Gene expression and organization of thylakoid protein complexes in the PSII-less mutant of Synechocystis sp. PCC 6803

Abstract

Photosystems I and II (PSI and PSII) are the integral components of the photosynthetic electron transport chain that utilize light to provide chemical energy for CO₂ fixation. In this study, we investigated how the deficiency of PSII affects the gene expression, accumulation, and organization of thylakoid protein complexes as well as physiological characteristics of Synechocystis sp. PCC 6803 by combining biochemical, biophysical, and transcriptomic approaches. RNA-seq analysis showed upregulated expression of genes encoding the PSII core proteins, and downregulation of genes associated with interaction between light-harvesting phycobilisomes and PSI. Two-dimensional separation of thylakoid protein complexes confirmed the lack of PSII complexes, yet unassembled PSII subunits were detected. The content of PsaB representing PSI was lower, while the content of cytochrome b₆f complexes was higher in the PSII-less strain as compared with control (CS). Application of oxygraph measurements revealed higher rates of dark respiration and lower PSI activity in the mutant. The latter likely resulted from the detected decrease in the accumulation of PSI, PSI monomerization, increased proportion of energetically decoupled phycobilisomes in PSII-less cultures, and low abundance of phycocyanin. Merging the functional consequences of PSII depletion with differential protein and transcript accumulation in the mutant, in comparison to CS, identified signal transduction from the photosynthetic apparatus to the genome level.

Keywords: D1 protein; RNA‐seq; Synechocystis sp. PCC 6803; photosynthesis; photosystem II; psbA2.

FIGURE 1

Content of photosynthetic proteins in CS and PSII‐less S. 6803. Total proteins isolated from the S. 6803 cells were separated by SDS‐PAGE and probed with antibodies to detect D1 (PSII core), PsaB (PSI core), Cyt f (Cyt b ₆ f), AtpB (ATP synthase), ferredoxin (Fdx), ferredoxin: NADP⁺ oxidoreductase (FNR), flavodiiron 3 (Flv3), rubisco large subunit (RbcL), orange carotenoid protein (OCP), allophycocyanin (APC), phycocyanin (PC), and the PSI‐associated PC linker (CpcL). The gels were loaded based on protein concentration. The figure shows representative results from three to six independent biological replicates

FIGURE 2

Organization and content of protein complexes in CS and PSII‐less S. 6803 thylakoids. (a) BN‐PAGE separation of protein complexes in thylakoids isolated from the CS and PSII‐less mutant strains. Bands representing visible protein complexes are indicated; 40 μg of proteins was loaded in the wells. (b) SDS‐PAGE separation of thylakoid complexes into individual protein subunits, following previous separation by BN‐PAGE in (a). Previously identified spots are labeled, including unassembled CP43, CP47, and PC and APC spots, according to identification by Herranen et al. (2004) and Zhang et al. (2004). PSI subunits are indicated in red, PSII subunits in blue, Cyt b₆f subunits in green and NDH‐1L subunits in cyan. The figure shows representative results from three independent biological replicates

FIGURE 3

Pigment composition and energy transfer in CS and PSII‐less cells. (a) Whole cell absorption spectra of CS (solid line) and PSII‐less (dashed line) cultures grown in BG‐11 media in the presence of 5‐mM Glc. Spectra were double‐normalized at 440 and 750 nm. Peaks corresponding to carotenoids, phycocyanin (PC) and chlorophyll a (Chl a) are indicated. Spectra shown are average of three independent biological replicates, shading indicates standard deviation. Inset shows PC peak in higher resolution to highlight the peak shift. (b) Oxygen uptake/evolution rates in CS (dark gray bars) and PSII‐less strain (light gray bars) of S. 6803. Rates of change in O₂ concentration were measured in darkness (respiration in darkness), under 1000 μmol m⁻² s⁻¹ light (PSII activity), and in light in the presence of DCMU, MV, 2,6‐dichlorophenolindophenol (DCPIP), ascorbate and sodium azide (NaN₃) (PSI activity). Values are average of three independent biological replicates. Error bars indicate standard deviation and asterisk statistically significant difference (p < .05). (c) 77‐K fluorescence emission spectra of CS (solid line) and PSII‐less strain (dotted line) S. 6803 cells were excited at 580 nm. Spectra shown are average of three independent biological replicates. Shading indicates standard deviation