Quiescent Center Research Articles

NatA is required for suspensor development in Arabidopsis

ABSTRACTSuspensor development is essential for early embryogenesis. The filamentous suspensor plays vital roles in supporting the embryo proper and in exchanging nutrients and information between the embryo proper and embryo sac. In addition, at the globular stage, the uppermost suspensor cell differentiates into the hypophysis, which generates the progenitors of the quiescent center and columella stem cells. In naa10 and naa15 mutant plants, suspensor cell identity was found to be abnormal and embryo development was disturbed, leading to embryonic lethality. Therefore, the NatA complex is required for proper suspensor development in Arabidopsis.

Changes in radical scavenging activity of normal, endoreduplicated and depolyploid root tip cells of Allium cepa.

The plant cell responds to abiotic stress conditions by adjusting its cellular metabolism and various defensive mechanisms. Cellular metabolism involves changes in the cell cycle, in which the cell undergoes repeated rounds of endocycles leading to polyploidization. Defense mechanisms such as role of antioxidants are a key to understand plant adaptation. The present work describes endoreduplication and radical scavenging activity as two different defense mechanisms adapted by plants for their survival under stress condition. The work describes linkage of these two processes with each other under abiotic stress. Endoreduplicated root tip cells of Allium cepa were depolyploidized by exogenous phytohormones. Further, free radical scavenging activity from normal, endoreduplicated and depolyploidized root tips cells was observed to understand the role of phytohormones. Elevated free radical scavenging potential was observed in endoreduplicated cells compared to normal and depolyploidized cells. Based on these results, it was concluded that endoreduplication and antioxidant pathways are linked with each other through phytohormonal activities. The concentration of auxin and cytokinin regulates the activity of ascorbate oxidase enzyme, which in turn maintains the concentration of AsA within the cell. AsA level directs the prolyl-hydroxylation process of cell division proteins in quiescent center cells either toward endoreduplication process or cell division process.

A P-Loop NTPase Regulates Quiescent Center Cell Division and Distal Stem Cell Identity through the Regulation of ROS Homeostasis in Arabidopsis Root.

Reactive oxygen species (ROS) are recognized as important regulators of cell division and differentiation. The Arabidopsis thaliana P-loop NTPase encoded by APP1 affects root stem cell niche identity through its control of local ROS homeostasis. The disruption of APP1 is accompanied by a reduction in ROS level, a rise in the rate of cell division in the quiescent center (QC) and the promotion of root distal stem cell (DSC) differentiation. Both the higher level of ROS induced in the app1 mutant by exposure to methyl viologen (MV), and treatment with hydrogen peroxide (H2O2) rescued the mutant phenotype, implying that both the increased rate of cell division in the QC and the enhancement in root DSC differentiation can be attributed to a low level of ROS. APP1 is expressed in the root apical meristem cell mitochondria, and its product is associated with ATP hydrolase activity. The key transcription factors, which are defining root distal stem niche, such as SCARECROW (SCR) and SHORT ROOT (SHR) are both significantly down-regulated at both the transcriptional and protein level in the app1 mutant, indicating that SHR and SCR are important downstream targets of APP1-regulated ROS signaling to control the identity of root QC and DSCs.

Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.

Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants.

HAWAIIAN SKIRT regulates the quiescent center-independent meristem activity in Arabidopsis roots.

Root apical meristem (RAM) drives post-embryonic root growth by constantly supplying cells through mitosis. It is composed of stem cells and their derivatives, the transit-amplifying (TA) cells. Stem cell organization and its maintenance in the RAM are well characterized, however, their relationships with TA cells remain unclear. SHORTROOT (SHR) is critical for root development. It patterns cell types and promotes the post-embryonic root growth. Defective root growth in the shr has been ascribed to the lack of quiescent center (QC), which maintains the surrounding stem cells. However, our recent investigation indicated that SHR maintains TA cells independently of QC by modulating PHABULOSA (PHB) through miRNA165/6. PHB controls TA cell activity by modulating cytokinin levels and type B Arabidopsis Response Regulator activity, in a dosage-dependent manner. To further understand TA cell regulation, we conducted a shr suppressor screen. With an extensive mutagenesis screen followed by genome sequencing of a pooled F2 population, we discovered two suppressor alleles with mutations in HAWAIIAN SKIRT (HWS). HWS, encoding an F-box protein with kelch domain, is expressed, partly depending on SHR, in the root cap and in the pericycle of the differentiation zone. Interestingly, root growth in the shr hws was more active than the wild-type roots for the first 7 days after germination, without recovering QC. Contrary to shr phb, shr hws did not show a recovery of cytokinin signaling. These indicate that HWS affects QC-independent TA cell activities through a pathway distinctive from PHB.

