A Jasmonate Signaling Network Activates Root Stem Cells and Promotes Regeneration.

Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.

PLETHORA transcription factors promote early embryo development through induction of meristematic potential.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential.

Arabidopsis ribosomal RNA processing meerling mutants exhibit suspensor-derived polyembryony due to direct reprogramming of the suspensor.

Embryo development in Arabidopsis (Arabidopsis thaliana) starts off with an asymmetric division of the zygote to generate the precursors of the embryo proper and the supporting extraembryonic suspensor. The suspensor degenerates as the development of the embryo proper proceeds beyond the heart stage. Until the globular stage, the suspensor maintains embryonic potential and can form embryos in the absence of the developing embryo proper. We report a mutant called meerling-1 (mrl-1), which shows a high penetrance of suspensor-derived polyembryony due to delayed development of the embryo proper. Eventually, embryos from both apical and suspensor lineages successfully develop into normal plants and complete their life cycle. We identified the causal mutation as a genomic rearrangement altering the promoter of the Arabidopsis U3 SMALL NUCLEOLAR RNA-ASSOCIATED PROTEIN 18 (UTP18) homolog that encodes a nucleolar-localized WD40-repeat protein involved in processing 18S preribosomal RNA. Accordingly, root-specific knockout of UTP18 caused growth arrest and accumulation of unprocessed 18S pre-rRNA. We generated the mrl-2 loss-of-function mutant and observed asynchronous megagametophyte development causing embryo sac abortion. Together, our results indicate that promoter rearrangement decreased UTP18 protein abundance during early stage embryo proper development, triggering suspensor-derived embryogenesis. Our data support the existence of noncell autonomous signaling from the embryo proper to prevent direct reprogramming of the suspensor toward embryonic fate.

A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division.

In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

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.

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules.

Continuous formation of somatic tissues in plants requires functional stem cell niches where undifferentiated cells are maintained. In Arabidopsis thaliana, PLETHORA (PLT) and SCARECROW (SCR) genes are outputs of apical-basal and radial patterning systems, and both are required for root stem cell specification and maintenance. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene is specifically expressed in and required for functions of a small group of root stem cell organizer cells, also called the quiescent center (QC). PLT and SCR are required for QC function, and their expression overlaps in the QC; however, how they specify the organizer has remained unknown. We show that PLT and SCR genetically and physically interact with plant-specific teosinte-branched cycloidea PCNA (TCP) transcription factors to specify the stem cell niche during embryogenesis and maintain organizer cells post-embryonically. PLT-TCP-SCR complexes converge on PLT-binding sites in the WOX5 promoter to induce expression.

The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is coordinated by the interaction with LXCXE-containing proteins.

Living organisms possess mechanisms to safeguard genome integrity. To avoid spreading mutations, DNA lesions are detected and cell division is temporarily arrested to allow repair mechanisms. Afterward, cells either resume division or respond to unsuccessful repair by undergoing programmed cell death (PCD). How the success rate of DNA repair connects to later cell fate decisions remains incompletely known, particularly in plants. The Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein and its partner E2FA, play both structural and transcriptional functions in the DNA damage response (DDR). Here we provide evidence that distinct RBR protein interactions with LXCXE motif-containing proteins guide these processes. Using the N849F substitution in the RBR B-pocket domain, which specifically disrupts binding to the LXCXE motif, we show that these interactions are dispensable in unchallenging conditions. However, N849F substitution abolishes RBR nuclear foci and promotes PCD and growth arrest upon genotoxic stress. NAC044, which promotes growth arrest and PCD, accumulates after the initial recruitment of RBR to foci and can bind non-focalized RBR through the LXCXE motif in a phosphorylation-independent manner, allowing interaction at different cell cycle phases. Disrupting NAC044-RBR interaction impairs PCD, but their genetic interaction points to opposite independent roles in the regulation of PCD. The LXCXE-binding dependency of the roles of RBR in the DDR suggests a coordinating mechanism to translate DNA repair success to cell survival. We propose that RBR and NAC044 act in two distinct DDR pathways, but interact to integrate input from both DDR pathways to decide upon an irreversible cell fate decision.

