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Investigation of Thylakoid Bleaching and Violaxanthin De-epoxidase Related Enzyme

Pålsson, Björn LU (2017) KEML13 20171
Department of Chemistry
Abstract
Photosynthetic organisms harvest and convert light energy into chemical energy and utilize it in their metabolism. The light is captured through pigments called chlorophylls and carotenoids, present in the thylakoid membranes inside the chloroplast. In addition to acting as auxiliary light harvesters, the carotenoids also act as antioxidants by 1) preventing the formation of reactive oxygen species (ROS) and 2) quenching formed ROS before it can cause damage to the cell.
ROS are reactive species, radicals and non-radicals, containing oxygen. They are naturally generated e.g. in the electron transport chains of chloroplasts and mitochondria and act as indicators of stress in the cell. Too high ROS concentration for a prolonged period of... (More)
Photosynthetic organisms harvest and convert light energy into chemical energy and utilize it in their metabolism. The light is captured through pigments called chlorophylls and carotenoids, present in the thylakoid membranes inside the chloroplast. In addition to acting as auxiliary light harvesters, the carotenoids also act as antioxidants by 1) preventing the formation of reactive oxygen species (ROS) and 2) quenching formed ROS before it can cause damage to the cell.
ROS are reactive species, radicals and non-radicals, containing oxygen. They are naturally generated e.g. in the electron transport chains of chloroplasts and mitochondria and act as indicators of stress in the cell. Too high ROS concentration for a prolonged period of time is lethal to the cell due to their high reactivity.
One way carotenoids are involved in preventing ROS formation is the non-photochemical quenching (NPQ) of excited singlet chlorophyll (1Chl*): the energy is transferred from 1Chl* to the carotenoid and is then dissipated to the solvent through vibrations and rotations. This prevents the conversion of 1Chl* into triplet state chlorophyll (3Chl*) which gives rise to ROS. Plants regulate the amount of carotenoids available to NPQ through the xanthophyll cycle: At high light intensity, the carotenoid violaxanthin is de-epoxidized into zeaxanthin, which more effectively participate in NPQ. The reaction is catalysed by violaxanthin de-epoxidase (VDE).
The closest relative of VDE is the not yet officially named protein nick-named ‘violaxanthin de-epoxidase related protein’ (VDE-R). It is present in the same context as VDE but its function is of yet unknown.
In this study, methods of bleaching photosynthetic pigments from Spinacia oleracea (spinach) using copper(II) phosphate (Cu3(PO4)2), UV light and hydrogen peroxide (H¬¬2O2) were investigated in order to identify experimental conditions under which it is possible to determine whether or not VDE-R is involved in repairing damaged photosynthetic pigments. Also the effect of VDE-R on unbleached thylakoids was briefly investigated.
The relative amount of pigments were determined using HPLC (high performance liquid chromatography) and UV-Vis spectroscopy.
It was concluded that the bleaching was most effective when H2O2, UV light and Cu2+ all were present rather than only one or two components. No connection between added VDE-R and the amount of major thylakoid pigments was observed, regardless of whether NADPH, FMN, ascorbic acid and Mg2+ were present or not.
The function of VDE-R is still highly unclear and further studies are needed. (Less)
Popular Abstract (Swedish)
Fotosyntetiserande växter, alger och bakterier fångar in och omvandlar solenergi till kemisk energi. Själva ljusinfångningen involverar olika pigment, varav de mest kända är de gröna klorofyllmolekylerna som förekommer i olika varianter (varav två varianter kallade klorofyll A respektive B förekommer hos högre växter). De ljusinfångande pigmenten inkluderar även de mer mångtaliga karotenoiderna, exempelvis β-karoten som ger morot dess orangea färg, vilka tillåter att mer varierande våglängder kan användas i fotosyntesen. Karotenoiderna skyddar även cellen från reaktiva syreföreningar som uppstår i samband med olika typer av stress, d.v.s. då miljöbetingelserna avviker för mycket från vad som är optimalt för organismen. Ett exempel på detta... (More)
Fotosyntetiserande växter, alger och bakterier fångar in och omvandlar solenergi till kemisk energi. Själva ljusinfångningen involverar olika pigment, varav de mest kända är de gröna klorofyllmolekylerna som förekommer i olika varianter (varav två varianter kallade klorofyll A respektive B förekommer hos högre växter). De ljusinfångande pigmenten inkluderar även de mer mångtaliga karotenoiderna, exempelvis β-karoten som ger morot dess orangea färg, vilka tillåter att mer varierande våglängder kan användas i fotosyntesen. Karotenoiderna skyddar även cellen från reaktiva syreföreningar som uppstår i samband med olika typer av stress, d.v.s. då miljöbetingelserna avviker för mycket från vad som är optimalt för organismen. Ett exempel på detta är för starkt ljus. Om ljusstyrkan överstiger vad cellen förmår använda i fotosyntesen finns det risk att en del av ljusenergin istället resulterar i att reaktiva syreföreningar uppstår. För att förhindra skada kan karotenoiderna avleda den ”extra energin” på ett säkert sätt i en process som kallas ”icke-fotokemisk utsläckning” (på engelska ”Non-photochemical quenching”, NPQ), alternativt reagera direkt med syreföreningarna och på så sätt ”offra sig” för resten av cellen. Den icke-fotokemiska utsläckningen är associerad med ett enzym kallat violaxantin-deepoxidas (eng. ”violaxanthin de-epoxidase”, VDE) som aktiveras vid starkt ljus och omvandlar en karotenoid kallad violaxantin till zeaxantin, vilken är effektivare på att avleda ljusenergin på ett säkert sätt.