The Protein Arginine Methylase 5 (PRMT5/SKB1) Gene Is Required for the Maintenance of Root Stem Cells in Response to DNA Damage

Plant root stem cells and their surrounding microenvironment, namely the stem cell niche, are hypersensitive to DNA damage. However, the molecular mechanisms that help maintain the genome stability of root stem cells remain elusive. Here we show that the root stem cells in the skb1 (Shk1 kinase binding protein 1) mutant undergoes DNA damage-induced cell death, which is enhanced when combined with a lesion of the Ataxia-telangiectasia mutated (ATM) or the ATM/RAD3-related (ATR) genes, suggesting that the SKB1 plays a synergistically effect with ATM and ATR in DNA damage pathway. We also provide evidence that SKB1 is required for the maintenance of quiescent center (QC), a root stem cell niche, under DNA damage treatments. Furthermore, we report decreased and ectopic expression of SHORTROOT (SHR) in response to DNA damage in the skb1 root tips, while the expression of SCARECROW (SCR) remains unaffected. Our results uncover a new mechanism of plant root stem cell maintenance under DNA damage conditions that requires SKB1.

TOPOISOMERASE1α Acts through Two Distinct Mechanisms to Regulate Stele and Columella Stem Cell Maintenance.

TOPOISOMERASE1 (TOP1), which releases DNA torsional stress generated during replication through its DNA relaxation activity, plays vital roles in animal and plant development. In Arabidopsis (Arabidopsis thaliana), TOP1 is encoded by two paralogous genes (TOP1α and TOP1β), of which TOP1α displays specific developmental functions that are critical for the maintenance of shoot and floral stem cells. Here, we show that maintenance of two different populations of root stem cells is also dependent on TOP1α-specific developmental functions, which are exerted through two distinct novel mechanisms. In the proximal root meristem, the DNA relaxation activity of TOP1α is critical to ensure genome integrity and survival of stele stem cells (SSCs). Loss of TOP1α function triggers DNA double-strand breaks in S-phase SSCs and results in their death, which can be partially reversed by the replenishment of SSCs mediated by ETHYLENE RESPONSE FACTOR115 In the quiescent center and root cap meristem, TOP1α is epistatic to RETINOBLASTOMA-RELATED (RBR) in the maintenance of undifferentiated state and the number of columella stem cells (CSCs). Loss of TOP1α function in either wild-type or RBR RNAi plants leads to differentiation of CSCs, whereas overexpression of TOP1α mimics and further enhances the effect of RBR reduction that increases the number of CSCs Taken together, these findings provide important mechanistic insights into understanding stem cell maintenance in plants.

Expression of MAX2 under SCARECROW promoter enhances the strigolactone/MAX2 dependent response of Arabidopsis roots to low-phosphate conditions.

MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partially sufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions. Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.

Environmental Nitrate Stimulates Abscisic Acid Accumulation in Arabidopsis Root Tips by Releasing It from Inactive Stores.

