Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems.

: Changes in environmental conditions like temperature and light critically influence crop production. To deal with these changes, plants possess various photoreceptors such as Phototropin (PHOT), Phytochrome (PHY), Cryptochrome (CRY), and UVR8 that work synergistically as a sensor and stress sensing receptor to different external cues. PHOTs are capable of regulating several functions like growth and development, chloroplast re-location, thermomorphogenesis, metabolite accumulation, stomatal opening, and phototropism in plants. PHOT plays a pivotal role in overcoming the damage caused by excess light and other environmental stresses (heat, cold, and salinity) and biotic stress. The crosstalk between photoreceptors and phytohormones contrib-utes to plant growth, seed germination, photo-protection, flowering, phototropism, and stomatal opening. Molecular genetic studies using gene targeting and synthetic biology approaches have revealed the potential role of different photoreceptor genes in the manipulation of various beneficial agronomic traits. Overexpression of PHOT2 in Fragaria ananassa leads to the increase in anthocyanin content in its leaves and fruits. Artificial illumination with blue light alone and in combination with red light influence the growth, yield, and secondary metabolite production in many plants while in algal species it affects growth, chlorophyll content, lipid produc-tion, and increase its bioremediation efficiency. Artificial illumination alters the morphological, developmental, and physiological characteristics of agronomic crops and algal species. This review focuses on PHOT modulat-ed signalosome and artificial illumination-based photo-biotechnological approaches for the development of Climate-Smart Crops.

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Rhodopsins have been extensively employed for optogenetic regulation of bioelectrical activity of excitable cells and other cellular processes across biological systems. Various strategies have been adopted to attune the cellular processes at the desired subcellular compartment (plasma membrane, endoplasmic reticulum, Golgi, mitochondria, lysosome) within the cell. These strategies include-adding signal sequences, tethering peptides, specific interaction sites, or mRNA elements at different sites in the optogenetic proteins for plasma membrane integration and subcellular targeting. However, a single approach for organelle optogenetics was not suitable for the relevant optogenetic proteins and often led to the poor expression, mislocalization, or altered physical and functional properties. Therefore, the current study is focused on the native subcellular targeting machinery of algal rhodopsins. The N-and C-terminus signal prediction led to the identification of rhodopsins with diverse organelle targeting signal sequences for the nucleus, mitochondria, lysosome, endosome, vacuole, and cilia. Several identified channelrhodopsins and ion-pumping rhodopsins possess effector domains associated with DNA metabolism (repair, replication, and recombination) and gene regulation. The identified algal rhodopsins with diverse effector domains and encoded native subcellular targeting sequences hold immense potential to establish expanded organelle optogenetic regulation and associated cellular signaling.

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In bacteria, cyclic adenosine monophosphate (cAMP) signaling plays an essential regulatory role whose modulation via optogenetic tools would provide researchers an immense opportunity to control biological processes simply by illumination. The cAMP signaling in bacteria is a complex network of regulatory pathways, which utilizes distinct proteomic resources under different nutrient environments. We established an optogenetic modulation of cAMP and studied important cellular process of growth, biofilm formation, and virulence in the model bacterium E. coli using a light-gated adenylate cyclase (LgAC) from Beggiatoa sp. Blue light-induced activation of LgAC elevated the cAMP level in a blue light-dependent manner in E. coli. Quantitative proteomics revealed a decrease in the level of certain proteins governing growth (PTS, Adk, AckA, GlnA, and EFP), biofilm formation (IhfA, flagellin, YajQ, YeaG, and HlfC), and virulence (ClpP, YebC, KatE, BtuE, and Zur) in E. coli cells expressing LgAC upon blue light illumination. This optogenetic modulation of cAMP would be useful for deciphering cAMP-associated host–pathogen signaling of bacterial systems. Proteome knowledge established by this research work would also be useful for the scientific community while adapting LgAC-based optogenetic modulation for studying other relevant cAMP-driven bacterial physiology (e.g., energy metabolism). The systematic utilization of the established method and more extensively designed experiments regarding bacterial growth, biofilm, survival, and virulence might provide a road map for the identification of new targets for developing novel antibacterial drugs.

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as: (1) they are a single-component system i.e., their sensor and effector functions are encoded by the 7-transmembrane domains and (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator and effector components are involved for attaining synchronous multicomponent regulation. Optical modulation of this network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanine cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish proof of concept. Light sensor coupled modular system could be expressed in a precise cell type and which holds great potential in the advancement of optogenetics 2.0. It would enable manipulating entire relevant cell signaling system. Here, we had identified 50 novels modular rhodopsins with variant rhodopsins domain and its diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized functionality based on their sequence and structural homology with previously characterized rhodopsins. Presented novel modular rhodopsins with various effector domains hold potential to expand optogenetics tool kit to regulate various cellular signaling pathways across the diverse biological model systems.