SCHIZORIZA domain-function analysis identifies requirements for its specific role in cell fate segregation.

Plant development continues postembryonically with a lifelong ability to form new tissues and organs. Asymmetric cell division, coupled with fate segregation, is essential to create cellular diversity during tissue and organ formation. Arabidopsis (Arabidopsis thaliana) plants harboring mutations in the SCHIZORIZA (SCZ) gene display fate segregation defects in their roots, resulting in the presence of an additional layer of endodermis, production of root hairs from subepidermal tissue, and misexpression of several tissue identity markers. Some of these defects are observed in tissues where SCZ is not expressed, indicating that part of the SCZ function is nonautonomous. As a class B HEAT-SHOCK TRANSCRIPTION FACTOR (HSFB), the SCZ protein contains several conserved domains and motifs. However, which domain(s) discriminates SCZ from its family members to obtain a role in development remains unknown. Here, we investigate how each domain contributes to SCZ function in Arabidopsis root patterning by generating altered versions of SCZ by domain swapping and mutation. We show that the SCZ DNA-binding domain is the main factor for its developmental function, and that SCZ likely acts as a nonmotile transcriptional repressor. Our results demonstrate how members of the HSF family can evolve toward functions beyond stress response.

Plant asymmetric cell division, vive la différence!

Although little is known about how asymmetric cell division in plants is regulated, recent discoveries provide a starting point for exploring the mechanisms underlying this process. These studies reveal parallels with asymmetric division in yeast and animals, but also point to regulated cell expansion as a new mechanism of asymmetric division in plants.

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.

RETINOBLASTOMA-RELATED interactions with key factors of the RNA-directed DNA methylation (RdDM) pathway and its influence on root development.

MAIN CONCLUSION: Our study presents evidence for a novel mechanism for RBR function in transcriptional gene silencing by interacting with key players of the RdDM pathway in Arabidopsis and several plant clades. Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double-stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs serve as guides to direct AGO4-siRNA complexes to chromatin-bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. The Arabidopsis Retinoblastoma protein homolog (RBR) is a master regulator of the cell cycle, stem cell maintenance, and development. We in silico predicted and explored experimentally the protein-protein interactions (PPIs) between RBR and members of the RdDM pathway. We found that the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2, and SUVR2 contain canonical and non-canonical RBR binding motifs and several of them are conserved since algae and bryophytes. We validated experimentally PPIs between Arabidopsis RBR and several of the RdDM pathway proteins. Moreover, seedlings from loss-of-function mutants in RdDM and RBR show similar phenotypes in the root apical meristem. We show that RdDM and SUVR2 targets are up-regulated in the 35S:AmiGO-RBR background.

The plant perceptron connects environment to development.

Plants cope with the environment in a variety of ways, and ecological analyses attempt to capture this through life-history strategies or trait-based categorization. These approaches are limited because they treat the trade-off mechanisms that underlie plant responses as a black box. Approaches that involve the molecular or physiological analysis of plant responses to the environment have elucidated intricate connections between developmental and environmental signals, but in only a few well-studied model species. By considering diversity in the plant response to the environment as the adaptation of an information-processing network, new directions can be found for the study of life-history strategies, trade-offs and evolution in plants.

In vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis roots.

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem.

The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

Mediator subunit MED31 is required for radial patterning of Arabidopsis roots.

Stem cell specification in multicellular organisms relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, in which the evolutionarily conserved Mediator coactivator complex plays an essential role. In Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) orchestrate a transcriptional program that determines the fate and asymmetrical divisions of stem cells generating the root ground tissue. The mechanism by which SHR/SCR relays context-specific regulatory signals to the Pol II general transcription machinery is unknown. Here, we report the role of Mediator in controlling the spatiotemporal transcriptional output of SHR/SCR during asymmetrical division of stem cells and ground tissue patterning. The Mediator subunit MED31 interacted with SCR but not SHR. Reduction of MED31 disrupted the spatiotemporal activation of CYCLIND6;1 (CYCD6;1), leading to defective asymmetrical division of stem cells generating ground tissue. MED31 was recruited to the promoter of CYCD6;1 in an SCR-dependent manner. MED31 was involved in the formation of a dynamic MED31/SCR/SHR ternary complex through the interface protein SCR. We demonstrate that the relative protein abundance of MED31 and SHR in different cell types regulates the dynamic formation of the ternary complex, which provides a tunable switch to strictly control the spatiotemporal transcriptional output. This study provides valuable clues to understand the mechanism by which master transcriptional regulators control organ patterning.