Abscisic acid (ABA) signaling plays a major role in root system development, regulating growth and root architecture. However, the precise localization of ABA remains undetermined. Here, we present a mechanism in which nitrate signaling stimulates the release of bioactive ABA from the inactive storage form, ABA-glucose ester (ABA-GE). We found that ABA accumulated in the endodermis and quiescent center of Arabidopsis thaliana root tips, mimicking the pattern of SCARECROW expression, and (to lower levels) in the vascular cylinder. Nitrate treatment increased ABA levels in root tips; this stimulation requires the activity of the endoplasmic reticulum-localized, ABA-GE-deconjugating enzyme b-GLUCOSIDASE1, but not de novo ABA biosynthesis. Immunogold labeling demonstrated that ABA is associated with cytoplasmic structures near, but not within, the endoplasmic reticulum. These findings demonstrate a mechanism for nitrate-regulated root growth via regulation of ABA accumulation in the root tip, providing insight into the environmental regulation of root growth.

Salt Stress Affects the Redox Status of Arabidopsis Root Meristems.

We report the redox status (profiles) for specific populations of cells that comprise the Arabidopsis root tip. For recently germinated, 3–5-day-old seedlings we show that the region of the root tip with the most reduced redox status includes the root cap initials, the quiescent center and the most distal portion of the proximal meristem, and coincides with (overlays) the region of the auxin maximum. As one moves basally, further into the proximal meristem, and depending on the growth conditions, the redox status becomes more oxidized, with a 5–10 mV difference in redox potential between the two borders delimiting the proximal meristem. At the point on the root axis at which cells of the proximal meristem cease division and enter the transition zone, the redox potential levels off, and remains more or less unchanged throughout the transition zone. As cells leave the transition zone and enter the zone of elongation the redox potentials become more oxidized. Treating roots with salt (50, 100, and 150 mM NaCl) results in marked changes in root meristem structure and development, and is preceded by changes in the redox profile, which flattens, and initially becomes more oxidized, with pronounced changes in the redox potentials of the root cap, the root cap initials and the quiescent center. Roots exposed to relatively mild levels of salt (<100 mM) are able to re-establish a normal, pre-salt treatment redox profile 3–6 days after exposure to salt. Coincident with the salt-associated changes in redox profiles are changes in the distribution of auxin transporters (AUX1, PIN1/2), which become more diffuse in their localization. We conclude that salt stress affects root meristem maintenance, in part, through changes in redox and auxin transport.

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

Main conclusionSCARECROW controls Arabidopsis root meristem size from the root endodermis tissue by regulating the DELLA protein RGA that in turn mediates the regulation ofARR1levels 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2471-0) contains supplementary material, which is available to authorized users.

The Essential Role of Cytokinin Signaling in Root Apical Meristem Formation during Somatic Embryogenesis.

The Essential Role of Cytokinin Signaling in Root Apical Meristem Formation during Somatic Embryogenesis.

Effects of Continuous Volumetric Direct-Coupled Nonequilibrium Atmospheric Microwave Plasma Discharge on Swirl-Stabilized Premixed Flames

The effect of continuous volumetric direct-coupled nonequilibrium atmospheric microwave plasma discharge on swirl-stabilized premixed methane–air flames was investigated using quantitative OH planar laser-induced fluorescence and spectrally resolved emission. The plasma discharge was found to influence the dynamics of flame stabilization, i.e., plasma-assisted flames stabilized in the quiescent center body wake were relatively stable while swirl flames stabilized in the active inner shear layer were prone to local extinction due to aerodynamic shear. At coupled plasma powers corresponding to less than 3% of the thermal power output, in addition to the improved flame stability, significant improvement in the lean blow-out limit ( $\sim 43$ %) and OH number density ( $\sim 150$ %) was observed. The enhancements are shown to be nonequilibrium plasma effects and not predominantly ohmic heating as significant equivalence ratio dependence of OH number density in plasma-assisted flames was observed. Spectrographic measurements indicated nitrogen vibrational temperatures as high as 6100 K, suggesting both vibrational and electronic excitation of nitrogen molecules in the presence of a plasma discharge. The activation of highly reactive species through vibrational–vibrational relaxation and direct impact dissociation accelerates the combustion chemistry. It is demonstrated that microwave direct plasma coupling can drastically enhance the dynamic flame stability of swirl-stabilized flames, especially at very lean operating conditions.

Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor

Lateral root formation is an important determinant of root system architecture. In Arabidopsis, lateral roots originate from pericycle cells, which undergo a program of morphogenesis to generate a new lateral root meristem. Despite its importance for root meristem organization, the onset of quiescent center (QC) formation during lateral root morphogenesis remains unclear. Here, we used live 3D confocal imaging to monitor cell organization and identity acquisition during lateral root development. Our dynamic observations revealed an early morphogenesis phase and a late meristem formation phase as proposed in the bi-phasic growth model. Establishment of lateral root QCs coincided with this developmental phase transition. QC precursor cells originated from the outer layer of stage II lateral root primordia, within which the SCARECROW (SCR) transcription factor was specifically expressed. Disrupting SCR function abolished periclinal divisions in this lateral root primordia cell layer and perturbed the formation of QC precursor cells. We conclude that de novo QC establishment in lateral root primordia operates via SCR-mediated formative cell division and coincides with the developmental phase transition.

Systems biology analysis of the WOX5 gene and its functions in the root stem cell niche

WUSCHEL RELATED HOMEOBOX 5 (WOX5) gene encodes the transcription factor, which is one of the key regulators, maintaining structure and functioning of the stem cell niche in plant root tips. Protein WOX5 is expressed in the quiescent center of the root apical meristem, preventing differentiation of columella initials and altogether with SCR, SHR, PLT1 and PLT2 participating in the control of differentiation of other root meristem initials. However, the details of WOX5 functioning are unclear. The WOX5 protein belongs to WUSCHEL related homeobox (WOX) family, the founder of which is the transcription factor WUSCHEL (WUS) providing maintenance of the stem cell niche in the shoot apical meristem. WOX5 and WUS diverged from a common ancestor at the base of angiosperms, which resulted in a specialization of shoot and root stem cell niches. However, the problem of WOX5 structural and functional divergence during angiosperm evolution was poorly addressed. In this review we present a systems biology analysis of the WOX5 gene to reveal specific features of its evolution and functioning. To this end, we performed a phylogenetic analysis on 62 publicly available WOX5 amino acid sequences, generalized published data about WOX5 expression domain in Arabidopsis and other species and its role in development, integrated the results of experiments on identification of primary and secondary targets for this transcription factor. Data on possible mechanisms of direct and indirect regulation of WOX5 expression were discussed. Particularly, we performed the analysis of WOX5 promoter regions from 30 species. Possible direct regulators of the WOX5 gene expression were proposed based on the presence of putative binding sites for the candidate transcription factors in conserved WOX5 promoter regions.

Shade avoidance 6encodes anArabidopsisflap endonuclease required for maintenance of genome integrity and development

Flap endonuclease-1 (FEN1) belongs to the Rad2 family of structure-specific nucleases. It is required for several DNA metabolic pathways, including DNA replication and DNA damage repair. Here, we have identified a shade avoidance mutant, sav6, which reduces the mRNA splicing efficiency of SAV6. We have demonstrated that SAV6 is an FEN1 homologue that shows double-flap endonuclease and gap-dependent endonuclease activity, but lacks exonuclease activity. sav6 mutants are hypersensitive to DNA damage induced by ultraviolet (UV)-C radiation and reagents that induce double-stranded DNA breaks, but exhibit normal responses to chemicals that block DNA replication. Signalling components that respond to DNA damage are constitutively activated in sav6 mutants. These data indicate that SAV6 is required for DNA damage repair and the maintenance of genome integrity. Mutant sav6 plants also show reduced root apical meristem (RAM) size and defective quiescent centre (QC) development. The expression of SMR7, a cell cycle regulatory gene, and ERF115 and PSK5, regulators of QC division, is increased in sav6 mutants. Their constitutive induction is likely due to the elevated DNA damage responses in sav6 and may lead to defects in the development of the RAM and QC. Therefore, SAV6 assures proper root development through maintenance of genome integrity.

MicroRNA miR396 Regulates the Switch between Stem Cells and Transit-Amplifying Cells in Arabidopsis Roots.

