We aim to engineer plant organoids using optogenetic circuits to reprogram lateral root pluripotent stem cells and redirect their developmental fate toward custom organogenic programs.

Organoids are miniaturized, three-dimensional models that recapitulate key structural and functional features. In animals, they serve as models for stem cell biology, regeneration, organogenesis, toxicology, drug discovery, and disease modeling. Plant organoids could enable the study of organogenic programs that are normally difficult to access or observe under standard growth conditions, particularly in species with long developmental cycles, and could serve as models to investigate how environmental factors influence organogenesis, which is especially relevant in the context of climate change. In vitro systems partly address these challenges but are limited to certain organs and species and require hormonal supplements that complicate developmental studies.

Organoid generation relies on reprogramming pluripotent cells, achievable in vitro or in vivo, although plant in vitro methods are constrained by the rigid cell wall. We propose two in vivo sources of reprogrammable cells: (1) lateral root primordia (LRP, stages IV–VI) and (2) the proliferative xylem-pole pericycle (XPP), inducible by exogenous auxin (Figure 1A). In these structures, cytokinin triggers reprogramming of specific populations, forming a converting structure that develops a shoot apical meristem (SAM) (Figure 1B), with root identity markers progressively replaced by SAM-specific ones (Figure 1C).

Figure 1. Our system. (A) We use lateral root primordia (LRP) at stages IV–VI and the proliferative xylem pole pericycle (XPP; induced by exogenous auxin treatment) as highly reprogrammable systems. (B) The addition of exogenous cytokinins (CK) triggers the conversion process toward a shoot apical meristem (SAM), ultimately leading to shoot formation on the roots. (C) At the molecular level, regulators defining the identity of LRP cell populations change their expression patterns, reflecting the progressive loss of LRP identity and the establishment of SAM-specific expression profiles. WOX5, WUSCHEL-RELATED HOMEOBOX 5; PLT4, PLETHORA 4; WUS, WUSCHEL; STM, SHOOT MERISTEMLESS. Scale bars: 25 µm.

Our research comprises several objectives:

1. To identify which cells are reprogrammable (Figure 2). Conversion to a SAM occurs within a narrow developmental window, requiring precise targeting of specific stem cell populations. External hormone treatments affect all cells indiscriminately, masking which LRP populations are reprogrammable. At the reprogrammable stage, LRPs contain several distinct cell populations, some shared with the proliferative XPP (Figure 2A–B). Markers of putative reprogrammable LRP populations are also expressed in the root apical meristem (RAM). To restrict expression, minimal cis-regulatory sequences driving LRP-specific transcription are combined with sequences excluding RAM expression, generating synthetic promoters targeting only reprogrammable LRP cells (Figure 2C–D). Expression patterns of known markers are analyzed by confocal microscopy, and single-cell sequencing during reprogramming will define markers at higher resolution and identify the cell populations and molecular mechanisms responsible for conversion.
   
   

   

   Figure 2. Directing expression to the cell population undergoing reprogramming. (A) Confocal images showing distinct cell populations at lateral root primordia (LRP) stages IV–VI. (B) Confocal images displaying specific cell populations within the proliferative xylem pole pericycle (XPP) region. (C) Confocal images illustrating the expression pattern of an LRP cell marker that is not expressed in the root apical meristem (RAM). (D) Schematic representation of an example strategy for constructing a synthetic promoter specifically active in the reprogrammable cells of the LRP. Scale bars: 25 µm.
    
2. To direct reprogramming circuits toward the reprogrammable cells of the LRP in order to induce organogenesis programs of interest (Figure 3). We aim to design synthetic genetic circuits activated by light-responsive artificial transcription factors (OPTO-ATFs) to modulate the transcription of stem cell reprogramming factors (Figure 3). Optogenetics, which converts light signals into biological activity through natural or engineered photoreceptors, provides reversible, quantitative, non-invasive, and spatiotemporally precise control. Roots, which typically develop underground in darkness, represent a promising platform for plant optogenetics. Artificial transcription factors that combine non-host DNA-binding domains with activation or repression modules are employed to minimize interference with the host’s endogenous transcriptional machinery.
   
   

   

   Figure 3. Diagram of the proposed synthetic genetic circuit. Art_TF_DB (Artificial transcription factor DNA-binding domain), Art_TF_AD (Artificial transcription factor activation domain), Art_TF_BS (Artificial transcription factor binding site), SCRF (stem cell reprogramming factor).
    
3. To study organoid development from early to advanced stages (Figure 4). Organoids generated from reprogrammed pluripotent LRP cells are analyzed for cellular anatomy and molecular marker dynamics (Figure 4A). Phenotypic comparisons are performed between regenerated organs and those developed naturally to assess similarity (Figure 4B).
   
   

   

   Figure 4. Morphological and molecular analysis of the generated organoids. We studied the dynamics of molecular markers and the cellular anatomy of the organoids generated by reprogramming the lateral root primordia (LRP) to a shoot apical meristem (SAM) (A), as well as the development of the resulting organs (B). Scale bars: 25 µm, 1cm.
    
4. To transfer this technology to species of agronomic interest (Figure 5). We aim to implement plant organoid generation in commercially relevant species. Focusing on legumes and solanaceous species, specifically Medicago truncatula and Solanum tuberosum. Additionally, we are developing a transformation protocol based on LRP-to-SAM conversion that could, in principle, be applied to any species capable of undergoing this transition (Figure 5).
   
   

   

   Figure 5. Optimization of a stable genetic transformation protocol based on the conversion of a lateral root primordium (LRP) into a shoot apical meristem (SAM). Confocal microscopy images show the presence of a green fluorescent recombinant protein in the Golgi apparatus of structures undergoing conversion (SAM in formation) following infection with Agrobacterium tumefaciens. The stem cell marker of a SAM confirms that the structure is in the process of converting from an LRP to a SAM. Scale bars: 25 µm.