Quiescent Center Cells Research Articles

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.

Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex.

Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

IAA and zeatin controls cell division and endoreduplication process in quiescent center cells of Allium cepa root

Role of endogenous IAA and zeatin level in regulation of cell division and endoreduplication processes was studied in quiescent center cells of Allium cepa using colchicine by considering morphological and cytological parameters. This resulted in formation of c-tumor at root tips, which contained endoreduplicated cells. Different concentrations of NAA and BAP (1–250 μM) were exogenously applied to these endoreduplicated root tips to depolyploidize endoreduplicated quiescent center cells. Exogenous NAA treatment showed negative response on depolyploidization of quiescent center cells of root tips. However, elongation of cells, and decrease in nucleus size was observed after exogenous NAA treatment. There was no change in length of endoreduplicated roots, and growth of root at the tip of c-tumor. Exogenous BAP showed positive response in depolyploidization of quiescent center cells of root tips. There was remarkable increase in length of roots, growth of root at tip of c-tumor and decrease in cell size and nucleus size after BAP treatment. It induced cytokinesis process in endoreduplicated meristematic cells and started cell division and differentiation process in quiescent center cells. Endogenous IAA and zeatin measured from normal, endoreduplicated and depolyploidized quiescent center root tip cells suggested that when endogenous IAA/zeatin ratio is high in meristematic cells then cell cycle shifts towards endoreduplication process but when it decreases cell cycle shifts towards cell division process.

Phytosulfokine peptide signalling.

Phytosulfokine (PSK) belongs to the group of plant peptide growth factors. It is a disulfated pentapeptide encoded by precursor genes that are ubiquitously present in higher plants, suggestive of universal functions. Processing of the preproprotein involves sulfonylation by a tyrosylprotein sulfotransferase in the trans-golgi and proteolytic cleavage in the apoplast. The secreted peptide is perceived at the cell surface by a membrane-bound receptor kinase of the leucine-rich repeat family. The PSK receptor PSKR1 from Arabidopsis thaliana is an active kinase and has guanylate cyclase activity resulting in dual-signal outputs. Receptor activity is regulated by calmodulin. While PSK may be an autocrine growth factor, it also acts non-cell autonomously by promoting growth of cells that are receptor-deficient. In planta, PSK has multiple functions. It promotes cell growth, acts in the quiescent centre cells of the root apical meristem, contributes to funicular pollen tube guidance, and differentially alters immune responses depending on the pathogen. It has been suggested that PSK integrates growth and defence signals to balance the competing metabolic costs of these responses. This review summarizes our current understanding of PSK synthesis, signalling, and activity.

PHABULOSA controls the quiescent center-independent root meristem activities in Arabidopsis thaliana.

Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in shr or scr mutants were significantly recovered in the shr phb or scr phb double mutant, respectively. This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain.

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.

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.

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.

Specific Expression of DR5 Promoter in Rice Roots Using a tCUP Derived Promoter-Reporter System

Variation of transgene expression caused by either position effect at the insertion site or the promoter/enhancer elements employed for the expression of selectable marker genes has complicated phenotype characterization and caused misinterpretation. We have developed a reporter system in rice to analyze the influence of vector configuration, spacer and selectable marker gene promoter on the expression of the promoterless GUS reporter and DR5 promoter. Our results indicate that a spacer inserted between the reversed 35S promoter and the GUS reporter could reduce leaky expression of the reporter but was unable to block the nonspecific expression of DR5::GUS. Stacking the selectable marker unit in head to tail with the GUS reporter aided the gene specific expression of the GUS reporter under the DR5 promoter even when the 35S promoter is used for expression of the selectable marker. Compared to 35S under this configuration, a quick and distinctive expression of DR5::GUS was observed in the root cap, quiescent center and xylem cells in the root apical meristem by using the tCUP derived promoter (tCUP1) for selection, that is similar to the pattern obtained by a sensitive DR5 variant (DR5rev) in Arabidopsis. These data suggest a conserved property of the tCUP promoter in preventing enhancer-promoter interactions in rice as it does in Arabidopsis, and also demonstrate that an analogous distal auxin maximum exists in roots of rice. Therefore, the tCUP promoter based selection system provides a new strategy for specific expression of transgenes in rice.

ERF115 Controls Root Quiescent Center Cell Division and Stem Cell Replenishment

The quiescent center (QC) plays an essential role during root development by creating a microenvironment that preserves the stem cell fate of its surrounding cells. Despite being surrounded by highly mitotic active cells, QC cells self-renew at a low proliferation rate. Here, we identified the ERF115 transcription factor as a rate-limiting factor of QC cell division, acting as a transcriptional activator of the phytosulfokine PSK5 peptide hormone. ERF115 marks QC cell division but is restrained through proteolysis by the APC/C(CCS52A2) ubiquitin ligase, whereas QC proliferation is driven by brassinosteroid-dependent ERF115 expression. Together, these two antagonistic mechanisms delimit ERF115 activity, which is called upon when surrounding stem cells are damaged, revealing a cell cycle regulatory mechanism accounting for stem cell niche longevity.