VDE:s närmaste släkting (ännu inte officiellt namngiven) förekommer i samma sammanhang som VDE men dess funktion är ännu okänd. Det är möjligt, med tanke på proteinernas släktskap och förekomst, att släktingen också har en funktion som involverar skydd mot stress och karotenoidpigment.
I denna studie undersöktes i första hand olika metoder för hur fotosyntes-pigment kan blekas och i andra hand huruvida det fanns något samband mellan halten pigment och det besläktade proteinet med hjälp av tylakoider, innanmätet av kloroplasterna (de organeller i cellen där fotosyntesen sker och där fotosyntes-pigmenten finns), från Spinacia oleracea (spenat). Metoderna som användes var spektroskopi och HPLC (”high performance liquid chromatography”) samt protein utvunnet och delvis renat från genmodifierade bakterier.
Det visade sig att effektiv blekning åstadkoms genom att tylakoiderna genomgick en behandling av kopparjoner, väteperoxid (H2O2) och UV-ljus och att behandling med väteperoxid och kopparjoner (med eller utan UV-ljus) resulterade i att ett ”nytt” pigment detekterades. Det ”nya” pigmentet absorberade ljus av samma våglängder som klorofyll A, men molekylen var mer hydrofil än klorofyll A. Däremot upptäcktes inget samband mellan pigment och tillsats av det aktuella proteinet, varför vidare studier krävs för att avgöra VDE-släktingens funktion. (Less)
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author
Pålsson, Björn LU
supervisor
organization
course
KEML13 20171
year
type
M2 - Bachelor Degree
subject
keywords
biokemi, biochemistry, Spinacia Oleracea, Photosynthetic pigments, Reactive oxygen species, Fenton chemistry, Violaxanthin de-epoxidase related enzyme, thylakoids
language
English
id
8913611
date added to LUP
2017-08-30 14:46:19
date last changed
2017-08-30 14:46:19
@misc{8913611,
  abstract     = {Photosynthetic organisms harvest and convert light energy into chemical energy and utilize it in their metabolism. The light is captured through pigments called chlorophylls and carotenoids, present in the thylakoid membranes inside the chloroplast. In addition to acting as auxiliary light harvesters, the carotenoids also act as antioxidants by 1) preventing the formation of reactive oxygen species (ROS) and 2) quenching formed ROS before it can cause damage to the cell. 
ROS are reactive species, radicals and non-radicals, containing oxygen. They are naturally generated e.g. in the electron transport chains of chloroplasts and mitochondria and act as indicators of stress in the cell. Too high ROS concentration for a prolonged period of time is lethal to the cell due to their high reactivity.
One way carotenoids are involved in preventing ROS formation is the non-photochemical quenching (NPQ) of excited singlet chlorophyll (1Chl*): the energy is transferred from 1Chl* to the carotenoid and is then dissipated to the solvent through vibrations and rotations. This prevents the conversion of 1Chl* into triplet state chlorophyll (3Chl*) which gives rise to ROS. Plants regulate the amount of carotenoids available to NPQ through the xanthophyll cycle: At high light intensity, the carotenoid violaxanthin is de-epoxidized into zeaxanthin, which more effectively participate in NPQ. The reaction is catalysed by violaxanthin de-epoxidase (VDE). 
The closest relative of VDE is the not yet officially named protein nick-named ‘violaxanthin de-epoxidase related protein’ (VDE-R). It is present in the same context as VDE but its function is of yet unknown. 
In this study, methods of bleaching photosynthetic pigments from Spinacia oleracea (spinach) using copper(II) phosphate (Cu3(PO4)2), UV light and hydrogen peroxide (H¬¬2O2) were investigated in order to identify experimental conditions under which it is possible to determine whether or not VDE-R is involved in repairing damaged photosynthetic pigments. Also the effect of VDE-R on unbleached thylakoids was briefly investigated. 
The relative amount of pigments were determined using HPLC (high performance liquid chromatography) and UV-Vis spectroscopy.
It was concluded that the bleaching was most effective when H2O2, UV light and Cu2+ all were present rather than only one or two components. No connection between added VDE-R and the amount of major thylakoid pigments was observed, regardless of whether NADPH, FMN, ascorbic acid and Mg2+ were present or not. 
The function of VDE-R is still highly unclear and further studies are needed.},
  author       = {Pålsson, Björn},
  keyword      = {biokemi,biochemistry,Spinacia Oleracea,Photosynthetic pigments,Reactive oxygen species,Fenton chemistry,Violaxanthin de-epoxidase related enzyme,thylakoids},
  language     = {eng},
  note         = {Student Paper},
  title        = {Investigation of Thylakoid Bleaching and Violaxanthin De-epoxidase Related Enzyme},
  year         = {2017},
}