Optogenetic and Chemical Induction Systems for Regulation of Transgene Expression in Plants: Use in Basic and Applied Research

Abstract

Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.

Keywords: agriculture; chemical induction; optogenetics; plants; transgene expression regulation.

Figure 1

General strategy for optogenetic systems in plants. In dark, the system is turned off, the target gene does not express. Upon illumination with the light of a certain wavelength, the optogenetic system is activated, resulting in the transcription of the target gene.

Figure 2

Domain structure and conformational changes of photoreceptors upon absorption of light. (A) Shown is the crystal structure of the LOV2 domain of A. sativa phototropin 1 (PDB ID 2V0U). The LOV2 domain is presented as ribbons (cyan) and the flavin chromophore as a stick model (red). (B) Schematic structure of the LOV2 domain from A. sativa fused with effector protein in darkness and under blue light. In dark, Jα helix docks onto the LOV2 core; under blue light it undocks, making the effector protein available for protein-protein interaction. (C) Homodimerization of the LOV domain containing protein EL222 from E. litoralis under blue light. The protein structure is presented as ribbons (cyan, blue, green) and the flavin chromophore is shown as a stick model (red). The crystal structure of the LOV domain from E. litoralis (PDB ID 3P7N) was used for visualization. HTH – DNA binding helix–turn–helix domain. (D) Typical domain structure of the photosensory module of the plant phytochromes. The PAS, GAF, and PHY domains are shown in cyan, purple, and orange ribbons, respectively; the C-terminal output module is not shown. The chromophore (PФB) is shown as a stick model (red). The crystal structure of A. thaliana PhyB photosensory module (PDB ID 4OUR) was used for visualization.

Figure 3

Light-driven changes in structure of tetrapyrrole chromophore PCB. Reversible Z/E isomerization of the C15/C16 double bond in bound PCB chromophore under illumination with far-red (640–680 nm) and NIR light (740–780 nm).

Figure 4

Application of optogenetic tools to control transgene expression in plants. (A) Application of the PhyB-PIF6 optogenetic pair in plant protoplasts. Far-red light (640–680 nm) induces heterodimerization of the PhyB and PIF6, resulting in activation of the reporter gene transcription. Incubation in darkness or illumination with NIR light (740–780 nm) inhibits this process. DBD – DNA-binding domain of the TetR, E or PiP proteins. (B) Green light-induced transcription inhibition in plants. In darkness, bacterial photoreceptor CarH fused to the VP16 AD and linked to the chromophore AdoB12 binds to the CarO operator resulting in transcription activation of the target gene. Illumination with 525 nm light induces dissociation of the CarH tetramers releasing CarO and subsequent inhibition of the reporter gene expression. (C) Blue light-induced gene expression system in alga C. reinhardtii. Blue light (460–480 nm) stimulates CIB1 and CRY2 heterodimerization and thus brings the VP16 AD closer to Gal4 DBD that results in the activation of the target gene transcription. In darkness, CRY2-VP16 complex dissociates from CIB1-Gal4 DBD. UAS—upstream activation sequence; Gal4—Gal4 DBD.

Figure 5

Control of transgene expression in plants using the PULSE system. Upon illumination with blue (460–480 nm) or white light, PhyB interacts with PIF6. However, transcription of the reporter gene is blocked by blue-off module, which is based on the blue light-induced homodimerization of the EL222 LOV domain fused to a plant transcriptional repressor domain SRDX. Under NIR light (740–780 nm) or in darkness, PhyB does not interact with PIF6 and blue-off module is not active. Under far-red illumination (660 nm), blue-off module is also not active, while PhyB and PIF6 form heterodimers, resulting in the transcription activation of the reporter gene. E—DBD of the macrolide repressor protein E. Pink and blue boxes are the far-red light activation and blue light inhibition (blue-off) modules, respectively.

Figure 6

Schematic representation of chemically-inducible systems for regulation of transgene expression in plants. (A) The Tet-derepressible system. In the absence of tetracycline (Tet), tetR binds to TRE sequences, and the target gene is not transcribed. Upon Tet addition, tetR cannot bind to TRE, allowing the target gene transcription. (B) The Tet-off system. In the absence of Tet, tetR-VP16 binds to TRE resulting in activation of the target gene. In the presence of Tet, tetR-VP16 cannot bind to TRE to induce the transgene expression. (C) The GVG/UAS system. In the absence of dexamethasone (Dex), GVG binds to the regulatory protein Hsp90 forming an inactive complex. Dex addition results in the dissociation of GVG from the Hsp90 protein and its binding to UAS upstream of the target gene. (D) The pOp6/LhGR system. In the absence of Dex, LhGR binds to the regulatory protein Hsp90, and, thus, is inactive. In the presence of Dex, LhGR binds to pOp6 sequence to activate the transgene expression. (E) ER-C1 system. In the absence of β-estradiol (E2), ER-C1 does not bind to ER elements (ERE). Once activated by E2, ER-C1 binds to ERE and activates target gene expression. (F) The XVE system. In the absence of E2, XVE does not bind to the LexA operator sequence (OlexA). After treatment with E2, XVE binds to OlexA and activates the target gene expression. (G) The XVE-controlled Cre/loxP system. The XVE and Cre expression cassettes are located between the constitutive promoter and the target gene flanked by two loxP sites. In uninduced condition, the target gene is not expressed. In the presence of inducer (E2), XVE activates the Cre transcription, resulting in the fusion of the constitutive promoter and the target gene due to Cre/loxP-mediated recombination. T—transcription terminator. (H) A dual-controlled TGV system. In the absence of Dex and Tet, TGV binds to the Hsp90 protein, forming an inactive complex. Upon Dex addition, TGV separates from Hsp90, binds to the Tet operator sequence (tetO7) and activates the target gene expression. In the presence of Tet, the TGV protein dissociates from the reporter gene promoter and the level of the target gene transcription drops to uninduced levels. (I) The GVEcR system. Without methoxyfenozide (Me), GVEcR does not bind to the LexA binding site (OlexA) and the target gene is not transcribed. In the presence of Me, GVEcR binds to OlexA and activates the target gene transcription. (J) The copper-inducible system. In the absence of copper (Cu²⁺), the transcription factor ACE1 does not bind to metal responsive element (MRE) and the transgene is not transcribed. In the presence of copper ions, ACE1 binds to MRE and activates the target gene transcription. (K) The ethanol-inducible system. Without ethanol (EtOH), the transcription factor AlcR does not bind to the promoter of the AlcA gene (AlcA) and the transgene is not transcribed. After addition of ethanol, AlcR binds to AlcA and activates the transgene expression.