Appreciating animal induced pluripotent stem cells to shape plant cell reprogramming strategies.

Animals and plants have developed resilience mechanisms to effectively endure and overcome physical damage and environmental challenges throughout their life span. To sustain their vitality, both animals and plants employ mechanisms to replenish damaged cells, either directly, involving the activity of adult stem cells, or indirectly, via dedifferentiation of somatic cells that are induced to revert to a stem cell state and subsequently redifferentiate. Stem cell research has been a rapidly advancing field in animal studies for many years, driven by its promising potential in human therapeutics, including tissue regeneration and drug development. A major breakthrough was the discovery of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells by expressing a limited set of transcription factors. This discovery enabled the generation of an unlimited supply of cells that can be differentiated into specific cell types and tissues. Equally, a keen interest in the connection between plant stem cells and regeneration has been developed in the last decade, driven by the demand to enhance plant traits such as yield, resistance to pathogens, and the opportunities provided by CRISPR/Cas-mediated gene editing. Here we discuss how knowledge of stem cell biology benefits regeneration technology, and we speculate on the creation of a universal genotype-independent iPSC system for plants to overcome regenerative recalcitrance.

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.

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.

The miR156 juvenility factor and PLETHORA 2 form a regulatory network and influence timing of meristem growth and lateral root emergence.

Plants develop throughout their lives: seeds become seedlings that mature and form fruits and seeds. Although the underlying mechanisms that drive these developmental phase transitions have been well elucidated for shoots, the extent to which they affect the root is less clear. However, root anatomy does change as some plants mature; meristems enlarge and radial thickening occurs. Here, in Arabidopsis thaliana, we show that overexpressing miR156A, a gene that promotes the juvenile phase, increased the density of the root system, even in grafted plants in which only the rootstock had the overexpression genotype. In the root, overexpression of miR156A resulted in lower levels of PLETHORA 2, a protein that affects formation of the meristem and elongation zone. Crossing in an extra copy of PLETHORA 2 partially rescued the effects of miR156A overexpression on traits affecting root architecture, including meristem length and the rate of lateral root emergence. Consistent with this, PLETHORA 2 also inhibited the root-tip expression of another miR156 gene, miR156C. We conclude that the system driving phase change in the shoot affects developmental progression in the root, and that PLETHORA 2 participates in this network.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Nature and Nurture: Genotype-Dependent Differential Responses of Root Architecture to Agar and Soil Environments.

Root development is crucial for plant growth and therefore a key factor in plant performance and food production. Arabidopsis thaliana is the most commonly used system to study root system architecture (RSA). Growing plants on agar-based media has always been routine practice, but this approach poorly reflects the natural situation, which fact in recent years has led to a dramatic shift toward studying RSA in soil. Here, we directly compare RSA responses to agar-based medium (plates) and potting soil (rhizotrons) for a set of redundant loss-of-function plethora (plt) CRISPR mutants with variable degrees of secondary root defects. We demonstrate that plt3plt7 and plt3plt5plt7 plants, which produce only a handful of emerged secondary roots, can be distinguished from other genotypes based on both RSA shape and individual traits on plates and rhizotrons. However, in rhizotrons the secondary root density and the total contribution of the side root system to the RSA is increased in these two mutants, effectively rendering their phenotypes less distinct compared to WT. On the other hand, plt3, plt3plt5, and plt5plt7 mutants showed an opposite effect by having reduced secondary root density in rhizotrons. This leads us to believe that plate versus rhizotron responses are genotype dependent, and these differential responses were also observed in unrelated mutants short-root and scarecrow. Our study demonstrates that the type of growth system affects the RSA differently across genotypes, hence the optimal choice of growth conditions to analyze RSA phenotype is not predetermined.

Root stem cell niche networks: it's complexed! Insights from Arabidopsis.

The presence of two meristematic cell populations in the root and shoot apex allows plants to grow indefinitely. Due to its simple and predictable tissue organization, the Arabidopsis root apical meristem remains an ideal model to study mechanisms such as stem cell specification, asymmetric cell division, and differentiation in plants. The root stem cell niche consists of a quiescent organizing centre surrounded by mitotically active stem cells, which originate all root tissues. The transcription factors PLETHORA, SCARECROW, and WOX5 form signalling hubs that integrate multiple inputs from an increasing number of proteins implicated in the regulation of stem cell niche function. Recently, locally produced auxin was added to the list of important mobile factors in the stem cell niche. In addition, protein-protein interaction data elegantly demonstrate how parallel pathways can meet in a common objective. Here we discuss how multiple networks converge to specify and maintain the root stem cell niche.

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.

Transcription factor dosage: more or less sufficient for growth.

