Quiescent Center Research Articles

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.

The concept of the quiescent centre and how it found support from work with X-rays. I. Historical perspectives

Within the tip of roots meristems of angiosperms and gymnosperms there is a small group of cells known as the quiescent centre (QC). The concept of the QC was developed 60 years ago by FAL Clowes, working in the Botany School, Oxford University, UK. To celebrate the Jubilee of the QC, a brief outline of the work that led to its demonstration by autoradiography was presented by Dubrovsky and Barlow (2015). The present article traces Clowes's subsequent experimental studies of the QC, especially with regard to how X-irradiation became an important tool for elucidating the properties and significance of the QC for root development. Also reviewed are some of the consequences that subsequently arose from this work with radiation, in particular the concerns over the use of radioisotopes in attempts to describe the kinetics of cell proliferation in the root meristem.

The concept of the quiescent centre and how it found support from work with X-rays. II. The molecular aftermath

The concept of the quiescent centre and how it found support from work with X-rays. II. The molecular aftermath

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.

PRL1 modulates root stem cell niche activity and meristem size through WOX5 and PLTs in Arabidopsis.

The stem cell niche in the root meristem maintains pluripotent stem cells to ensure a constant supply of cells for root growth. Despite extensive progress, the molecular mechanisms through which root stem cell fates and stem cell niche activity are determined remain largely unknown. In Arabidopsis thaliana, the Pleiotropic Regulatory Locus 1 (PRL1) encodes a WD40-repeat protein subunit of the spliceosome-activating Nineteen Complex (NTC) that plays a role in multiple stress, hormone and developmental signaling pathways. Here, we show that PRL1 is involved in the control of root meristem size and root stem cell niche activity. PRL1 is strongly expressed in the root meristem and its loss of function mutation results in disorganization of the quiescent center (QC), premature stem cell differentiation, aberrant cell division, and reduced root meristem size. Our genetic studies indicate that PRL1 is required for confined expression of the homeodomain transcription factor WOX5 in the QC and acts upstream of the transcription factor PLETHORA (PLT) in modulating stem cell niche activity and root meristem size. These findings define a role for PRL1 as an important determinant of PLT signaling that modulates maintenance of the stem cell niche and root meristem size.

Developmentally distinct activities of the exocyst enable rapid cell elongation and determine meristem size during primary root growth in Arabidopsis.

BackgroundExocytosis is integral to root growth: trafficking components of systems that control growth (e.g., PIN auxin transport proteins) to the plasma membrane, and secreting materials that expand the cell wall to the apoplast. Spatiotemporal regulation of exocytosis in eukaryotes often involves the exocyst, an octameric complex that tethers selected secretory vesicles to specific sites on the plasma membrane and facilitates their exocytosis. We evaluated Arabidopsis lines with mutations in four exocyst components (SEC5, SEC8, EXO70A1 and EXO84B) to explore exocyst function in primary root growth.ResultsThe mutants have root growth rates that are 82% to 11% of wild-type. Even in lines with the most severe defects, the organization of the quiescent center and tissue layers at the root tips appears similar to wild-type, although meristematic, transition, and elongation zones are shorter. Reduced cell production rates in the mutants are due to the shorter meristems, but not to lengthened cell cycles. Additionally, mutants demonstrate reduced anisotropic cell expansion in the elongation zone, but not the meristematic zone, resulting in shorter mature cells that are similar in shape to wild-type. As expected, hypersensitivity to brefeldin A links the mutant root growth defect to altered vesicular trafficking. Several experimental approaches (e.g., dose–response measurements, localization of signaling components) failed to identify aberrant auxin or brassinosteroid signaling as a primary driver for reduced root growth in exocyst mutants.ConclusionsThe exocyst participates in two spatially distinct developmental processes, apparently by mechanisms not directly linked to auxin or brassinosteroid signaling pathways, to help establish root meristem size, and to facilitate rapid cell expansion in the elongation zone.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0386-0) contains supplementary material, which is available to authorized users.

