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The xanthophyll cycle, its enzymes, pigments and regulation

Eskling Carlsson, Marie LU (1998)
Abstract (Swedish)
Popular Abstract in Swedish

Xantofyll cykeln, dens enzym, pigment och reglering
Abstract
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.... (More)
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. (Less)
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author
opponent
  • Prof Aro, Eva-Mari, Dept. of Biology, University of Turku, FIN-20014 Turku, Finland
organization
publishing date
type
Thesis
publication status
published
subject
keywords
zeaxanthin, xanthophyll cycle, violaxanthin de-epoxidase, thylakoid, spinach, regulation, purification, pigments, photoprotection, pH dependence, ascorbate, light stress, Plant biochemistry, Växtbiokemi
pages
121 pages
publisher
Plant Biochemistry Department, Box 117, Lund University, S-221 00 Lund, Sweden,
defense location
Lund University, Solvegatan 35A, Lund, Sweden
defense date
1998-05-20 13:15
external identifiers
  • other:ISRN: LUNKDL/NKVK-98/1012
ISBN
91-628-2988-2
language
English
LU publication?
yes
id
f4c93c6a-80cb-4887-aad9-a2062c73e9e3 (old id 38640)
date added to LUP
2007-06-20 14:50:31
date last changed
2016-09-19 08:45:03
@phdthesis{f4c93c6a-80cb-4887-aad9-a2062c73e9e3,
  abstract     = {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.<br/><br>
<br/><br>
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.},
  author       = {Eskling Carlsson, Marie},
  isbn         = {91-628-2988-2},
  keyword      = {zeaxanthin,xanthophyll cycle,violaxanthin de-epoxidase,thylakoid,spinach,regulation,purification,pigments,photoprotection,pH dependence,ascorbate,light stress,Plant biochemistry,Växtbiokemi},
  language     = {eng},
  pages        = {121},
  publisher    = {Plant Biochemistry Department, Box 117, Lund University, S-221 00 Lund, Sweden,},
  school       = {Lund University},
  title        = {The xanthophyll cycle, its enzymes, pigments and regulation},
  year         = {1998},
}