Recent findings highlight three instances in which major aspects of plant development are controlled by dosage-dependent protein levels. In the shoot apical meristem the mobile transcription factor WUS displays an intricate function with respect to target regulation that involves WUS dosage, binding site affinity and protein dimerization. The size of the root meristem is controlled by dosage-dependent PLT protein activity. Recent identification of targets and feedbacks provide new insights and entry into possible mechanisms of dosage read-out. Finally, HD-ZIPIII dosage, enforced by a gradient of mobile miRNAs, presents a relatively unexplored case in the radial patterning of vasculature and ground tissue. We evaluate our current knowledge of these three examples and address molecular mechanisms of dosage translation where possible.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

Cis-regulatory PLETHORA promoter elements directing root and nodule expression are conserved between Arabidopsis thaliana and Medicago truncatula.

Nodules are unique organs formed on roots of legumes by soil-borne bacteria, collectively known as rhizobium. Recently, we have shown that orthologs of the AINTEGUMENTA-like (AIL) AP2 transcription factors PLETHORA (PLT) 1 to 4, that redundantly regulate Arabidopsis thaliana root development are involved in root and nodule growth in Medicago truncatula. Hence, it is conceivable that rhizobium has co-opted these genes for nodule development. Whether this co-option requires the presence of specific cis-elements in the promoters and/or specialization of PLT protein function is not clear. Here, we analyzed the qualitative expression patterns of the Arabidopsis PLT1 to 4 promoters in Medicago roots and nodules and compared these with the described expression patterns of the Medicago PLT genes. Our studies reveal that the expression patterns of the investigated promoters and their Medicago orthologs are very similar, indicating that at least all cis-elements regulating spatial PLT expression are conserved among the Arabidopsis and Medicago PLT1 to 4 promoters.

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.

A SCARECROW-based regulatory circuit controls Arabidopsis thaliana meristem size from the root endodermis.

MAIN CONCLUSION: SCARECROW controls Arabidopsis root meristem size from the root endodermis tissue by regulating the DELLA protein RGA that in turn mediates the regulation of ARR1 levels at the transition zone. Coherent organ growth requires a fine balance between cell division and cell differentiation. Intriguingly, plants continuously develop organs post-embryonically thanks to the activity of meristems that allow growth and environmental plasticity. In Arabidopsis thaliana, continued root growth is assured when division of the distal stem cell and their daughters is balanced with cell differentiation at the meristematic transition zone (TZ). We have previously shown that at the TZ, the cytokinin-dependent transcription factor ARR1 controls the rate of differentiation commitment of meristematic cells and that its activities are coordinated with those of the distal stem cells by the gene SCARECROW (SCR). In the stem cell organizer (the quiescent center, QC), SCR directly suppresses ARR1 both sustaining stem cell activities and titrating non-autonomously the ARR1 transcript levels at the TZ via auxin. Here, we show that SCR also exerts a fine control on ARR1 levels at the TZ from the endodermis by sustaining gibberellin signals. From the endodermis, SCR controls the RGA REPRESSOR OF ga1-3 (RGA) DELLA protein stability throughout the root meristem, thus controlling ARR1 transcriptional activation at the TZ. This guarantees robustness and fineness to the control of ARR1 levels necessary to balance cell division to cell differentiation in sustaining coherent root growth. Therefore, this work advances the state of the art in the field of root meristem development by integrating the activity of three hormones, auxin, gibberellin, and cytokinin, under the control of different tissue-specific activities of a single root key regulator, SCR.

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.

SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement.

Intercellular signaling through trafficking of regulatory proteins is a widespread phenomenon in plants and can deliver positional information for the determination of cell fate. In the Arabidopsis root meristem, the cell fate determinant SHORT-ROOT (SHR), a GRAS domain transcription factor, acts as a signaling molecule from the stele to the adjacent layer to specify endodermal cell fate. Upon exiting the stele, SHR activates another GRAS domain transcription factor, SCARCROW (SCR), which, together with several BIRD/INDETERMINATE DOMAIN proteins, restricts movement of SHR to define a single cell layer of endodermis. Here we report that endodermal cell fate also requires the joint activity of both SCR and its closest homologue SCARECROW-LIKE23 (SCL23). We show that SCL23 protein moves with zonation-dependent directionality. Within the meristem, SCL23 exhibits short-ranged movement from ground tissue to vasculature. Away from the meristem, SCL23 displays long-range rootward movement into meristematic vasculature and a bidirectional radial spread, respectively. As a known target of SHR and SCR, SCL23 also interacts with SCR and SHR and can restrict intercellular outspread of SHR without relying on nuclear retention as SCR does. Collectively, our data show that SCL23 is a mobile protein that controls movement of SHR and acts redundantly with SCR to specify endodermal fate in the root meristem.

Root developmental programs shape the Medicago truncatula nodule meristem.

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.