The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching

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Rodney Johnston
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1 In the formt provided y the uthors nd unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: The xnthophyll cycle ffects reversile interctions etween PsS nd light-hrvesting complex II to control non-photochemicl quenching Jonn Schrz, Vsco Giovgnetti, Petr Ungerer, Giuli Mstroinni nd Alexnder V. Run* School of Biologicl nd Chemicl Sciences, Queen Mry University of London, Mile End Rod, London E1 4NS, UK. *e-mil:.run@qmul.c.uk NATURE PLANTS DOI: /nplnts

2 Supplementry Figure 1 Cross-linker effects on quenching nd PsS oligomeristion Control 0.5mM DSP 1.0mM DSP Pull-down ssy DSP [mm] 2.5mM DSP Figure S1. Effect of the hydrophoic cross-linker DSP on NPQ induction nd recovery on intct chloroplsts of spinch. Impct of DSP on memrne kinetics ws tested y ddition to the chloroplsts suspension (concentrtion of chlorophyll 35ug/mL) of DSP t the finl concentrtions of 0.5, 1.0 nd 2.5 mm, 10 min prior to illumintion. PAM fluorescence ws used to ssess NPQ induction nd recovery. Smples were illuminted with ctinic light (447 µmol photons m -2 s -1 ) for 10 min followed y 10 min in the drk (). Visuliztion of PsS protein in thylkoid memrnes (Th) nd mteril isolted y pull-down ssys from illuminted chloroplsts incuted with incresing DSP concentrtions (0, 0.5, 1.0, 1.5, 2.0 nd 2.5 mm). Identifiction of PsS y western lotting. NATURE PLANTS DOI: /nplnts

3 Supplementry Figure 2 PsS interctions in Aridopsis thlin plnts overexpressing PsS treted with lincomycin DSP Th - + Th - + -DSP α-pss α-lhc1 +DSP Violxnthin α-lhc2 α-lhc3 29- α-lhc4 c Chlorophyll Fluorescence α-lhc5 α-lhc6 Fluorescence Drk NPQ Time [s] Figure S2. Pull-down ffinity ssy of PsS protein from violxnthin-contining chloroplst of Aridopsis thlin L17 treted for 2 weeks with lincomycin. Identifiction of proteins in thylkoid memrnes (Th) nd co-purified with PsS vi SDS-PAGE nd western lotting (proed ginst Lhc1-6) (). Quntifiction of proteins isolted with PsS without nd with ddition of the cross-linker DSP (0.5 mm). All dt normlized to the totl PsS isolted in ech condition (). Representtive fluorescence trce nd quenching for violxthin-contining chloroplsts from L17 mutnt plnts (C). NATURE PLANTS DOI: /nplnts

Supplementry Figure 3 Immunogold lelling controls -αpss npq4 A Immunogold-lelling of PsS: Procedure without primry ntiody (αpss) B Immunogold-lelling of PsS: Both primry (αpss) nd

(A) Immunogold lelling of PsS in S. olerce chloroplsts where no primry ntiodies (α-pss) ws used followed y incution with 1:100 secondry ntiody.

4 Supplementry Figure 3 Immunogold lelling controls -αpss npq4 A Immunogold-lelling of PsS: Procedure without primry ntiody (αpss) B Immunogold-lelling of PsS: Both primry (αpss) nd secondry ntiody were used in dilutions: 1:50 nd 1:100 respecitively Figure S3. Control smples for immunogold lelling of PsS protein in Spince olerce nd A. thlin chloroplsts. (A) Immunogold lelling of PsS in S. olerce chloroplsts where no primry ntiodies (α-pss) ws used followed y incution with 1:100 secondry ntiody. (B) Control of PsS ntiody inding specificity performed using oth primry (PsS) nd secondry ntiody on A. thlin npq4 mutnt chloroplsts (not expressing PsS) (B). NATURE PLANTS DOI: /nplnts

5 Supplementry Figure 4 SUPPLEMENTARY INFORMATION npq4 NPQ [+DSP]* NPQ= B α-pss α-lhc1 α-lhc4 Figure S4. Aridopsis thlin npq4 line (not expressing PsS protein) ws used s control in pull-down ssys. Proteins from thylkoid memrnes (Th) nd elution were seprted on SDS-PAGE nd proed ginst α-pss, α-lhc1 nd α-lhc4 ntiodies respectively (). PAM fluorescence trce on npq4 mutnt chloroplsts illuminted with ctinic light (447 µmol photons m -2 s -1 ) (). NATURE PLANTS DOI: /nplnts

Supplementry Figure 5 Visuliztion of pulled down mteril Pull-down ssy from soluilised thylkoid memrnes

6% β-dm) Control (-αcp29, -αpss) Control (αcp29, -αpss) c Memrne frgment Immunogold lelled CP29 d 50nm

Controls without PsS nd CP29 ntiodies (), nd with CP29 ut without PsS ntiody ().

Blue rrows indicte elements distinguishle under TEM: 50 nm mgnetic ed to which PsS ntiodies hve een

6 Supplementry Figure 5 Visuliztion of pulled down mteril Pull-down ssy from soluilised thylkoid memrnes from Aridopsis (0.6% β-dm) Control (-αcp29, -αpss) Control (αcp29, -αpss) c Memrne frgment Immunogold lelled CP29 d 50nm Drk Mgnetic ed + αpss Drk + DSP Figure S5. Immunogold lelling of CP29 in PsS pull-down elutions. Controls without PsS nd CP29 ntiodies (), nd with CP29 ut without PsS ntiody (). Pull-down ssy of PsS from spinch thylkoid memrnes immunogold leled with CP29 ntiody (-DSP). Blue rrows indicte elements distinguishle under TEM: 50 nm mgnetic ed to which PsS ntiodies hve een coupled, nd secondry ntiodies ttched to 5nm gold prticles which inds to CP29 primry ntiodies (c). Enlrged imge of the sme experiment in presence of DSP (0.5 mm) (d). NATURE PLANTS DOI: /nplnts

NORTH GATE BLVD. STRUTHERS RD Denver Region A B ELEVEN 60X49 45'--0" PAGE ' 24'-0" PAGE 5 PAGE

NORTH GATE BLVD. STRUTHERS RD Denver Region A B ELEVEN 60X49 45'--0 PAGE ' 24'-0 PAGE 5 PAGE SITE PLN NOT TO SCLE Region 0 E. rd venue, Suite 00, CO 809 0-7-99.' 8 '--0" '-0" 0 List Drive, CO 8099 79-8-00 07 9 0 9 STRUTHERS RD..98' 7 #807 9 GLENEGLE GTE VIEW EL PSO COUNTY, CO JOHN CURRN LNGFORD