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The roles of Lhcb1 och Lhcb2 in regulation of photosynthetic light harvesting
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
2015 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Lhcb1 och Lhcb2s olika roller i regleringen av fotosyntesens ljusinfångning (Swedish)
Abstract [en]

Photosynthesis in higher plants relies upon collection of light by chlorophyll molecules associated with light harvesting chlorophyll a/b-binding (LHC) proteins. The two most abundant of these are Lhcb1 and Lhcb2, which make up light harvesting complex (LHC) II trimers. They are also involved in facilitating state transitions, a process during which energy balancing between photosystem (PS) II and I is achieved. Overexcitation of PSII reduces the plastoquinone pool which activates STN7, a kinase, that phosphorylates a threonine residue on Lhcb1 and Lhcb2. In order to studythe kinetics of this we developed antibodies capable of recognizingphosphorylated forms of each of these proteins. This showed that Lhcb2 is more rapidly phosphorylated than Lhcb1, that there are no differences in the migration of phosphorylated and non-phosphorylated forms of Lhcb1 and Lhcb2 and that the majority of phosphorylated LHCII (P-Lhcb1 and PLhcb2) are associated with super- and megacomplexes. Furthermore, a state 2-specific LHCII-PSI-LHCI band contains P-Lhcb2 but almost no P-Lhcb1, and a band corresponding to M trimers (band 4 from sucrose gradients, composed of LHCII, CP24 and CP29), contains only P-Lhcb1 but no P-Lhcb2. We also developed artificial microRNA lines specifically depleted in either Lhcb1 or Lhcb2, amiLhcb1 and amiLhcb2 respectively. We show that the roles of Lhcb1 and Lhcb2 in state transitions are complementary. Lhcb1 modulate the size of grana stacks. In the absence of Lhcb1 only a few LHC trimers are formed, while in the absence of Lhcb2, the antenna looks like in the wild type although the plants cannot perform state transitions normally. Trimers containing P-Lhcb2 functionally detach from PSII and connect to PSI to balance the relative excitation pressure. State transitions only occur when both Lhcb1 and Lhcb2 are present, presumably in a (Lhcb1)2 Lhcb2 heterotrimer. In absence of Lhcb2, the LHCII-PSI-LHCI supercomplex is not formed indicting that P-Lhcb2 mediates attachment of LHCII to PSI.

We tried complementing amiLhcb2 with modified Lhcb2 genes coding for proteins with altered amino acids, Arg2 to Lys or the phosphorylatable Thr3 residue to Asn or Ser. Introduction of the additional gene often causes loss of amiRNA-inhibition, however we could confirm that substitution of the Thr3 with Asn led to the absence of Lhcb2 phosphorylation and thus no state transition.

Abstract [sv]

Klorofyll a/b-bindande proteiner (s k light harvesting chlorophyll a/b-binding proteins eller LHC proteiner) är viktiga för högre växters fotosyntes, då deras klorofyllmolekyler skördar solljuset. Två av dessa proteiner, Lhcb1 och Lhcb2, bygger upp ”LHCII trimerer” och finns i större mängd än de andra och dessa är även viktiga för s k ”state transtions”, en process som ser till att fotosystem (PS) I och PSII exciteras lika mycket. Om PSII exciteras för mycket reduceras plastoquinon-poolen som i sin tur aktiverar ett proteinkinas, STN7, som fosforylerar en av Lhcb1/Lhcb2s treoniner. För att studera denna fosforylering har vi utvecklat antikroppar som är specifika för dessa fosforylerade former av proteinerna, och vi använde dem för att visa att Lhcb2 fosforyleras snabbare än Lhcb1, och att största delen av det fosforylerade proteinerna (P-Lhcb1 och P-Lhcb2) finns i s k super- eller megakomplex. Ett komplex som bara finns finns i ”state 2” består av LHCII, PSI och LHCI, och det innehåller endast P-Lhcb2 men nästan inget P-Lhcb1, och ett band som består av LHCII, CP24 och CP29 innehåller endast PLhcb1. Vi skapade artificiella mikro-RNA-linjer, amiLhcb1 och amiLhcb2, som saknade antingen Lhcb1 eller Lhcb2. Lhcb1 påverkar höjden av grana stackarna. Med hjälp av dessa visade vi att Lhcb1 och Lhcb2 har komplementära roller för state transitions, saknas Lhcb1 gör växten bara få LHCII trimerer, medan om Lhcb2 gör växten antennener som liknar vildtypens, men den kan inte utföra state transitions som den. Mängden Lhcb1 påverkar storleken av ”grana stacks”. Trimerer som innehåller PLhcb2 kopplas över från PSII till PSI för att balansera excitationstrycket. Både Lhcb1 och Lhcb2, antagligen i trimerer bestående av en Lhcb2 och the Lhcb1, behövs för state transitions. Saknas Lhcb2 bildas inga komplex bestående av LHCII, PSI och LHCI, vilket visar att P-Lhcb2 antagligen möjliggör LHCIIs bindning till PSI. Vi försökte komplementera amiLhcb2 med Lhcb2 gener där amino syror bytts ut, Arg2 till Lys eller den fosforylerbara Thr3 till Asn eller Ser. När denna gen introducerades försvann dock ofta amiRNA-inhiberingen, men vi kunde visa att om Thr3 ersattes med Asn skedde inga state transitions.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 54 p.
Keyword [en]
Arabidopsis, Lhcb1, Lhcb2, LHCII, state transitions, phosphorylation
National Category
Natural Sciences Biological Sciences
Research subject
biology
Identifiers
URN: urn:nbn:se:umu:diva-97987ISBN: 978-91-7601-208-6 (print)OAI: oai:DiVA.org:umu-97987DiVA: diva2:779204
Public defence
2015-02-06, BiA201, Biologihuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2015-01-16 Created: 2015-01-12 Last updated: 2015-11-06Bibliographically approved
List of papers
1. Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis
Open this publication in new window or tab >>Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis
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2013 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 76, no 2, 236-246 p.Article in journal (Refereed) Published
Abstract [en]

