Dissection Of Arabidopsis CURT1 Complex Functions In Thylakoid Ultrastructure For Mation And Photosynthesis

Grana,cylindrical structures of stacked thylakoid membranes,are ubiquitous in land plant chloroplasts.Their role in photosynthesis remains controversially discussed and little is known about the mechanism underlying their formation.In Arabidopsis thaliana,the CURT1 protein family,containing four members(CURT1A-D),is involved in the grana formation by facilitating membrane bending in the grana margins.This effect is CURT1 dosage-dependent,as overexpression of CURT1A(oeCURT1A)or loss of CURT1 proteins(curtlabcd)leads to opposite altered grana phenotypes.In this thesis,the photosynthetic performance and growth rate of oeCURT1A and curtlabcd lines were compared to WT under different light conditions.Both lines exhibited deficiency in photosynthesis and moderately retarded growth rate,suggesting that the grana formation is not essential,but beneficial for photosynthesis and growth.Regarding the mechanism of membrane curvature,CURT1 proteins were shown to insert into the thylakoid membrane and to form oligomeric structures.The aims of this project were to study:(i)the effects of altered grana stacking on photosynthetic performance and growth rate,and(ii)to uncover the roles of CURT1 proteins and reversible phosphorylation in regulating grana formation in Arabidopsis,as well as the interplay between these two mechanisms.To address the first part,oeCURT1A and curtlabcd mutants,which show distinct alternation in grana formation,were characterized in several aspects:(i)Photosynthetic performances and growth rates under various light conditions,(ii)D1 turnover kinetics under high light conditions,(iii)Distribution patterns of CURT1 and major thylakoid complexes under different light conditions.The second part focuses on the effects of CURT 1 amounts and reversible phosphorylation on grana formation.Regarding the CURT1 proteins,the effects of CURT1 dosage and the functional redundancy were addressed.To this end,the expression levels of different CURT1 members were assessed in oeCURTIA line.Moreover,curtlabc mutants were complemented with CURT1 proteins from.Arabidopsis or Synechocystis and thylakoid ultrastructure was analyzed by transmission electron microscopy(TEM).In the light of phosphorylation,the goal was to check the role of phosphorylation on CURT1 proteins and grana stacking.The experimental approaches involved:(i)Assessment of CURT 1 levels in PK/PP mutant lines,and(ii)Examination of the thylakoid ultrastructure of crossed mutants(e.g.curtlabc tap38)and curtl mutants complemented with phospho-site exchange(PSE)proteins.In this study,CURT1A was found to be the major component of the CURT1 protein complexes as(i)in the oeCURTIA line,overexpression of CURT1A induced shorter but higher grana stacking without simultaneously increased expression levels of CURT1B and C,and(ii)in 35S:CURT1A curtlabc,CURT1A reconstituted grana stacking in the absence of CURT1B and C.Besides a dependence of the bending mechanism on CURT1 protein amounts,thylakoid phosphorylation was a suggested mechanism to regulate grana stacking.In this study,we showed that the effects of the CURT1 protein amounts dominated thylakoid phosphorylation in affecting grana formation,as curtlabc tap38 mutants exhibited a similar grana phenotype as the curtlabc mutant line.Thus we propose that thylakoid phosphorylation rather functions on a regulatory level influencing grana plasticity.Besides changing the membrane charge directly by phsphorylating thylakoid proteins,this regulation could be also accomplished either by phosphorylating CURT1 proteins or a phosphorylation cascade.In this respect,the analysis of the assembly behavior of CURT1 proteins was initiated by investigating both PSE lines(lines complemented with CURT 1 phospho-site exchange variants)and protein kinase/phosphatase mutant lines(PK/PP lines).Finally,based on an altered migration behavior of CURT1 proteins under HL conditions,a possible interplay between CURT 1 protein phosphorylation and CURT1 complex formation is discussed with respect to the modulation of grana formation.