Control of plant stem cell function by conserved interacting transcriptional regulators.

SUMMARYPlant stem cells in the shoot apical meristem (SAM) and root apical meristem (RAM) provide for postembryonic development of above-ground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development1–4. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the SAM, is a key regulatory factor controlling stem cell populations in the Arabidopsis SAM5–6 and is thought to establish the shoot stem cell niche via a feedback circuit with the CLAVATA3 (CLV3) peptide signaling pathway7. WUSCHEL-RELATED HOMEOBOX5 (WOX5), specifically expressed in root quiescent center (QC), defines QC identity and functions interchangeably with WUS in control of shoot and root stem cell niches8. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche9–11. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom12 and emerge as key actors in the specification and maintenance of stem cells within all meristems13. However, the nature of the genetic regime in stem cell niches that centers on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM)family transcription regulators act as conserved interacting co-factors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.

Precise control of plant stem cell activity through parallel regulatory inputs.

The regulation of columella stem cell activity in the Arabidopsis root cap by a nearby organizing centre, the quiescent centre, has been a key example of the stem cell niche paradigm in plants. Here, we investigate interactions between transcription factors that have been shown to regulate columella stem cells using a simple quantification method for stem cell activity in the root cap. Genetic and expression analyses reveal that the RETINOBLASTOMA-RELATED protein, the FEZ and SOMBRERO NAC-domain transcription factors, the ARF10 and ARF16 auxin response factors and the quiescent centre-expressed WOX5 homeodomain protein each provide independent inputs to regulate the number of columella stem cells. Given the tight control of columella development, we found that these inputs act in a surprisingly parallel manner. Nevertheless, important points of interaction exist; for example, we demonstrate the repression of SMB activity by non-autonomous action of WOX5. Our results suggest that the developmental progression of columella stem cells may be quantitatively regulated by several more broadly acting transcription factors rather than by a single intrinsic stem cell factor, which raises questions about the special nature of the stem cell state in plants.

ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1) is required for cell production, patterning, and morphogenesis in root development

Arabidopsis homolog of trithorax1 (ATX1/SDG27), a known regulator of flower development, encodes a H3K4histone methyltransferase that maintains a number of genes in an active state. In this study, the role of ATX1 in root development was evaluated. The loss-of-function mutant atx1-1 was impaired in primary root growth. The data suggest that ATX1 controls root growth by regulating cell cycle duration, cell production, and the transition from cell proliferation in the root apical meristem (RAM) to cell elongation. In atx1-1, the quiescent centre (QC) cells were irregular in shape and more expanded than those of the wild type. This feature, together with the atypical distribution of T-divisions, the presence of oblique divisions, and the abnormal cell patterning in the RAM, suggests a lack of coordination between cell division and cell growth in the mutant. The expression domain of QC-specific markers was expanded both in the primary RAM and in the developing lateral root primordia of atx1-1 plants. These abnormalities were independent of auxin-response gradients. ATX1 was also found to be required for lateral root initiation, morphogenesis, and emergence. The time from lateral root initiation to emergence was significantly extended in the atx1-1 mutant. Overall, these data suggest that ATX1 is involved in the timing of root development, stem cell niche maintenance, and cell patterning during primary and lateral root development. Thus, ATX1 emerges as an important player in root system architecture.

WOX5 Suppresses CYCLIN D Activity to Establish Quiescence at the Center of the Root Stem Cell Niche

