach
  • regulation
  • purification
  • pigments
  • photoprotection
  • pH dependence
  • ascorbate
  • light stress
  • Plant biochemistry
  • Växtbiokemi


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