Cellular and molecular mechanisms of development and regeneration.

Regeneration involves a highly coordinated interplay of intricate cellular processes, enabling living organisms to renew and repair themselves, from individual cells to entire ecosystems. Further, regeneration offers profound insights into developmental biology, tissue engineering and regenerative medicine. The Cellular and Molecular Mechanisms of Development and Regeneration (CMMDR) 2024 conference, which took place at the Shiv Nadar Institute of Eminence and University (India), gathered together an international array of researchers studying a wide variety of organisms across both plant and animal kingdoms. In this short Meeting Review, we highlight some of the outstanding research presented at this conference and draw together some of the common themes that emerged.

A PLETHORA-auxin transcription module controls cell division plane rotation through MAP65 and CLASP.

Despite their pivotal role in plant development, control mechanisms for oriented cell divisions have remained elusive. Here, we describe how a precisely regulated cell division orientation switch in an Arabidopsis stem cell is controlled by upstream patterning factors. We show that the stem cell regulatory PLETHORA transcription factors induce division plane reorientation by local activation of auxin signaling, culminating in enhanced expression of the microtubule-associated MAP65 proteins. MAP65 upregulation is sufficient to reorient the cortical microtubular array through a CLASP microtubule-cell cortex interaction mediator-dependent mechanism. CLASP differentially localizes to cell faces in a microtubule- and MAP65-dependent manner. Computational simulations clarify how precise 90° switches in cell division planes can follow self-organizing properties of the microtubule array in combination with biases in CLASP localization. Our work demonstrates how transcription factor-mediated processes regulate the cellular machinery to control orientation of formative cell divisions in plants.

Species-specific function of conserved regulators in orchestrating rice root architecture.

Shoot-borne adventitious/crown roots form a highly derived fibrous root system in grasses. The molecular mechanisms controlling their development remain largely unknown. Here, we provide a genome-wide landscape of transcriptional signatures - tightly regulated auxin response and in-depth spatio-temporal expression patterns of potential epigenetic modifiers - and transcription factors during priming and outgrowth of rice (Oryza sativa) crown root primordia. Functional analyses of rice transcription factors from WUSCHEL-RELATED HOMEOBOX and PLETHORA gene families reveal their non-redundant and species-specific roles in determining the root architecture. WOX10 and PLT1 regulate both shoot-borne crown roots and root-borne lateral roots, but PLT2 specifically controls lateral root development. PLT1 activates local auxin biosynthesis genes to promote crown root development. Interestingly, O. sativa PLT genes rescue lateral root primordia outgrowth defects of Arabidopsis plt mutants, demonstrating their conserved role in root primordia outgrowth irrespective of their developmental origin. Together, our findings unveil a molecular framework of tissue transdifferentiation during root primordia establishment, leading to the culmination of robust fibrous root architecture. This also suggests that conserved factors have evolved their transcription regulation to acquire species-specific function.

Model systems for regeneration: Arabidopsis.

Plants encompass unparalleled multi-scale regenerative potential. Despite lacking specialized cells that are recruited to injured sites, and despite their cells being encased in rigid cell walls, plants exhibit a variety of regenerative responses ranging from the regeneration of specific cell types, tissues and organs, to the rebuilding of an entire organism. Over the years, extensive studies on embryo, shoot and root development in the model plant species Arabidopsis thaliana have provided insights into the mechanisms underlying plant regeneration. These studies highlight how Arabidopsis, with its wide array of refined molecular, genetic and cell biological tools, provides a perfect model to interrogate the cellular and molecular mechanisms of reprogramming during regeneration.

Multiple feedbacks on self-organized morphogenesis during plant regeneration.

Decades of research have primarily emphasized genetic blueprint as the driving force behind plant regeneration. The flow of information from genetics, which manifests as biochemical properties, including hormones, has been extensively implicated in plant regeneration. However, recent advancements have unveiled additional intrinsic modules within this information flow. Here, we explore the three core modules of plant regeneration: biochemical properties, mechanical forces acting on cells, and cell geometry. We debate their roles and interactions during morphogenesis, emphasizing the potential for multiple feedbacks between these core modules to drive pattern formation during regeneration. We propose that de novo organ regeneration is a self-organized event driven by multidirectional information flow between these core modules.

Gametophytic epigenetic regulators, MEDEA and DEMETER, synergistically suppress ectopic shoot formation in Arabidopsis.