SummaryIn Arabidopsis, stem cells maintain the provision of new cells for root growth. They surround a group of slowly dividing cells named the quiescent center (QC), and, together, they form the stem cell niche (SCN). The QC acts as the signaling center of the SCN, repressing differentiation of the surrounding stem cells [1] and providing a pool of cells able to replace damaged stem cells [2, 3]. Maintenance of the stem cells depends on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the QC [4]. However, the molecular mechanisms by which WOX5 promotes stem cell fate and whether WOX5 regulates proliferation of the QC are unknown. Here, we reveal a new role for WOX5 in restraining cell division in the cells of the QC, thereby establishing quiescence. In contrast, WOX5 and CYCD3;3/CYCD1;1 both promote cell proliferation in the nascent columella. The additional QC divisions occurring in wox5 mutants are suppressed in mutant combinations with the D type cyclins CYCD3;3 and CYCD1;1. Moreover, ectopic expression of CYCD3;3 in the QC is sufficient to induce cell division in the QC. WOX5 thus suppresses QC divisions that are otherwise promoted by CYCD3;3 and CYCD1;1, in part by interacting with the CYCD3;3 promoter to repress CYCD3;3 expression in the QC. Therefore, we propose a specific role for WOX5 in initiating and maintaining quiescence of the QC by excluding CYCD activity from the QC.

The ASH1-RELATED3 SET-domain protein controls cell division competence of the meristem and the quiescent center of the Arabidopsis primary root.

The stem cell niche of the Arabidopsis (Arabidopsis thaliana) primary root apical meristem is composed of the quiescent (or organizing) center surrounded by stem (initial) cells for the different tissues. Initial cells generate a population of transit-amplifying cells that undergo a limited number of cell divisions before elongating and differentiating. It is unclear whether these divisions occur stochastically or in an orderly manner. Using the thymidine analog 5-ethynyl-2'-deoxyuridine to monitor DNA replication of cells of Arabidopsis root meristems, we identified a pattern of two, four, and eight neighboring cells with synchronized replication along the cortical, epidermal, and endodermal cell files, suggested to be daughters, granddaughters, and great-granddaughters of the direct progeny of each stem cell. Markers of mitosis and cytokinesis were not present in the region closest to the transition zone where the cells start to elongate, suggesting that great-granddaughter cells switch synchronously from the mitotic cell cycle to endoreduplication. Mutations in the stem cell niche-expressed ASH1-RELATED3 (ASHR3) gene, encoding a SET-domain protein conferring histone H3 lysine-36 methylation, disrupted this pattern of coordinated DNA replication and cell division and increased the cell division rate in the quiescent center. E2Fa/E2Fb transcription factors controlling the G1-to-S-phase transition regulate ASHR3 expression and bind to the ASHR3 promoter, substantiating a role for ASHR3 in cell division control. The reduced length of the root apical meristem and primary root of the mutant ashr3-1 indicate that synchronization of replication and cell divisions is required for normal root growth and development.

Regulation of Plant Stem Cell Quiescence by a Brassinosteroid Signaling Module

The quiescent center (QC) maintains the activity of the surrounding stem cells within the root stem cell niche, yet specific molecular players sustaining the low rate of QC cell division remain poorly understood. Here, we identified a R2R3-MYB transcription factor, BRAVO (BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER), acting as a cell-specific repressor of QC divisions in the primary root of Arabidopsis. Ectopic BRAVO expression restricts overall root growth and ceases root regeneration upon damage of the stem cells, demonstrating the role of BRAVO in counteracting Brassinosteroid (BR)-mediated cell division in the QC cells. Interestingly, BR-regulated transcription factor BES1 (BRI1-EMS SUPRESSOR 1) directly represses and physically interacts with BRAVO in vivo, creating a switch that modulates QC divisions at the root stem cell niche. Together, our results define a mechanism for BR-mediated regulation of stem cell quiescence in plants.

L-Cysteine inhibits root elongation through auxin/PLETHORA and SCR/SHR pathway in Arabidopsis thaliana.

L-Cysteine plays a prominent role in sulfur metabolism of plants. However, its role in root development is largely unknown. Here, we report that L-cysteine reduces primary root growth in a dosage-dependent manner. Elevating cellular L-cysteine level by exposing Arabidopsis thaliana seedlings to high L-cysteine, buthionine sulphoximine, or O-acetylserine leads to altered auxin maximum in root tips, the expression of quiescent center cell marker as well as the decrease of the auxin carriers PIN1, PIN2, PIN3, and PIN7 of primary roots. We also show that high L-cysteine significantly reduces the protein level of two sets of stem cell specific transcription factors PLETHORA1/2 and SCR/SHR. However, L-cysteine does not downregulate the transcript level of PINs, PLTs, or SCR/SHR, suggesting that an uncharacterized post-transcriptional mechanism may regulate the accumulation of PIN, PLT, and SCR/SHR proteins and auxin transport in the root tips. These results suggest that endogenous L-cysteine level acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1/2 and SCR/SHR. L-Cysteine may serve as a link between sulfate assimilation and auxin in regulating root growth.

