GENERATION OF PLANT ORGANOIDS USING OPTOGENETIC CIRCUITS


Group leader: Pablo Pérez García - Young Investigator Researcher (YIR)
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Personnel:

 

 

Research description and objectives

Our primary aim is to engineer plant organoids through the implementation of synthetic genetic circuits that reprogram pluripotent stem cells derived from lateral root primordia, redirecting their intrinsic developmental trajectories toward custom-designed organogenic programs.


Although organoid technology is still in its early stages, it has already emerged as a transformative research tool in human and animal developmental biology, biotechnology, and regenerative medicine. Synthetic biology tools, such as synthetic promoters and optogenetic switches, have demonstrated significant utility in dissecting developmental processes in animal and human systems with high spatiotemporal precision. However, the application of these technologies in plant systems remains largely unexplored, presenting an untapped potential for advancing our understanding of plant development and driving the next frontier in plant organoid generation.


Our main objectives are

  1. Design synthetic promoters to precisely regulate gene expression in pluripotent stem cells within lateral root primordia.
  2. Utilize optogenetic switches to modulate gene expression with high spatiotemporal resolution.
  3. Engineer artificial transcription factors to ensure functional autonomy of synthetic circuits introduced into plant cells.
  4. Reprogram pluripotent stem cells from lateral root primordia to direct organogenesis toward the formation of plant organoids.

 


1. Synthetic promoters for driving gene expression in pluripotent stem cells of lateral root primordia


The precise regulation of gene expression is largely governed by promoters, which orchestrate transcriptional activity by recruiting specific transcription factors. Modifying the cis-regulatory elements within promoter regions can modulate downstream gene expression. Native promoters typically exhibit a diverse and variable arrangement of cis elements, which can be systematically reconstituted to create synthetic promoters with tailored functionalities.


Synthetic promoters offer the advantage of controlling gene expression during specific developmental stages, generating precise spatiotemporal and stage-specific expression patterns. Their design involves characterizing the cis-regulatory elements of native promoters, constructing minimal promoters by excluding non-essential elements, designing new synthetic configurations, and validating their functionality through reporter gene assays and other experimental approaches.


Compared to native promoters, synthetic promoters can overcome limitations related to transcriptional efficiency, strength, and cell-type specificity. By reducing complexity, optimal minimal synthetic promoters mitigate issues such as background expression and cross-talk between synthetic circuits and endogenous regulatory networks.


We leveraged knowledge of the transcriptional regulatory networks active during lateral root primordia formation to identify native promoters of interest. We analyzed the cis-regulatory elements of these promoters to design minimal synthetic promoters that specifically direct gene expression to pluripotent stem cells within lateral root primordia.

 


Fig1. Scheme of the synthetic promoter generation process. GFP: GREEN FLUORESCENT PROTEIN.


2. Optogenetic switches for the precise regulation of gene expression with high spatiotemporal resolution


Optogenetics utilizes engineered photoreceptors expressed in transgenic organisms to control biological processes through light-based activation. A diverse array of photoreceptors, derived from bacteria, fungi, and plants, has been engineered to harness the natural variation in photobiological properties. One of the key advantages of optogenetic systems is their reversibility, as light can be switched on or off to modulate gene expression dynamically. Furthermore, by adjusting light intensity and exposure duration, optogenetic systems allow for fine-tuned, quantitative control of biological responses, offering a non-invasive approach with exceptional spatiotemporal precision.


Although optogenetic tools have been extensively applied in various model organisms, their implementation in plants remains limited. This is largely due to the challenge of disentangling the light-dependent regulatory mechanisms required for plant growth from optogenetic activation. To address this, we propose utilizing optogenetic switches within the lateral root primordia to precisely modulate gene expression in the pluripotent stem cells. Since roots naturally grow in darkness, we will cultivate the roots under dark conditions to simulate their natural environment. This approach ensures that optogenetic activation within the lateral root primordia does not interfere with the plant’s intrinsic light-dependent developmental processes.

 

 

Fig 2. Examples of optogenetic switches. PHYB: PHYTOCHROME B. PIF3: PHYTOCHROME INTERACTING FACTOR 3. CRY2: CRYPTOCHROME 2. CIB1: CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1. POI: Protein of interest

 


3. Engineering of artificial transcription factors to minimize interactions with the plant genome


Transcriptional regulation is primarily mediated by transcription factors, proteins that recognize specific sequences within gene promoters and directly interact with DNA to modulate gene expression levels. Artificial transcription factors (ATFs) are engineered constructs composed of two functional units: one for DNA binding and another for transcriptional activation or repression. Traditionally, DNA-binding domains are derived from naturally occurring transcription factors, though novel approaches, such as CRISPR-based systems, have emerged as powerful alternatives.

ATFs are pivotal in synthetic biology for linking distinct modules of synthetic genetic circuits, while minimizing cross-talk or interference with the plant’s endogenous regulatory machinery. In our lab, we employ diverse strategies, ranging from incorporating species-specific transcription factor domains to leveraging CRISPR technologies for the precise construction of ATFs, ensuring efficient and controlled transcriptional modulation within plant systems.

 

4. Reprogramming pluripotent stem cells for plant organoid generation


Technologies centered on the generation of organ systems from pluripotent stem cells have garnered significant attention in animal biology. These systems, referred to as organoids, are three-dimensional, miniaturized, and simplified models of organs that recapitulate key aspects of native micro-anatomy. Organoids serve as developmental models to study both physiological and pathological processes, offering a platform for generating functional tissues or structured biological assemblies in vitro. Despite their transformative impact in animal systems, the application of organoid technology in plants remains largely unexplored.