To ensure an adequate organ mass, the daughters of stem cells progress through a transit-amplifying phase displaying rapid cell division cycles before differentiating. Here, we show that Arabidopsis thaliana microRNA miR396 regulates the transition of root stem cells into transit-amplifying cells by interacting with GROWTH-REGULATING FACTORs (GRFs). The GRFs are expressed in transit-amplifying cells but are excluded from the stem cells through inhibition by miR396. Inactivation of the GRFs increases the meristem size and induces periclinal formative divisions in transit-amplifying cells. The GRFs repress PLETHORA (PLT) genes, regulating their spatial expression gradient. Conversely, PLT activates MIR396 in the stem cells to repress the GRFs. We identified a pathway regulated by GRF transcription factors that represses stem cell-promoting genes in actively proliferating cells, which is essential for the progression of the cell cycle and the orientation of the cell division plane. If unchecked, the expression of the GRFs in the stem cell niche suppresses formative cell divisions and distorts the organization of the quiescent center. We propose that the interactions identified here between miR396 and GRF and PLT transcription factors are necessary to establish the boundary between the stem cell niche and the transit-amplifying region.

Genotoxicity and Anatomical Root Changes in Allium cepa L. (Amaryllidaceae) Caused by the Effluent of the Processing of Ornamental Rocks

The effluent of the ornamental rock industry is characterized by presenting great concentrations of total solids, high contents of iron, and elevated pH, all responsible for the contamination of the superficial and ground waters, destruction of the soil, the vegetation, and the silting of the rivers. The purpose of this study is to assess the cytotoxic and genotoxic effects and the anatomical changes caused by the effluents arising from the ornamental rock polishing industry in root apex cells of Allium cepa L. (Amaryllidaceae). The samples of the effluent were collected in a polishing industry located in Nova Venecia, State of Espirito Santo, and were analyzed by mass spectrometry and atomic emission. Bulbs of A. cepa were exposed to the effluent at 12.5, 25, 37.5, 50, 75, and 100 % concentrations (residue in raw form) (v/v) for a period of 20 days. For the positive control, metilmethanesulfonate (MMS) at 4 × 10−4-M concentration was used, and distilled water was used for the negative control. The experiment was assessed taking into consideration the following parameters: mitotic index, frequency of chromosomal and nuclear abnormalities in the root apical meristem, and root anatomy. The mitotic index suffered a decrease proportional to the increase in the concentration of effluent. All the concentrations of the effluent led to chromosomal and nuclear abnormalities being stickiness and nuclear shoots the most frequent. The root apex evidenced changes that reflected on the decrease of the percentage area of the protoderm and the fundamental meristem and the increase in the areas of the cap and quiescent center. The symptoms of toxicity are related to the high frequency of cell in cellular death process observed in the roots exposed to the higher concentrations and to the decrease in the mitotic index of the apical root meristem.

Origin of the concept of the quiescent centre of plant roots.

Concepts in biology feed into general theories of growth, development and evolution of organisms and how they interact with the living and non-living components of their environment. A well-founded concept clarifies unsolved problems and serves as a focus for further research. One such example of a constructive concept in the plant sciences is that of the quiescent centre (QC). In anatomical terms, the QC is an inert group of cells maintained within the apex of plant roots. However, the evidence that established the presence of a QC accumulated only gradually, making use of strands of different types of observations, notably from geometrical-analytical anatomy, radioisotope labelling and autoradiography. In their turn, these strands contributed to other concepts: those of the mitotic cell cycle and of tissue-related cell kinetics. Another important concept to which the QC contributed was that of tissue homeostasis. The general principle of this last-mentioned concept is expressed by the QC in relation to the recovery of root growth following a disturbance to cell proliferation; the resulting activation of the QC provides new cells which not only repair the root meristem but also re-establish a new QC.

WOX5 is Shining in the Root Stem Cell Niche.

The WUS-RELATED HOMEOBOX 5 (WOX5) gene is expressed in the quiescent center (QC) to regulate the columella stem cell (CSC) identity. Three recent reports not only show how WOX5 is controlled but also highlight the key role of WOX5 in root stem cell niche maintenance.