Origin and development of the root cap in rice.

The tip of the root is covered by a thimble-shaped root cap that is the site of perception and transduction for many environmental stimuli. Until now, little was known about how the root cap of rice (Oryza sativa) develops and functions to regulate the adaptive behavior of the root. To address this, we examined the formation of the rice root cap during embryogenesis and characterized the anatomy and structure of the rice radicle root cap. We further investigated the role of the quiescent center in the de novo origin of the root cap. At the molecular level, we found that shoot-derived auxin was absolutely needed to trigger root cap regeneration when the quiescent center was removed. Our time-course analysis of transcriptomic dynamics during the early phases of root cap regeneration indicated that changes in auxin signaling and appropriate levels of cytokinin are critical for root cap regeneration after the removal of the root cap. Moreover, we identified 152 genes that produce root cap-specific transcripts in the rice root tip. These findings together offer, to our knowledge, new mechanistic insights into the cellular and molecular events inherent in the formation and development of the root cap in rice and provide a basis for future research on the developmental and physiological function of the root cap of monocot crops.

A quiescent path to plant longevity

The giant sequoia and the bristlecone pine trees are capable of living up to several hundreds or even thousands of years. Plants achieve this longevity by regenerating stem cells capable of giving rise to all differentiated cells. Plant stem cells reside in specific niches with high mitotic activity that are known as meristems. Remarkably, at the center of the root stem cell niche (SCN) resides a group of mitotically inactive cells known as the quiescent center (QC). Recent studies suggest that stress-related phytohormones and DNA damage can initiate QC cell division, resulting in the replenishment of stem cells surrounding the QC. We therefore propose that the QC represents a pool of backup cells that serve to replace damaged stem cells, thereby contributing to plant longevity.

Normal root elongation requires arginine produced by argininosuccinate lyase in rice

Plant roots play an important role in the uptake of water and nutrients, structural support and environmental sensing, but the molecular mechanisms involved in root development are poorly understood in rice (Oryza sativa), which is characterized by a dense fibrous root system. Here we report a rice mutant (red1 for root elongation defect 1) with short roots. Morphological and physiological analyses showed that the mutant had a shorter length from the quiescent center (QC) to the starting point of the elongation zone but a similar cell size and number of lateral and crown roots compared with the wild type. Furthermore, the mutant had similar radial structure and nutrient uptake patterns to the wild type. Map-based cloning revealed that the mutant phenotype was caused by a point mutation of a gene encoding an argininosuccinate lyase (ASL), catalyzing the last step of arginine biosynthesis. The OsASL1 gene has two distinct transcripts, OsASL1.1 and OsASL1.2, which result from different transcription start sites, but only OsASL1.1 was able to complement the mutant phenotype. OsASL1.1 was expressed in both the roots and shoots. The protein encoded by OsASL1.1 showed ASL activity in yeast. OsALS1.1 was localized to the plastid. The short root of the mutant was rescued by exogenous addition of arginine, but not by other amino acids. These results indicate that arginine produced by ASL is required for normal root elongation in rice.

