Anti-Lhcb1 | LHCII type I chlorophyll a/b-binding protein

Thylakoid protein phosphorylation and state transitions are disturbed after high light treatment in pgr6 background. a Total protein extracts of 4-week-old wild type (WT), pgr6-1, pgr6?2, sps2 and stn7/stn8 analysed by immunoblotting with anti-phosphothreonine antibody; the principal thylakoid phospho proteins are indicated on the right according to their size. Core photosystem II proteins D1 (PsbA) and D2 (PsbD) are indicated as a single band due to their poor resolution. Actin was used as a loading control. b Lhcb1 and Lhcb2 phosphorylation levels were visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), stn7/stn8 double mutant is a non-phosphorylated control. c Average transient of the variable room temperature chlorophyll fluorescence measured during the transition from red (660nm) supplemented with far-red light (720nm) state 1 to pure red light state 2 (n?=?4 independent pots containing 2–3 plants). The fluorescence curves from pgr6 and sps2 are shifted on the x-axis to allow visualising the FMST1 and FMST2 values. The x-axis time scale refers to the wild-type curve. d Calculated quenching related to state transition (qT?=?(FMST1?–?FMST2)/FM), expressed as the percentage of FM that is dissipated by the state 1 to state 2 transition, of wild type (WT), pgr6?1 and sps2 under moderate light (120?mol?m?2?s?1) (ML) and after 3?h of high light (500?mol?m?2?s?1) (HL). Whiskers and box plot shows the minimum, first quartile, median, average, third quartile and maximum of each dataset (n?=?4 biologically independent samples); p-values are calculated via a two-tailed Student’s t test. e STN7 phosphorylation level visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), a protein sample from stn7/stn8 double mutant was loaded as a control for the antibody specificity. Uncropped images of the membranes displayed in a, b and e are available as Supplementary Fig. 11. Data points for items c, d are available as Supplementary data 2

Characterization of BCM1.a Co-expression analysis of BCM1. BCM1 together with the CBGs, GLK2, the carotenoid biosynthesis gene DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE (DXS), and the gene encoding the chloroplast protein HIGH CHLOROPHYLL FLUORESCENT 107 (HCF107) were hierarchically clustered based on pairwise levels of similarity between their expression profiles, using the gene co-expression database ATTEDII (http://atted.jp/)76. Low distance values indicate high degrees of co-expression. b Accumulation of BCM1, CBEs, and LHCb1 in different organs of Arabidopsis. c Light-induced accumulation of BCM1, CBEs, and LHCb1 in 5-day-old etiolated WT seedlings, following illumination (80??mol photons?m?2?s?1) for 0, 3, 6, 12, and 24?h. d Schematic overview of the domain structure of BCM1. The cTP is shown in gray, while the six predicted TMDs are shown in red. e Subcellular localization of the BCM1-YFP fusion protein. Both BCM1-YFP and cTPBCM1-YFP fluorescence coincides with Chl autofluorescence, confirming chloroplast targeting of BCM1. In the control, YFP itself accumulates in the cytosol and nucleus. Scale bars, 5??m. f Suborganellar localization of BCM1. Chloroplasts from WT seedlings were subfractionated into envelope, stroma, and thylakoid fractions. For comparison, the proteins TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF CHLOROPLAST 40 (Tic40), GSAT, and LHC were specifically located in the envelope, stroma, and thylakoid, respectively. g Distribution of BCM1 across the different thylakoid membrane regions. Thylakoid proteins were solubilized with digitonin and fractionated into grana core, grana margins, and stroma lamellae. h Salt washing of thylakoid membranes. The WT thylakoids were sonicated in the presence of 1?M NaCl, 200?mM Na2CO3, 0.1?M NaOH, and 6?M urea on ice for 30?min before centrifugation to separate membrane (P) from soluble (S) fractions. Untreated thylakoids served as the control. LHCb1 and the CF1 ? subunit of ATP synthase, representing an intrinsic membrane protein and a peripheral thylakoid-associated protein, respectively, were used as positive controls for binding affinity to thylakoid membrane fractions. In b, c, f–h, immunoblot analyses were conducted using the indicated antibodies. Ponceau S-stained membrane strips bearing the large subunit of Rubisco (RbcL) or LHC proteins of PSII (LHCII) were used as loading controls.

BCM2 plays a conserved role in Chl biosynthesis.a Representative images of 35-day-old VIGS-GFP (as the negative control) and VIGS-BCM2 seedlings, which were infected with the pTRV2-GFP and pTRV2-BCM2 vectors via agroinfiltration in the WT and bcm1-3 background, respectively. The VIGS seedlings were grown under long-day normal light (120??mol photon?m?2?s?1) conditions. The old leaves were indicated by red arrows. b qRT-PCR analysis of BCM1 and BCM2 transcripts in the seedlings shown in a. c Representative images of detached leaves from seedlings shown in a. The old and young mature leaves selected for further analyses were marked by red and white dotted frames, respectively. d Levels of Chl in the old and young leaves shown in c. e Steady-state levels of the indicated proteins in the old and young leaves analyzed in c were determined by immunoblot analysis using the indicated antibodies. f Representative images of 28-day-old WT, bcm1-2, and three independent BCM2-OX/bcm1 transgenic lines overexpressing BCM2 in the bcm1-2 background grown under short-day normal light (120??mol photon?m?2?s?1) conditions. g qRT-PCR analysis of BCM1 and BCM2 transcripts in the seedlings shown in f. Expression levels are presented relative to those in WT seedlings. h, i Levels of Chl (h) and Mg-porphyrin (i) in 18-day-old WT, bcm1-2 and BCM2-OX/bcm1 seedlings grown under short-day normal light (120??mol photon?m?2?s?1) conditions. j Steady-state levels of the indicated proteins in seedlings analyzed in h were determined by immunoblot analysis using the indicated antibodies. In a, c, f, scale bars, 1?cm. In b, g, expression levels are presented relative to those in the VIGS-GFP/WT and WT seedlings, respectively. ND, not detected. In b, d, g–i, error bars represent SD of three biological replicates. Letters above histograms indicate significant differences determined by Tukey’s HSD method (P?<?0.05). In e, j, Ponceau S-stained RbcL was used as the loading control. Numbers below immunoblots represent normalized protein abundances relative to WT seedlings. Asterisks indicate nonspecific signals on the immunoblots.