Reconstruction of lateral root formation through single-cell RNA sequencing reveals order of tissue initiation.

Postembryonic organogenesis is critical for plant development. Underground, lateral roots (LRs) form the bulk of mature root systems, yet the ontogeny of the LR primordium (LRP) is not clear. In this study, we performed the single-cell RNA sequencing through the first four stages of LR formation in Arabidopsis. Our analysis led to a model in which a single group of precursor cells, with a cell identity different from their pericycle origins, rapidly reprograms and splits into a mixed ground tissue/stem cell niche fate and a vascular precursor fate. The ground tissue and stem cell niche fates soon separate and a subset of more specialized vascular cells form sucrose transporting phloem cells that appear to connect to the primary root. We did not detect cells resembling epidermis or root cap, suggesting that outer tissues may form later, preceding LR emergence. At this stage, some remaining initial precursor cells form the primordium flanks, while the rest create a reservoir of pluripotent cells that are able to replace the LR if damaged. Laser ablation of the central and lateral LRP regions showed that remaining cells restart the sequence of tissue initiation to form a LR. Collectively, our study reveals an ontological hierarchy for LR formation with an early and sequential split of main root tissues and stem cells.

An auxin-regulable oscillatory circuit drives the root clock in Arabidopsis.

In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. 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. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis.

Body regeneration through formation of new organs is a major question in developmental biology. We investigated de novo root formation using whole leaves of Arabidopsis (Arabidopsis thaliana). Our results show that local cytokinin biosynthesis and auxin biosynthesis in the leaf blade followed by auxin long-distance transport to the petiole leads to proliferation of J0121-marked xylem-associated tissues and others through signaling of INDOLE-3-ACETIC ACID INDUCIBLE28 (IAA28), CRANE (IAA18), WOODEN LEG, and ARABIDOPSIS RESPONSE REGULATORS1 (ARR1), ARR10, and ARR12. Vasculature proliferation also involves the cell cycle regulator KIP-RELATED PROTEIN2 and ABERRANT LATERAL ROOT FORMATION4, resulting in a mass of cells with rooting competence that resembles callus formation. Endogenous callus formation precedes specification of postembryonic root founder cells, from which roots are initiated through the activity of SHORT-ROOT, PLETHORA1 (PLT1), and PLT2. Primordia initiation is blocked in shr plt1 plt2 mutant. Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved.

The transcription factor OBP4 controls root growth and promotes callus formation.

Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.