KEY MESSAGE: The gametophytic epigenetic regulators, MEA and DME, extend their synergistic role to the sporophytic development by regulating the meristematic activity via restricting the gene expression in the shoot apex. The gametophyte-to-sporophyte transition facilitates the alternation of generations in a plant life cycle. The epigenetic regulators DEMETER (DME) and MEDEA (MEA) synergistically control central cell proliferation and differentiation, ensuring proper gametophyte-to-sporophyte transition in Arabidopsis. Mutant alleles of DME and MEA are female gametophyte lethal, eluding the recovery of recessive homozygotes to examine their role in the sporophyte. Here, we exploited the paternal transmission of these mutant alleles coupled with CENH3-haploid inducer to generate mea-1;dme-2 sporophytes. Strikingly, the simultaneous loss of function of MEA and DME leads to the emergence of ectopic shoot meristems at the apical pole of the plant body axis. DME and MEA are expressed in the developing shoot apex and regulate the expression of various shoot-promoting factors. Chromatin immunoprecipitation (ChIP), DNA methylation, and gene expression analysis revealed several shoot regulators as potential targets of MEA and DME. RNA interference-mediated transcriptional downregulation of shoot-promoting factors STM, CUC2, and PLT5 rescued the twin-plant phenotype to WT in 9-23% of mea-1-/-;dme-2-/- plants. Our findings reveal a previously unrecognized synergistic role of MEA and DME in restricting the meristematic activity at the shoot apex during sporophytic development.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

PLETHORA gradient formation mechanism separates auxin responses.

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip. In addition, auxin is also pivotal for tropic responses. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.

Genome-Wide Transcript Profiling Reveals an Auxin-Responsive Transcription Factor, OsAP2/ERF-40, Promoting Rice Adventitious Root Development.

Unlike dicots, the robust root system in grass species largely originates from stem base during postembryonic development. The mechanisms by which plant hormone signaling pathways control the architecture of adventitious root remain largely unknown. Here, we studied the modulations in global genes activity in developing rice adventitious root by genome-wide RNA sequencing in response to external auxin and cytokinin signaling cues. We further analyzed spatiotemporal regulations of key developmental regulators emerged from our global transcriptome analysis. Interestingly, some of the key cell fate determinants such as homeodomain transcription factor (TF), OsHOX12, no apical meristem protein, OsNAC39, APETALA2/ethylene response factor, OsAP2/ERF-40 and WUSCHEL-related homeobox, OsWOX6.1 and OsWOX6.2, specifically expressed in adventitious root primordia. Functional analysis of one of these regulators, an auxin-induced TF containing AP2/ERF domain, OsAP2/ERF-40, demonstrates its sufficiency to confer the adventitious root fate. The ability to trigger the root developmental program is largely attributed to OsAP2/ERF-40-mediated dose-dependent transcriptional activation of genes that can facilitate generating effective auxin response, and OsERF3-OsWOX11-OsRR2 pathway. Our studies reveal gene regulatory network operating in response to hormone signaling pathways and identify a novel TF regulating adventitious root developmental program, a key agronomically important quantitative trait, upstream of OsERF3-OsWOX11-OsRR2 pathway.

The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

PtWOX11 acts as master regulator conducting the expression of key transcription factors to induce de novo shoot organogenesis in poplar.

KEY MESSAGE: WUSCHEL-RELATED HOMEOBOX 11 establishes the acquisition of pluripotency during callus formation and accomplishes de novo shoot formation by regulating key transcription factors in poplar. De novo shoot regeneration is a prerequisite for propagation and genetic engineering of elite cultivars in forestry. However, the regulatory mechanism of de novo organogenesis is poorly understood in tree species. We previously showed that WUSCHEL (WUS)-RELATED HOMEOBOX 11 (PtWOX11) of the hybrid poplar clone 84K (Populus alba × P. glandulosa) promotes de novo root formation. In this study, we found that PtWOX11 also regulates de novo shoot regeneration in poplar. The overexpression of PtWOX11 enhanced de novo shoot formation, whereas overexpression of PtWOX11 fused with the transcriptional repressor domain (PtWOX11-SRDX) or reduced expression of PtWOX11 inhibited this process, indicating that PtWOX11 promotes de novo shoot organogenesis. Although PtWOX11 promotes callus formation, overexpression of PtWOX11 and PtWOX11-SRDX also produced increased and decreased numbers of de novo shoots per unit weight, respectively, implying that PtWOX11 promotes de novo shoot organogenesis partially by regulating the intrinsic mechanism of shoot development. RNA-seq and qPCR analysis further revealed that PtWOX11 activates the expression of PLETHORA1 (PtPLT1) and PtPLT2, whose Arabidopsis paralogs establish the acquisition of pluripotency, during incubation on callus-inducing medium. Moreover, PtWOX11 activates the expression of shoot-promoting factors and meristem regulators such as CUP-SHAPED COTYLEDON2 (PtCUC2), PtCUC3, WUS and SHOOT MERISTEMLESS to fulfill shoot regeneration during incubation on shoot-inducing medium. These results suggest that PtWOX11 acts as a central regulator of the expression of key genes to cause de novo shoot formation. Our studies further provide a possible means to genetically engineer economically important tree species for their micropropagation.

