Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical-and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi-and fully synthetic open-and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

that derive from clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques, currently positively reshapes biological studies. [2] Key applications of the specific DNA-recognizing CRISPR/Cas9 systems encompass for example genome editing and transcriptional regulation with high sequence specificity. [2][3][4][5][6] In recent years, these technologies led to major advances in synthetic biology, gene therapy, and gene modification in almost every model organism. Various Cas-variants were subsequently derived from different microorganisms and utilized to overcome some of the limitations restricting in vivo applications, or enabled the recognition of RNA instead of DNA. [2,[7][8][9] Among those, the discovery of the RNA-targeting Cas13 proteins yielded in powerful RNA-editing tools. [9,10] Cas13 belongs to type VI CRISPR effectors and has been utilized for specific knockdown of endogenous RNAs in human cells and manipulation of alternative RNA splicing. [9,11] While the DNAtargeting CRISPR/Cas9 systems require the presence of a short protospacer adjacent motif (PAM) sequence at the editing site, Cas13 is PAM-independent. [9,10] Recent engineering efforts of CRISPR/Cas tools for optogenetic regulation expanded their capabilities including high spatial and temporal control precision, [12][13][14][15][16][17][18][19] however, these systems exclusively target DNA. Only few light-regulated RNA modification tools currently exist, mostly based on RNA interference or short regulatory RNAs, which are CRISPR-independent. [20][21][22][23] We devised here an optogenetic tool to destabilize cellular mRNA by enabling optical control of RNA-targeting CRISPR/ Cas13 systems. For this, we combined a blue light-inducible gene expression switch [24] with the Prevotella sp.-derived Cas13b effector (PspCas13b), [9] resulting in a system termed Lockdown (blue light-operated CRISPR/Cas13b-mediated mRNA knockdown). Blue light activates the gene switch to induce the expression of PspCas13b which, in the presence of a gRNA targeting the mRNA of interest, leads to downregulation of said RNA. Our results demonstrate how Lockdown can be used to regulate cellular processes with blue light through specific mRNA degradation. In our tests, these induced G2 cell cycle arrest and inhibition of cell growth under blue light. [25,26] We further combined Lockdown with the recently published "Blue-OFF" system for synergistic triple-targeted downregulation of proteins. [27,28] To engineer the Lockdown system, we used a split transcription factor based on a modified light-oxygen-voltage domain The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introd...

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors maybe also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, a genetically encoded, ratiometric sensor, StrigoQuant, was developed and allowed the quantification of the activity of a wide concentration range of SLs.StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL GR24. We then show the recording of the luminescence signal and how obtained data are processed and used to assess/determine SL perception.