Tic40 | Inner envelope membrane translocon complex protein (chloroplast)

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.

The second, chloroplast growth phase involves greening and is supported by protein accumulation profiles. a Chlorophyll content, quantified in each of three independent biological replicates per sample. Error bars represent standard error of the mean. See Additional file 5: Table S6 for calculations. b, c, d Expression (Z-scores) of pigment biosynthesis and thylakoid biogenesis (b), light reactions (c) and carbon fixation-associated genes (d). e Expression (Z-scores) of chloroplast development-associated transcripts reflecting two stages of plastid development, peaking in the early plastid phase (RCB, ARC5 and TIC40) and, second, chloroplast phase (PSBO2, LHCB1.4 and SBPAse). f Immunoblot analysis of the protein products of the genes displayed in e. In total, 20??g of protein of samples 1–14 (for PSBO2, LHCB1.4 and SBPAse), 40??g (for RCB, ARC5 and TIC40) or 10??g (for Histone H3 as a constitutive control) was separated on denaturing SDS-PAGE gels, transferred to blots and probed with antibodies against the protein indicated. A Coomassie-stained total protein replica gel is also shown. Molecular weights (KDa) are indicated on the left. The results show one typical example from among three independent protein extraction and immunoblot experiments