Theophylline-dependent synthetic riboswitches enable tightly regulated inducible gene expression in tobacco plastids

Plastid transformation offers significant advantages for biotechnological applications, such as the cost-effective production of high-value proteins and secure transgene containment, owing to the predominantly maternal inheritance of plastid DNA. However, its broader application is limited by the lack of tightly controlled and inducible gene expression systems. Previously, a theophylline-dependent synthetic riboswitch (s.theo-RS) enabled translational regulation in plastids but exhibited leaky expression in the absence of the inducer, theophylline. To address this limitation, we engineered transplastomic tobacco plants expressing firefly luciferase under the control of a distinct class of synthetic riboswitches (RS-D, RS-E, and RS-E*). All three variants showed lower basal expression than s.theo-RS and achieved ON/OFF expression ratios exceeding ten-fold after theophylline treatment. RS-E supported the highest expression levels, whereas RS-E* exhibited stringent repression. Despite effective inducibility, RS-E-driven expression remained below 2% of that achieved with the constitutively active plastid psbA 5′ untranslated region (UTR), even when transcription was driven by a highly active plastid psbA promoter. Although further improvements, including optimization of the cis-regulatory sequence, are required, these results suggest that RS-E and/or RS-E* provide promising foundations for plastid-inducible expression systems requiring stringent OFF-state control, particularly for applications involving toxic proteins or products that impose substantial metabolic burdens on plants.