The root system is essential for the survival of terrestrial plants and environmental adaptation, instructing whole plant growth and production. Most plant organs are newly formed after embryogenesis, providing nutrition, anchorage, reproduction, and more. Additionally, plants can regenerate almost any organ. To further understand organ formation and regeneration, we use the root of the plant Arabidopsis thaliana as a model, allowing us to study in vivo morphogenic processes through biomarker imaging, genetics, and RNA-sequencing. We use these data to learn intrinsic system properties through Gene Regulatory Network (GRN) inference and computer simulations.

The development of root traits that increase aerial plant biomass and nutrition has not been systematically exploited. We are translating our knowledge from model species into crops (poplar and tomato) to identify new breeding traits, markers and natural eco-friendly bio-stimulants.

 Figure 1: Root formation and development in Arabidopsis (A), Poplar (B) and Tomato (C)

Understanding the Root Clock Mechanism and Exploiting it to Develop Novel Root Traits and Biostimulants

In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression, while the hormone auxin plays an important role in maintaining and generating gene oscillations.

Figure 2: The root clock generates periodic gene expression (bright colors in movies) in the oscillation zone (OZ) to create new branching sites (PBS), where lateral roots (LR) are formed along the primary root (PR). The OZ is located above the stem cell niche (SCN). A multilevel computer simulation shows how gene expression oscillation coordinates with cell division and growth

Our work has identified the core molecular mechanism driving the root clock and how it is modified by exogenous cues. We identified auxin response factors, auxin-sensitive inhibitors, and auxin forming a negative regulatory loop circuit driving gene expression oscillations. Through multilevel computer modeling fitted to experimental data we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. We also show that external auxin stimuli can lead to entrainment of the root clock.

We are currently investigating

• Novel regulation of periodic auxin signaling and its memorization to generate new root branches.
• Unveiling the root clock in poplar and tomato to improve root vigor.
• Characterizing novel natural molecules targeting root clock components and its potential use as biostimulants.

Figure 3: A) Unraveling novel root clock regulators of periodic auxin signaling through analyses of mutants (e.g. OZ#8) with expanded oscillation zone (OZ). B) Conservation of the root clock in crops. C, D) Identification of natural compounds (C.N.) targeting the root clock and characterization of their potential use as biostimulants.

Stem Cell Induction and the Effect of Environmental Conditions

Root systems, as observed in Arabidopsis thaliana, undergo lateral root formation, a developmental mechanism requiring the establishment of stem cell lineages.

We have dissected the formative phase of lateral root development through single-cell RNA sequencing (scRNA-seq), computational approaches, and confocal microscopy, defining the associated cell types and developmental trajectories. Our analysis reveals an ontological hierarchy for lateral root formation with an early and sequential split of main root tissues and stem cells. We have also reconstructed an Arabidopsis spatiotemporal cell-type-specific transcriptional map of the root stem cell lineage from precursor to fully developed stem cells through RNA sequencing (RNA-seq) of single and double fluorescent markers.

Figure 4: A) Computed developmental trajectories for early root organogenesis based on single-cell transcriptomic analyses. B) Fluorescent markers used to profile stem cell induction through RNA-sequencing. C) Reconstructed gene regulatory network for early organogensis. D) A cold response regulator is required for stem cell induction.

Employing dynamic Bayesian gene regulatory network inference alongside tree-based methods, we are:

• Understanding lineage developmental progression during root organogenesis.
• Unveiling stem cell induction mechanisms and associated core transcriptional signatures.

Environmental cues affect plant development; and although plants are exposed to disparate environmental conditions, how these cues affect stem cell specification during organogenesis is unknown. Intriguingly, we have found that cold response regulators are required for stem cell induction. We are investigating:

• How cold and other environmental cues regulate stem cell induction and specification during root organogenesis.

Understanding Stem Cell Regeneration Under Adverse Environmental Conditions

Plant development involves the specification and regeneration of stem cells, and we have observed that moderate cold induces expression of stem biomarkers, while acute cold is detrimental for development. The activity of stem cells involves telomerase, although its role in regeneration is not fully understood. Telomerase is crucial for maintaining telomere length, which protects chromosome ends from degradation.

Figure 5: A) Scheme for known identity transitions during root regeneration based on literature. B) Stem cell specification is enhanced upon moderate cold treatment. C) Stem cell regeneration requires cold response regulators and is promoted by moderate cold treatments. D) Telomere measurements through confocal laser microscopy. E) Research approach for understanding cross regulation between telomerase/telomere length and environmental cues during stem cell regeneration.

We are investigating

• How ABA-independent and ABA-dependent responses to environmental cues are integrated into regenerative programs and stem cell regeneration.

• Understanding how telomerase and telomere length influence the capacity of stem cells to regenerate and how this impacts overall plant growth and adaptability.

• Cross-regulation between telomerase/telomere length and environmental cues during stem cell regeneration.