Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools.

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice.

INTRODUCTION: Ambient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB-PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. OBJECTIVES: This study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. METHODS: The thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. RESULTS: At an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. CONCLUSION: Transient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4-auxin-mediated thermomorphogenic responses in plants.

Color-Tuning Mechanism of the Lit Form of Orange Carotenoid Protein.

Orange carotenoid protein (OCP) of photosynthetic cyanobacteria binds to ketocarotenoids noncovalently and absorbs excess light to protect the host organism from light-induced oxidative damage. Herein, we found that mutating valine 40 in the α3 helix of Gloeocapsa sp. PCC 7513 (GlOCP1) resulted in blue- or red-shifts of 6-20 nm in the absorption maxima of the lit forms. We analyzed the origins of absorption maxima shifts by integrating X-ray crystallography, homology modeling, molecular dynamics simulations, and hybrid quantum mechanics/molecular mechanics calculations. Our analysis suggested that the single residue mutations alter the polar environment surrounding the bound canthaxanthin, thereby modulating the degree of charge transfer in the photoexcited state of the chromophore. Our integrated investigations reveal the mechanism of color adaptation specific to OCPs and suggest a design principle for color-specific photoswitches.

Color Sensing and Signal Transmission Diversity of Cyanobacterial Phytochromes and Cyanobacteriochromes.

To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes.

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Genomic Survey of Salt Acclimation-Related Genes in the Halophilic Cyanobacterium Euhalothece sp. Z-M001.

Like other halophilic cyanobacterial genomes, the de novo-assembled genome of Euhalothece sp. Z-M001 lacks genes encoding keto-carotenoid biosynthesis enzymes, despite the presence of genes encoding carotenoid-binding proteins (CBPs). Consistent with this, HPLC analysis of carotenoids identified ß-carotene and zeaxanthin as the dominant carotenoids. CBPs coexpressed with the zeaxanthin biosynthesis gene increased the survival rates of Escherichia coli strains by preventing antibiotic-induced accumulation of reactive oxygen species (ROS). RNA-seq analysis of Euhalothece revealed that among various salt resistance-related genes, those encoding the Na+ transporting multiple resistance and pH adaptation (Mrp) systems, glycine betaine biosynthesis enzymes, exopolysaccharide metabolic enzymes, and CBPs were highly upregulated, suggesting their importance in hypersaline habitats. During the early phase of salt deprivation, the amounts of ß-carotene and zeaxanthin showed a negative correlation with ROS content. Overall, we propose that in some halophilic cyanobacteria, ß-carotene and zeaxanthin, rather than keto-carotenoids, serve as the major chromophores for CBPs, which in turn act as effective antioxidants.