Quiescent Center Research Articles

OP22 - Glutathione transport into the nucleus

The low molecular weight thiol antioxidant glutathione is present in the nucleus of plant cells, where it functions as an important redox buffer. The glutathione redox potential of the nuclei determined using redox-sensitive (ro-) green fluorescent protein was found to be the same at that of the cytosol (-295 /- 2.5 mV) in the first two layers of diving cells above the quiescent centre in roots over the first 48h period after germination. Using 3mM hydroxyurea to synchronise the cell cycle, together with a CYCB1:1GUS marker for the G2 phase of the cell cycle, we established that the cells accumulated at G2 between 16 and 18h after the start of incubation with the inhibitor. At this point the glutathione redox potential was the same in the nuclei and the cytosol, which were both highly reduced. However, the redox potential of the nuclei can become oxidised relative to the cytosol under different conditions. This talk will focus on the glutathione redox potential of the nuclei and on how it might be controlled.

Aluminum-Dependent Terminal Differentiation of the Arabidopsis Root Tip Is Mediated through an ATR-, ALT2-, and SOG1-Regulated Transcriptional Response.

By screening for suppressors of the aluminum (Al) hypersensitive Arabidopsis thaliana mutant als3-1, it was found that mutational loss of the Arabidopsis DNA damage response transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) confers increased Al tolerance similar to the loss-of-function mutants for the cell cycle checkpoint genes ATAXIA TELANGIECTASIA AND RAD3 RELATED (ATR) and ALUMINUM TOLERANT2 (ALT2). This suggests that Al-dependent terminal differentiation of the root tip is an active process resulting from activation of the DNA damage checkpoint by an ATR-regulated pathway, which functions at least in part through SOG1. Consistent with this, ATR can phosphorylate SOG1 in vitro. Analysis of SOG1's role in Al-dependent root growth inhibition shows that sog1-7 prevents Al-dependent quiescent center differentiation and endoreduplication in the primary root tip. Following Al exposure, SOG1 increases expression of several genes previously associated with DNA damage, including BRCA1 and PARP2, with gel-shift analysis showing that SOG1 can physically associate with the BRCA1 promoter in vitro. Al-responsive expression of these SOG1-regulated genes requires ATR and ALT2, but not ATAXIA TELANGIECTASIA MUTATED, thus demonstrating that in response to chronic Al exposure, ATR, ALT2, and SOG1 function together to halt root growth and promote terminal differentiation at least in part in a transcription-dependent manner.

Spatial Regulation of Root Growth: Placing the Plant TOR Pathway in a Developmental Perspective

Plant cells contain specialized structures, such as a cell wall and a large vacuole, which play a major role in cell growth. Roots follow an organized pattern of development, making them the organs of choice for studying the spatio-temporal regulation of cell proliferation and growth in plants. During root growth, cells originate from the initials surrounding the quiescent center, proliferate in the division zone of the meristem, and then increase in length in the elongation zone, reaching their final size and differentiation stage in the mature zone. Phytohormones, especially auxins and cytokinins, control the dynamic balance between cell division and differentiation and therefore organ size. Plant growth is also regulated by metabolites and nutrients, such as the sugars produced by photosynthesis or nitrate assimilated from the soil. Recent literature has shown that the conserved eukaryotic TOR (target of rapamycin) kinase pathway plays an important role in orchestrating plant growth. We will summarize how the regulation of cell proliferation and cell expansion by phytohormones are at the heart of root growth and then discuss recent data indicating that the TOR pathway integrates hormonal and nutritive signals to orchestrate root growth.

Function and regulation of transcription factors involved in root apical meristem and stem cell maintenance.

Plant roots are essential for overall plant development, growth, and performance by providing anchorage in the soil and uptake of nutrients and water. The primary root of higher plants derives from a group of pluripotent, mitotically active stem cells residing in the root apical meristem (RAM) which provides the basis for growth, development, and regeneration of the root. The stem cells in the Arabidopsis thaliana RAM are surrounding the quiescent center (QC), which consists of a group of rarely dividing cells. The QC maintains the stem cells in a non-cell-autonomous manner and prevents them from differentiation. The necessary dynamic but also tight regulation of the transition from stem cell fate to differentiation most likely requires complex regulatory mechanisms to integrate external and internal cues. Transcription factors play a central role in root development and are regulated by phytohormones, small signaling molecules, and miRNAs. In this review we give a comprehensive overview about the function and regulation of specific transcription factors controlling stem cell fate and root apical meristem maintenance and discuss the possibility of TF complex formation, subcellular translocations and cell-to-cell movement functioning as another level of regulation.