Several challenges must be addressed in the in vitro generation of organoids. The microenvironment in which stem cells reside exerts significant influence on their function, with both biochemical signals and the physical properties of surrounding tissues playing crucial roles in the spatial configuration and functionality of the resulting organoids. Alternatively, in vivo direct reprogramming seeks to modulate stem cell fate by externally modifying differentiation pathways while leveraging the organism's native microenvironment. Although in vivo reprogramming of animal cells involves stable genetic alterations, the process is often complex, time-intensive, and accompanied by bioethical concerns.

In plant sciences, conventional cell culture techniques face additional limitations due to the rigidity of the cell wall, which presents a substantial barrier to the establishment of robust in vitro organoid systems. Despite these challenges, in vivo cell reprogramming remains largely unexplored in plants and presents a promising avenue for generating plant organoids, potentially bypassing the limitations of in vitro culture systems.

The elucidation of gene regulatory networks governing developmental transitions during lateral root primordia formation enables the prediction of specific developmental trajectories, including the pathways leading to stem cell emergence within the primordia. The well-defined differentiation program that guides the formation of a root meristem in the lateral root primordia can be reprogrammed through the precise temporal application of exogenous factors, redirecting organogenesis toward the formation of shoots instead of roots. This demonstrates the pluripotent nature of stem cells within the lateral root primordia, which can be reprogrammed to generate alternative organs when provided with appropriate developmental cues.

Using synthetic biology tools, we aim to precisely engineer or introduce genetic circuits that drive desired self-organizing programs within the lateral root primordia, a structure characterized by its high pluripotency and reprogramming potential.

Fig 3. Example of a designed optogenetic circuit to produce shoot organoids in the lateral root primordia. (A) Oversimplified model of the gene regulatory network of the stem cell trajectory operating within the lateral root primordia. (B) Segmentation of a promotor into minimal cis elements (C) Red-light dependent activation of a photo-switch to activate the expression of a stem cell reprograming factor (SCRF) (D) Schematic representation of a possible optogenetic circuit to promote the conversion of the root apical meristem (RAM) to a shoot apical meristem (SAM). Square boxes: open reading frames. Circles: gene products. Numbers: Minimal cis elements. LRP: lateral root primordia. GFP: GREEN FLUORESCENT PROTEIN. PHYB: PHYTOCHROME B. PIF3: PHYTOCHROME INTERACTING FACTOR 3. TF: Transcription factor. CKs: Cytokinins. WUS: WUSCHEL.

  


Representative Publications

Perez-Garcia, P., Pucciariello, O., Sanchez-Corrionero, A., Cabrera, J., del Barrio, C., Del Pozo, J.C., Perales, M., Wabnik, K., Moreno-Risueno, M.A. 2023. The cold-induced factor CBF3 mediates root stem cell activity, regeneration and developmental responses to cold. Plant Communications 100737. DOI: 10.1016/j.xplc.2023.100737


Perez-Garcia, P., Serrano-Ron, L., Moreno-Risueno, M.A. 2022. The nature of the root clock at single cell resolution: Principles of communication and similarities with plant and animal pulsatile and circadian mechanisms. Current Opinion in Cell Biology 77, 102102. DOI: 10.1016/j.ceb.2022.102102


Serrano-Ron, L., Perez-Garcia, P., Sanchez-Corrionero, A., Gude, I., Cabrera, J., Ip, P.-L., Birnbaum, K.D., Moreno-Risueno, M.A. 2021. Reconstruction of lateral root formation through single-cell RNA sequencing reveals order of tissue initiation. Molecular Plant 14, 1362–1378. DOI: 10.1016/j.molp.2021.05.028


Serrano-Ron, L., Cabrera, J., Perez-Garcia, P., Moreno-Risueno, M.A. 2021. Unraveling Root Development Through Single-Cell Omics and Reconstruction of Gene Regulatory Networks. Frontiers in Plant Science 12, 671. DOI: 10.3389/fpls.2021.661361


Perez-Garcia, P., Moreno-Risueno, M.A. 2018. Stem cells and plant regeneration. Developmental Biology 442, 3–12. DOI: 10.1016/j.ydbio.2018.06.021


Pérez-García, P., Ma, Y., Yanovsky, M.J., Mas, P. 2015. Time-dependent sequestration of RVE8 by LNK proteins shapes the diurnal oscillation of anthocyanin biosynthesis. Proceedings of the National Academy of Sciences 112, 5249–5253. DOI: 10.1073/pnas.1420792112


Albornos, L., Martín, I., Pérez, P., Marcos, R., Dopico, B., Labrador, E. 2012. Promoter activities of genes encoding β-galactosidases from Arabidopsis a1 subfamily. Plant physiology and biochemistry: PPB 60, 223–232. DOI: 10.1016/j.plaphy.2012.08.012


Huang, W., Pérez-García, P., Pokhilko, A., Millar, A.J., Antoshechkin, I., Riechmann, J.L., Mas, P. 2012. Mapping the Core of the Arabidopsis Circadian Clock Defines the Network Structure of the Oscillator. Science 336, 75–79. DOI: 10.1126/science.1219075