Light-harvesting complex II (LHCII) contains three highly homologous chlorophyll-a/b-binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo- and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so-called state-transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10sec of state 2 light' results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non-phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super- and mega-complexes that comprised both photosystem (PS)I and PSII, and the state 2-specific PSI-LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keyword
Arabidopsis, Lhcb2, LHCII, phosphorylation, state transitions
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-83099 (URN)10.1111/tpj.12297 (DOI)000325367300006 ()
Funder
Swedish Research Council
Note

This work was supported by the European Union project FP7-PEO-PLE-ITN-2008 'HARVEST: Control of Light Use Efficiency in Plants and Algae From Light to Harvest', the Swedish Research Council (VR), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas), the Academy of Finland (project Nos 118637 and 138703) and C. Leoni by a 1-year fellowship from the University of Bari (Rectoral Decree No. 1598). The authors declare no conflict of interest.

Available from: 2013-11-18 Created: 2013-11-18 Last updated: 2017-12-06Bibliographically approved
2. The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in Arabidopsis
Open this publication in new window or tab >>The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in Arabidopsis
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2014 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 26, no 9, 3646-3660 p.Article in journal (Refereed) Published
Abstract [en]

Photosynthetic light harvesting in plants is regulated by phosphorylation-driven state transitions: functional redistributions of the major trimeric light-harvesting complex II (LHCII) to balance the relative excitation of photosystem I and photosystem II. State transitions are driven by reversible LHCII phosphorylation by the STN7 kinase and PPH1/TAP38 phosphatase. LHCII trimers are composed of Lhcb1, Lhcb2, and Lhcb3 proteins in various trimeric configurations. Here, we show that despite their nearly identical amino acid composition, the functional roles of Lhcb1 and Lhcb2 are different but complementary. Arabidopsis thaliana plants lacking only Lhcb2 contain thylakoid protein complexes similar to wild-type plants, where Lhcb2 has been replaced by Lhcb1. However, these do not perform state transitions, so phosphorylation of Lhcb2 seems to be a critical step. In contrast, plants lacking Lhcb1 had a more profound antenna remodeling due to a decrease in the amount of LHCII trimers influencing thylakoid membrane structure and, more indirectly, state transitions. Although state transitions are also found in green algae, the detailed architecture of the extant seed plant light-harvesting antenna can now be dated back to a time after the divergence of the bryophyte and spermatophyte lineages, but before the split of the angiosperm and gymnosperm lineages more than 300 million years ago.

Keyword
state transitions, phosphorylation, Lhcb1, Lhcb2
National Category
Biological Sciences
Research subject
biology
Identifiers
urn:nbn:se:umu:diva-97916 (URN)10.1105/tpc.114.127373 (DOI)000345919700015 ()
Available from: 2015-01-08 Created: 2015-01-08 Last updated: 2017-12-05Bibliographically approved
3. Phosphorylation of Lhcb2 at Thr3 is important for state transitions
Open this publication in new window or tab >>Phosphorylation of Lhcb2 at Thr3 is important for state transitions
2015 (English)Manuscript (preprint) (Other academic)
Publisher
17 p.
Keyword
Arabidopsis thaliana, state transitions, phosphorylation, artificial microRNA, complementation
National Category
Natural Sciences
Research subject
biology
Identifiers
urn:nbn:se:umu:diva-97920 (URN)
Available from: 2015-01-08 Created: 2015-01-08 Last updated: 2015-09-22

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