A synthetic switch based on orange carotenoid protein to control blue–green light responses in chloroplasts

Abstract Synthetic biology approaches to engineer light-responsive systems are widely used, but their applications in plants are still limited due to the interference with endogenous photoreceptors and the intrinsic requirement of light for photosynthesis. Cyanobacteria possess a family of soluble carotenoid-associated proteins named orange carotenoid proteins (OCPs) that, when activated by blue–green light, undergo a reversible conformational change that enables the photoprotection mechanism that occurs on the phycobilisome. Exploiting this system, we developed a chloroplast-localized synthetic photoswitch based on a protein complementation assay where two nanoluciferase fragments were fused to separate polypeptides corresponding to the OCP2 domains. Since Arabidopsis (Arabidopsis thaliana) does not possess the prosthetic group needed for the assembly of the OCP2 complex, we first implemented the carotenoid biosynthetic pathway with a bacterial β-carotene ketolase enzyme (crtW) to generate keto-carotenoid-producing plants. The photoswitch was tested and characterized in Arabidopsis protoplasts and stably transformed plants with experiments aimed to uncover its regulation by a range of light intensities, wavelengths, and its conversion dynamics. Finally, we applied the OCP-based photoswitch to control transcriptional responses in chloroplasts in response to green light illumination by fusing the two OCP fragments with the plastidial SIGMA FACTOR 2 and bacteriophage T4 anti-sigma factor AsiA. This pioneering study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses upon exposure to specific light spectra.

A, Comparison in plant size, calculated as the Projected Leaf Area (PLA) and, B, comparison in the HUEvalue between Col-0, crtW#11 and crtW#19. The analysis was made on 20 replicates, and repeated at different time points: 17, 21, 24 and 28 days after germination. Asterisks indicate statistical differences calculated from one-way ANOVA followed by Tukey's post-hoc test (****, p≤ 0.0001).
Supplemental Figure S3. Comparison of split-NlucOCP2 activity in two different crtW lines.
Light response in Arabidopsis crtW#11 and crtW#19 isolated protoplasts. Protoplasts were treated with either dark or blue light (350 µmol μm -2 s -1 ) for 20 min. In the box plots, dots represent biological replicates, the black line marks the median, and the box indicates the interquartile range (IQR). Whiskers extend to data points below 1.5 X IQR away from the box extremities. A statistic Student t-test was used to assess significant differences. Asterisks indicate statistical differences (***, p≤ 0.001, **, 0.001<p≤0.01, n=4). Figure S4. Effect of different light spectra on the activity of a synthetic NanoLuc.

Supplemental
The activity of a synthetic Nanoluc was assessed in crtW#11 isolated protoplasts under different light regimes. Samples were treated with 350 µmol μm -2 s -1 light for 20 min. A, Green and blue light treated protoplasts compared with dark treated samples. B, Comparison between dark and red light treated samples. In the box plots, dots represent biological replicates, the black line marks the median, and the box indicates the interquartile range (IQR). Whiskers extend to data points below 1.5 X IQR away from the box extremities. A statistic Student t-test was used to assess significant differences (p≤ 0.05, n=4) and they were indicated marked with different letters, when present. Figure S5. Comparison between protoplast vitality and Nanoluc activity in light treated samples.

Supplemental
Protoplasts were treated with either dark or blue light (350 µmol μm -2 s -1 ) for 20, 40 and 80 min. A, Protoplast viability, expressed as % Live/Dead, in dark and light treated samples. B, Nanoluc activity measured in the samples used for vitality check. In the box plots, dots represent biological replicates, the black line marks the median, and the box indicates the interquartile range (IQR). Whiskers extend to data points below 1.5 X IQR away from the box extremities. Different letters indicate statistical differences (P ≤ 0.05) calculated from one-way ANOVA (n=6) followed by Tukey's post-hoc test. RpoD residues involved in AsiA contact are shown in red. Asterisks below a residue indicate identity between the two sequences; ':' and '.'indicate similar physical-chemical properties of residues.
Supplemental Table S1. Comparison of the carotenoid content in crtW and wild-type Arabidopsis plants.
Data are percentages of each pigment relative to the total carotenoid content per each line. Data are reported as average of three independent replicates. Errors are reported as standard deviation, shown per each group of lines. A Student T-test was used to assess significant differences between crtW and Col-0 genotypes. Different letters between rows represent differences in carotenoid content (p < 0.05).