Brassinazole resistant 1 (BZR1)-dependent brassinosteroid signalling pathway leads to ectopic activation of quiescent cell division and suppresses columella stem cell differentiation.

Previous publications have shown that BRI1 EMS suppressor 1 (BES1), a positive regulator of the brassinosteroid (BR) signalling pathway, enhances cell divisions in the quiescent centre (QC) and stimulates columella stem cell differentiation. Here, it is demonstrated that BZR1, a BES1 homologue, also promotes cell divisions in the QC, but it suppresses columella stem cell differentiation, opposite to the action of BES1. In addition, BR and its BZR1-mediated signalling pathway are shown to alter the expression/subcellular distribution of pin-formed (PINs), which may result in changes in auxin movement. BR promotes intense nuclear accumulation of BZR1 in the root tip area, and the binding of BZR1 to the promoters of several root development-regulating genes, modulating their expression in the root stem cell niche area. These BZR1-mediated signalling cascades may account for both the ectopic activation of QC cell divisions as well as the suppression of the columella stem cell differentiation. They could also inhibit auxin-dependent distal stem cell differentiation by antagonizing the auxin/WOX5-dependent pathway. In conclusion, BZR1-/BES1-mediated BR signalling pathways show differential effects on the maintenance of root apical meristem activities: they stimulate ectopic QC division while they show opposite effects on the differentiation of distal columella stem cells in a BR concentration- and BZR1-/BES1-dependent manner.

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.

BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

Organizer-Derived WOX5 Signal Maintains Root Columella Stem Cells through Chromatin-Mediated Repression of CDF4 Expression

Stem cells in plants and animals are maintained pluripotent by signals from adjacent niche cells. In plants, WUSCHEL HOMEOBOX (WOX) transcription factors are central regulators of stem cell maintenance in different meristem types, yet their molecular mode of action has remained elusive. Here we show that in the Arabidopsis root meristem, the WOX5 protein moves from the root niche organizer, the quiescent center, into the columella stem cells, where it directly represses the transcription factor gene CDF4. This creates a gradient of CDF4 transcription, which promotes differentiation opposite to the WOX5 gradient, allowing stem cell daughter cells to exit the stem cell state. We further show that WOX5 represses CDF4 transcription by recruiting TPL/TPR co-repressors and the histone deacetylase HDA19, which consequently induces histone deacetylation at the CDF4 regulatory region. Our results show that chromatin-mediated repression of differentiation programs is a common strategy in plant and animal stem cell niches.

Mathematical modelling ofWOX5- andCLE40-mediated columella stem cell homeostasis inArabidopsis

The root meristem of Arabidopsis thaliana harbours a pool of stem cells, which divide to give rise to the differentiated cells of the various root tissues. Regulatory networks of inter-cellular signalling molecules control the homeostasis of stem cell number and position so that both stem and differentiated cells are consistently available. This work focuses on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), the signalling peptide CLAVATA3/EMBRYO-SURROUNDING REGION 40 (CLE40) and the feedback loops involving them, which maintain the columella stem cells (CSCs). WOX5 signals from the quiescent centre (QC) to promote stem cell fate, while CLE40 is secreted from the differentiated columella cells (CCs) to promote differentiation. Our analyses of mathematical models of this network show that, when cell fate is controlled primarily by antagonistic factors, homeostasis requires a spatial component and inter-cellular signalling. We have also found that WOX5 contributes to, but is not absolutely necessary for, CSC maintenance. Furthermore, our modelling led us to postulate an additional signalling molecule that promotes CSC maintenance. We propose that this WOX5-independent signal originates in the QC, is targeted by CLE40 signalling and is capable of maintaining CSCs.

The Arabidopsis thaliana elongator complex subunit 2 epigenetically affects root development.

The elongator complex subunit 2 (ELP2) protein, one subunit of an evolutionarily conserved histone acetyltransferase complex, has been shown to participate in leaf patterning, plant immune and abiotic stress responses in Arabidopsis thaliana. Here, its role in root development was explored. Compared to the wild type, the elp2 mutant exhibited an accelerated differentiation of its root stem cells and cell division was more active in its quiescent centre (QC). The key transcription factors responsible for maintaining root stem cell and QC identity, such as AP2 transcription factors PLT1 (PLETHORA1) and PLT2 (PLETHORA2), GRAS transcription factors such as SCR (SCARECROW) and SHR (SHORT ROOT) and WUSCHEL-RELATED HOMEOBOX5 transcription factor WOX5, were all strongly down-regulated in the mutant. On the other hand, expression of the G2/M transition activator CYCB1 was substantially induced in elp2. The auxin efflux transporters PIN1 and PIN2 showed decreased protein levels and PIN1 also displayed mild polarity alterations in elp2, which resulted in a reduced auxin content in the root tip. Either the acetylation or methylation level of each of these genes differed between the mutant and the wild type, suggesting that the ELP2 regulation of root development involves the epigenetic modification of a range of transcription factors and other developmental regulators.

Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots

The spatiotemporal balance between stem cell maintenance, proliferation, and differentiation determines the rate of root growth and is controlled by hormones, including auxin and brassinosteroid (BR). However, the spatial actions of BR and its interactions with auxin remain unclear in roots. Here, we show that oppositely patterned and antagonistic actions of BR and auxin maintain the stem cell balance and optimal root growth. We discover a pattern of low levels of nuclear-localized BR-activated transcription factor BZR1 in the stem cell niche and high BZR1 levels in the transition-elongation zone. This BZR1 pattern requires local BR catabolism and auxin synthesis, as well as BR signaling. Cell-type-specific expression of a constitutively active form of BZR1 confirms that the high and low levels of BZR1 are required for the normal cell behaviors in the elongation zone and quiescent center (QC), respectively. Comparison between BR-responsive, BZR1-targeted, auxin-responsive, and developmental zone-specific transcriptomes indicates that BZR1 mostly activates its target genes expressed in the transition-elongation zone, but represses genes in the QC and surrounding stem cells, and that BR and auxin have overall opposite effects on gene expression. Genetic and physiological interactions support that a balance between the antagonistic actions of BR and auxin is required for optimal root growth. These results demonstrate that the level and output specificity of BR signaling are spatially patterned and that, in contrast to their synergism in shoots, BR and auxin interact antagonistically in roots to control the spatiotemporal balance of stem cell dynamics required for optimal root growth.

Use of Antagonistic Peptide Technology to Determine the CLE Peptides that Regulate Root Growth in Arabidopsis

Plants constantly produce cells that form tissues and organs from their meristems. During development, patterned cell divisions and cell fate specification through cell-cell communication play a key role in organ shape and organization. The root apical meristem (RAM) of Arabidopsis is the source of many cell types in the root and requires intrinsic and extrinsic factors for development and maintenance; bidirectional signaling between stem and mature cells contributes to RAM activity, and its organization in Arabidopsis is a useful model to determine discrete signaling mechanisms in plant growth and morphogenesis. CLE peptides are intercellular signals in plant development. They are encoded by members of the CLE gene family, which produce small proteins believed to play a role in stem cell maintenance. The Tax lab demonstrated that CLE peptides have a differential regional effect on the root, promoting proliferation in the quiescent center and inhibiting cell division in the transit amplifying region, overall suppressing root growth (unpublished data). The goal is to determine which CLE peptides contribute to these differential effects in Arabidopsis roots. With antagonistic peptide technology, single residues in CLE peptides can be modified and cause specific phenotypes, such as disruption of stem cell maintenance, as seen by Song et al when replacing the sixth glycine residue to a threonine in CLV3. This technology can be used to characterize these signals, and ultimately better evaluate the role of CLE peptides in plant development. We plan to alter residues in CLE peptides and treat roots with the mutant peptides to determine what effect they have on different regions of the root and in mutant backgrounds. Song et al Plant Physiology 161.3 2013

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.

Role of ethylene in activation of cell division in quiescent center of excised maize roots

In the present work, two maize cultivars in which root meristem responded differently to root tip excision were used: in Interkras-375 MW, meristem opening was observed due to the activation of cell divisions in the quiescent center (QC); in Krasnodar-194 MW, the meristem remained closed. Excised root tips of Interkras MB-375 were shown to produce much more ethylene than excised root tips of Krasnodar-194 MW. The inhibitor of ethylene biosynthesis L-α-(2-amino-ethoxyvinyl)glycine-HCl (AVG) and inhibitors of ethylene action AgNO3 and 1-methyl-cyclopropene (MCP) prevented meristem opening in excised root tips of Intekras-375 MW. The obtained results allow us to conclude that ethylene plays an important role in the activation of cell divisions in the QC of excised root tips.

Endophytic microbes Bacillus sp. LZR216-regulated root development is dependent on polar auxin transport in Arabidopsis seedlings.