Non-canonical WOX11-mediated root branching contributes to plasticity in Arabidopsis root system architecture.

Lateral roots (LRs), which originate from the growing root, and adventitious roots (ARs), which are formed from non-root organs, are the main contributors to the post-embryonic root system in Arabidopsis However, our knowledge of how formation of the root system is altered in response to diverse inductive cues is limited. Here, we show that WOX11 contributes to root system plasticity. When seedlings are grown vertically on medium, WOX11 is not expressed in LR founder cells. During AR initiation, WOX11 is expressed in AR founder cells and activates LBD16LBD16 also functions in LR formation and is activated in that context by ARF7/19 and not by WOX11 This indicates that divergent initial processes that lead to ARs and LRs may converge on a similar mechanism for primordium development. Furthermore, we demonstrated that when plants are grown in soil or upon wounding on medium, the primary root is able to produce both WOX11-mediated and non-WOX11-mediated roots. The discovery of WOX11-mediated root-derived roots reveals a previously uncharacterized pathway that confers plasticity during the generation of root system architecture in response to different inductive cues.

Geometric cues forecast the switch from two- to three-dimensional growth in Physcomitrella patens.

During land colonization, plants acquired a range of body plan adaptations, of which the innovation of three-dimensional (3D) tissues increased organismal complexity and reproductivity. In the moss, Physcomitrella patens, a 3D leafy gametophore originates from filamentous cells that grow in a two-dimensional (2D) plane through a series of asymmetric cell divisions. Asymmetric cell divisions that coincide with different cell division planes and growth directions enable the developmental switch from 2D to 3D, but insights into the underlying mechanisms coordinating this switch are still incomplete. Using 2D and 3D imaging and image segmentation, we characterized two geometric cues, the width of the initial cell and the angle of the transition division plane, which sufficiently distinguished a gametophore initial cell from a branch initial cell. These identified cues were further confirmed in gametophore formation mutants. The identification of a fluorescent marker allowed us to successfully predict the gametophore initial cell with > 90% accuracy before morphological changes, supporting our hypothesis that, before the transition division, parental cells of the gametophore initials possess different properties from those of the branch initials. Our results suggest that the cell fate decision of the initial cell is determined in the parental cell, before the transition division.

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.

PLETHORA transcription factors orchestrate de novo organ patterning during Arabidopsis lateral root outgrowth.

Plant development is characterized by repeated initiation of meristems, regions of dividing cells that give rise to new organs. During lateral root (LR) formation, new LR meristems are specified to support the outgrowth of LRs along a new axis. The determination of the sequential events required to form this new growth axis has been hampered by redundant activities of key transcription factors. Here, we characterize the effects of three PLETHORA (PLT) transcription factors, PLT3, PLT5, and PLT7, during LR outgrowth. In plt3plt5plt7 triple mutants, the morphology of lateral root primordia (LRP), the auxin response gradient, and the expression of meristem/tissue identity markers are impaired from the "symmetry-breaking" periclinal cell divisions during the transition between stage I and stage II, wherein cells first acquire different identities in the proximodistal and radial axes. Particularly, PLT1, PLT2, and PLT4 genes that are typically expressed later than PLT3, PLT5, and PLT7 during LR outgrowth are not induced in the mutant primordia, rendering "PLT-null" LRP. Reintroduction of any PLT clade member in the mutant primordia completely restores layer identities at stage II and rescues mutant defects in meristem and tissue establishment. Therefore, all PLT genes can activate the formative cell divisions that lead to de novo meristem establishment and tissue patterning associated with a new growth axis.