The root indeterminacy‐to‐determinacy developmental switch is operated through a folate‐dependent pathway in Arabidopsis thaliana

Roots have both indeterminate and determinate developmental programs. The latter is preceded by the former. It is not well understood how the indeterminacy-to-determinacy switch (IDS) is regulated. We isolated a moots koom2 (mko2; 'short root' in Mayan) Arabidopsis thaliana mutant with determinate primary root growth and analyzed the root apical meristem (RAM) behavior using various marker lines. Deep sequencing and genetic and pharmacological complementation permitted the identification of a point mutation in the FOLYLPOLYGLUTAMATE SYNTHETASE1 (FPGS1) gene responsible for the mko2 phenotype. Wild-type FPGS1 is required to maintain the IDS in the 'off' state. When FPGS1 function is compromised, the IDS is turned on and the RAM becomes completely consumed. The polyglutamate-dependent pathway of the IDS involves activation of the quiescent center independently of auxin gradients and regulatory modules participating in RAM maintenance (WUSCHEL-RELATED HOMEOBOX5 (WOX5), PLETHORA, and SCARECROW (SCR)). The mko2 mutation causes drastic changes in folate metabolism and also affects lateral root primordium morphogenesis but not initiation. We identified a metabolism-dependent pathway involved in the IDS in roots. We suggest that the root IDS represents a specific developmental pathway that regulates RAM behaviour and is a different level of regulation in addition to RAM maintenance.

Osmotic adjustment and maintenance of the redox balance in root tissue may be key points to overcome a mild water deficit during the early growth of wheat

In this investigation we analyzed in detail the consequences of water deficit during the first 4 days of wheat development, focusing on root growth as affected by eventual changes in cell cycle regulation and oxidative processes. Root elongation decreased under water restriction in correlation with the intensity of this limitation, but the total number of cells between the quiescent center and the start of the rapid elongation zone in the root apex did not vary. Neither lipid peroxidation nor protein carbonylation increased in the roots of water-starved seedlings (ψw: −0.6 MPa); accordingly, catalase activity increased, and transcript levels of cat2 gene were enhanced. Superoxide dismutase activity rose at day 2 and 3 and, unlike catalase, displayed quite similar levels on comparing roots and coleoptiles. Proline and total soluble carbohydrates increased in the roots of water-starved seedling. Total conductivity and osmolality were also augmented. No changes in the transcript levels of the markers related to G1-S transition phase of cell cycle could be detected. However, two expansin genes (TaEXPB8 and TaEXPA5) were up-regulated in roots under water deficit. We conclude that wheat root elongation in water-deprived seedlings was simply hampered by lack of water income to cells. The enhanced expression of two root expansin genes is probably related to the eventual need of a quick cell wall expansion to allow the existing root cells to recover normal turgor, in case of sudden rewatering. Maintenance of redox balance, osmotic adjustment and modification of cell wall plasticity may be key points for wheat seedlings to overcome a water restriction during early growth.

The iRoCS Toolbox – 3D analysis of the plant root apical meristem at cellular resolution

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three-dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single-cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open-source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem.

WOX5–IAA17 Feedback Circuit-Mediated Cellular Auxin Response Is Crucial for the Patterning of Root Stem Cell Niches in Arabidopsis

In plants, the patterning of stem cell-enriched meristems requires a graded auxin response maximum that emerges from the concerted action of polar auxin transport, auxin biosynthesis, auxin metabolism, and cellular auxin response machinery. However, mechanisms underlying this auxin response maximum-mediated root stem cell maintenance are not fully understood. Here, we present unexpected evidence that WUSCHEL-RELATED HOMEOBOX 5 (WOX5) transcription factor modulates expression of auxin biosynthetic genes in the quiescent center (QC) of the root and thus provides a robust mechanism for the maintenance of auxin response maximum in the root tip. This WOX5 action is balanced through the activity of indole-3-acetic acid 17 (IAA17) auxin response repressor. Our combined genetic, cell biology, and computational modeling studies revealed a previously uncharacterized feedback loop linking WOX5-mediated auxin production to IAA17-dependent repression of auxin responses. This WOX5-IAA17 feedback circuit further assures the maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of distal stem cell (DSC) populations. Our experimental studies and in silico computer simulations both demonstrate that the WOX5-IAA17 feedback circuit is essential for the maintenance of auxin gradient in the root tip and the auxin-mediated root DSC differentiation.