Environmental Tuning of Homologs of the Orange Carotenoid Protein-Encoding Gene in the Cyanobacterium Fremyella diplosiphon

Abstract

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs - two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.

Keywords: cyanobacteria; non-photochemical quenching (NPQ); orange carotenoid protein (OCP); photosynthesis; stress.

FIGURE 1

Expression of orange carotenoid protein (ocp) homologs under varying light conditions. Cultures of wild type (A) and ΔrcaE (B) Fremyella diplosiphon were grown under green light (GL) and red light (RL) at light intensities of 10 (dark green and dark red), 50 (green and red), and 100 (light green and light red) μmol m^–2 s^–1. Relative expression of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using quantitative real-time PCR (qPCR). Expression under each condition is shown relative to expression under 10 μmol m^–2 s^–1 of green light. Error bars represent the standard deviation of each measurement. *p < 0.05, with dark gray stars indicating a difference for a strain in GL compared to RL at a specific light intensity, and red or green stars indicating a difference between different intensities of RL or GL, respectively; ^#p < 0.05 representing a difference between WT and ΔrcaE in identical light conditions as determined by a ANOVA with Tukey-Kramer HSD post hoc test.

FIGURE 2

Expression of ocp homologs in the ΔrcaC strain of F. diplosiphon. Wild type and ΔrcaC strains of F. diplosiphon cultures were grown under green and red light at light intensities of 20 μmol m^–2 s^–1. Relative expression of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using qPCR, comparing samples from the ΔrcaC mutant to samples from SF33. (±SD, n = 4). *p < 0.05, with dark gray stars indicating a difference for a strain in GL compared to RL, and green stars indicating a difference between WT and the ΔrcaC mutant grown in GL as determined by a ANOVA with Tukey-Kramer HSD post hoc test.

FIGURE 3

Cold-dependent expression of ocp homologs. Relative expression of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using qPCR, comparing samples incubated for 24 h at (A) 8°C or (B) 15°C to control samples maintained at 28°C (±SD, n = 6). *p < 0.05 as determined by a two tailed Student’s t-test for the comparison of the treated and untreated samples.

FIGURE 4

Expression of ocp homologs in response to salt stress. The expression levels of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using qPCR, comparing samples incubated for 24 h in BG-11/HEPES + 0.2 M NaCl to control samples grown in BG-11/HEPES (±SD, n = 6). *p < 0.05 as determined by a two tailed Student’s t-test for the comparison of the treated and untreated samples.

FIGURE 5

Expression of ocp homologs in response to nitrogen limitation stress. The expression levels of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using qPCR, comparing samples incubated for 24 h in BG-11/HEPES without nitrogen to control samples maintained in BG-11/HEPES with nitrogen (±SD, n = 6). *p < 0.05 as determined by a two tailed Student’s t-test for the comparison of the treated and untreated samples.

FIGURE 6

Expression of ocp homologs in the presence of methyl viologen or DCMU. The expression levels of ocp1, ocp2, frp, hcp1, hcp2, hcp3, and ccp2 were measured using qPCR, comparing samples incubated in the presence of 0.03 μM methyl viologen (A) or 10 μM DCMU (B) for 24 h to untreated samples (±SD, n = 4–6). *p < 0.05 as determined by a two tailed Student’s t-test for the comparison of the treated and untreated samples.

FIGURE 7

Promoter elements found in ocp homologs. The intergenic region upstream of each ocp homolog was analyzed using BPROM (Solovyev and Salamov, 2011) to predict transcription start positions (–10 and –35 sites) and transcription factor binding sites for each predicted promoter. Distinct binding sites are denoted with identical shapes and colors across distinct promoters.