Intermediate Developmental Phases During Regeneration.

The initial view that regeneration can be a continuum in terms of regulatory mechanisms is gradually changing, and recent evidence points towards the presence of discrete regulatory steps and intermediate phases. Furthermore, regeneration presents an excellent example of a process generating order and pattern, i.e. a self-organization process. It is likely that the process traverses a set of intermediate phases before reaching an endpoint. Although some progress has been made in deciphering the identity of these intermediate phases, a lot more work is needed to derive a comprehensive and complete picture. Here, we discuss the intermediate developmental phases in plant regeneration and compare them with the possible intermediate developmental phases in animal regeneration.

The WOX11-LBD16 Pathway Promotes Pluripotency Acquisition in Callus Cells During De Novo Shoot Regeneration in Tissue Culture.

De novo shoot regeneration in tissue culture undergoes at least two phases. Explants are first cultured on auxin-rich callus-inducing medium (CIM) to produce a group of pluripotent cells termed callus; the callus is then transferred to cytokinin rich shoot-inducing medium (SIM) to promote the formation of shoot progenitor cells, from which adventitious shoots may differentiate. Here, we show that the Arabidopsis thaliana transcription factor gene LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) is involved in pluripotency acquisition in callus cells. LBD16, which is activated by WUSCHEL RELATED HOMEOBOX11 (WOX11), is specifically expressed in the newly formed callus on CIM and its expression decreases quickly when callus is moved to SIM. Blocking the WOX11-LBD16 pathway results in the loss of pluripotency in callus cultured on CIM, leading to shooting defects on SIM. Further analysis showed that LBD16 may function in the establishment of the root primordium-like identity in the newly formed callus, indicating that the root primordium-like identity is the cellular nature of pluripotency in callus cells. Additionally, LBD16 promotes cell division during callus initiation. Our study clarified that the WOX11-LBD16 pathway promotes pluripotency acquisition in callus cells.

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants.

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

Local auxin biosynthesis regulation by PLETHORA transcription factors controls phyllotaxis in Arabidopsis.

Lateral organ distribution at the shoot apical meristem defines specific and robust phyllotaxis patterns that have intrigued biologists and mathematicians for centuries. In silico studies have revealed that this self-organizing process can be recapitulated by modeling the polar transport of the phytohormone auxin. Phyllotactic patterns change between species and developmental stages, but the processes behind these variations have remained unknown. Here we use regional complementation experiments to reveal that phyllotactic switches in Arabidopsis shoots can be mediated by PLETHORA-dependent control of local auxin biosynthesis.

A mathematical basis for plant patterning derived from physico-chemical phenomena.

The position of leaves and flowers along the stem axis generates a specific pattern, known as phyllotaxis. A growing body of evidence emerging from recent computational modeling and experimental studies suggests that regulators controlling phyllotaxis are chemical, e.g. the plant growth hormone auxin and its dynamic accumulation pattern by polar auxin transport, and physical, e.g. mechanical properties of the cell. Here we present comprehensive views on how chemical and physical properties of cells regulate the pattern of leaf initiation. We further compare different computational modeling studies to understand their scope in reproducing the observed patterns. Despite a plethora of experimental studies on phyllotaxis, understanding of molecular mechanisms of pattern initiation in plants remains fragmentary. Live imaging of growth dynamics and physicochemical properties at the shoot apex of mutants displaying stable changes from one pattern to another should provide mechanistic insights into organ initiation patterns.

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions.

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.