Endophytic microbes Bacillus sp. LZR216 isolated from Arabidopsis root promoted Arabidopsis seedlings growth. It may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. Plant roots are colonized by an immense number of microbes, including epiphytic and endophytic microbes. It was found that they have the ability to promote plant growth and protect roots from biotic and abiotic stresses. But little is known about the mechanism of the endophytic microbes-regulated root development. We isolated and identified a Bacillus sp., named as LZR216, of endophytic bacteria from Arabidopsis root. By employing a sterile experimental system, we found that LZR216 promoted the Arabidopsis seedlings growth, which may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. By testing the cell type-specific developmental markers, we demonstrated that Bacillus sp. LZR216 increases the DR5::GUS and DR5::GFP expression but decreases the CYCB1;1::GUS expression in Arabidopsis root tips. Further studies indicated that LZR216 is able to inhibit the meristematic length and decrease the cell division capability but has little effect on the quiescent center function of the root meristem. Subsequently, it was also shown that LZR216 has no significant effects on the primary root length of the pin2 and aux1-7 mutants. Furthermore, LZR216 down-regulates the levels of PIN1-GFP, PIN2-GFP, PIN3-GFP, and AUX1-YFP. In addition, the wild-type Arabidopsis seedlings in the present of 1 or 5µM NPA (an auxin transport inhibitor) were insensitive to LZR216-inhibited primary root elongation. Collectively, LZR216 regulates the development of root system architecture depending on polar auxin transport. This study shows a new insight on the ability of beneficial endophytic bacteria in regulating postembryonic root development.

Mathematical modeling and experimental validation of the spatial distribution of boron in the root of Arabidopsis thaliana identify high boron accumulation in the tip and predict a distinct root tip uptake function.

Boron, an essential micronutrient, is transported in roots of Arabidopsis thaliana mainly by two different types of transporters, BORs and NIPs (nodulin26-like intrinsic proteins). Both are plasma membrane localized, but have distinct transport properties and patterns of cell type-specific accumulation with different polar localizations, which are likely to affect boron distribution. Here, we used mathematical modeling and an experimental determination to address boron distributions in the root. A computational model of the root is created at the cellular level, describing the boron transporters as observed experimentally. Boron is allowed to diffuse into roots, in cells and cell walls, and to be transported over plasma membranes, reflecting the properties of the different transporters. The model predicts that a region around the quiescent center has a higher concentration of soluble boron than other portions. To evaluate this prediction experimentally, we determined the boron distribution in roots using laser ablation-inductivity coupled plasma-mass spectrometry. The analysis indicated that the boron concentration is highest near the tip and is lower in the more proximal region of the meristem zone, similar to the pattern of soluble boron distribution predicted by the model. Our model also predicts that upward boron flux does not continuously increase from the root tip toward the mature region, indicating that boron taken up in the root tip is not efficiently transported to shoots. This suggests that root tip-absorbed boron is probably used for local root growth, and that instead it is the more mature root regions which have a greater role in transporting boron toward the shoots.

ROW1 maintains quiescent centre identity by confining WOX5 expression to specific cells.

The quiescent centre (QC) in the Arabidopsis root apical meristem is essential for stem cell organization. Here we show that the loss of REPRESSOR OF WUSCHEL1 (ROW1), a PHD domain-containing protein, leads to QC failure, defects in cell differentiation and ectopic expression of WUSCHEL-RELATED HOMEOBOX 5 (WOX5) in cells that normally express ROW1. The wox5-1/row1-3 double mutants show similar phenotypes to wox5-1 indicating that WOX5 is epistatic to ROW1. ROW1 specifically binds trimethylated histone H3 lysine 4 (H3K4me3) in the WOX5 promoter region to repress its transcription. QC expression of ROW1 results in a wox5-1-like phenotype with undetectable WOX5 transcripts. We propose that ROW1 is essential for QC maintenance and for stem cell niche development through the repression of WOX5 in the proximal meristem.

Plant development. Genetic control of distal stem cell fate within root and embryonic meristems.

The root meristem consists of populations of distal and proximal stem cells and an organizing center known as the quiescent center. During embryogenesis, initiation of the root meristem occurs when an asymmetric cell division of the hypophysis forms the distal stem cells and quiescent center. We have identified NO TRANSMITTING TRACT (NTT) and two closely related paralogs as being required for the initiation of the root meristem. All three genes are expressed in the hypophysis, and their expression is dependent on the auxin-signaling pathway. Expression of these genes is necessary for distal stem cell fate within the root meristem, whereas misexpression is sufficient to transform other stem cell populations to a distal stem cell fate in both the embryo and mature roots.