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

The BRASSINOSTEROID INSENSITIVE1–LIKE3 Signalosome Complex Regulates Arabidopsis Root Development

Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase brassinosteroid insensitive1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 associated kinase1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein-protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.

A CLE–WOX signalling module regulates root meristem maintenance and vascular tissue development in rice

CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins belong to a small peptide family conserved in plants. Recent studies in Arabidopsis and rice have revealed a key role for CLEs in mediating cell-cell communication and stem cell maintenance during plant development, but how CLE signalling controls root development in the rice remains largely unknown. Here it is shown that exogenous application of a synthetic dodeca-amino acid peptide corresponding to the CLE motif of the rice FON2-LIKE CLE PROTEIN2 (FCP2p) protein or overexpression of FCP2 terminates root apical meristem (RAM) activity and impairs late metaxylem formation. FCP2p treatment suppresses the expression of the rice QUIESCENT-CENTER-SPECIFIC HOMEOBOX (QHB) gene, a putative orthologue of Arabidopsis WUSCHEL (WUS)-RELATED HOMEOBOX 5 (WOX5) gene, in both quiescent centre and late metaxylem cells; whereas inducible overexpression of QHB reduces the sensitivity of rice to FCP2p treatment. These results together suggest that in rice RAM maintenance and late metaxylem development are probably controlled by the mutual regulation between FCP2 and QHB. Moreover, a cross-species peptide treatment experiment in Arabidopsis implies that FCP2 has both evolutionarily conserved and species-specific roles in root development.

Moderation of Arabidopsis Root Stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 Receptor Kinase Complexes

The root system of higher plants originates from the activity of a root meristem, which comprises a group of highly specialized and long-lasting stem cells. Their maintenance and number is controlled by the quiescent center (QC) cells and by feedback signaling from differentiated cells. Root meristems may have evolved from structurally distinct shoot meristems; however, no common player acting in stemness control has been found so far. We show that CLAVATA1 (CLV1), a key receptor kinase in shoot stemness maintenance, performs a similar but distinct role in root meristems. We report that CLV1 is signaling, activated by the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION40 (CLE40), together with the receptor kinase ARABIDOPSIS CRINKLY4 (ACR4) to restrict root stemness. Both CLV1 and ACR4 overlap in their expression domains in the distal root meristem and localize to the plasma membrane (PM) and plasmodesmata (PDs), where ACR4 preferentially accumulates. Using multiparameter fluorescence image spectroscopy (MFIS), we show that CLV1 and ACR4 can form homo- and heteromeric complexes that differ in their composition depending on their subcellular localization. We hypothesize that these homo- and heteromeric complexes may differentially regulate distal root meristem maintenance. We conclude that essential components of the ancestral shoot stemness regulatory system also act in the root and that the specific interaction of CLV1 with ACR4 serves to moderate and control stemness homeostasis in the root meristem. The structural differences between these two meristem types may have necessitated this recruitment of ACR4 for signaling by CLV1.

RNA interference-mediated gene knockdown within specific cell types

In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types.

Calcium Could Be Involved in Auxin-Regulated Maintenance of the Quiescent Center in the Arabidopsis Root

Ca2+ transduces hormone and environmental signals to the Ca2+ sensors and relays them to the downstream target effectors in cells. During the post-embryonic developmental process, auxin plays a critical role in maintaining the mitotically inactive status of the quiescent center (QC) and the root growth and development that follows. In this report, we demonstrate that Ca2+ plays an important role in the maintenance of the QC, probably by regulating PIN1-mediated auxin transport. Perturbation of the intracellular Ca2+ levels with chemicals that modify the Ca2+ level decreases the endogenous auxin level and the size of the auxin maximum in the root tip and, at the same time, activates QC cell division and expansion. This decreased level of auxin is almost completely restored to the control level by the treatment of exogenous auxin. Interestingly, treatment with Ca2+ level modifying chemicals significantly decreased the PIN1 expression in the root vasculature. Taken together, we suggest that balancing Ca2+ homeostasis is one of contributing factors in establishing the proper auxin maximum in the root tip and maintaining the QC identity.

Quantification of the brassinosteroid insensitive1 receptor in planta.

In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors μm⁻² in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors μm⁻². Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor density.

Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.

Brassinosteroid perception in the epidermis controls root meristem size

Multiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3 and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.