Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to function in the context of horticultural light environments. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter, a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system. Analysis of a re-engineered CcaS in Escherichia coli demonstrated green/red photoswitching with phytochromobilin, a chromophore endogenous to plants, but also revealed a blue light response likely derived from a flavin-binding LOV-like domain. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We implemented the system to demonstrate optogenetic control over plant immunity and pigment production through modulation of the spectral composition of broadband visible (white) light. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.