PLETHORA‐WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance
2022; Springer Nature; Volume: 23; Issue: 6 Linguagem: Inglês
10.15252/embr.202154105
ISSN1469-3178
AutoresRebecca C Burkart, Vivien I. Strotmann, Gwendolyn K. Kirschner, Abdullah Akinci, Laura Czempik, Anika Dolata, Alexis Maizel, Stefanie Weidtkamp‐Peters, Yvonne Stahl,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle4 April 2022Open Access Source DataTransparent process PLETHORA-WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance Rebecca C Burkart Rebecca C Burkart orcid.org/0000-0003-0015-5312 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Vivien I Strotmann Vivien I Strotmann orcid.org/0000-0003-4822-6706 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Gwendolyn K Kirschner Gwendolyn K Kirschner Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Abdullah Akinci Abdullah Akinci Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Laura Czempik Laura Czempik Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Anika Dolata Anika Dolata Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation, Visualization Search for more papers by this author Alexis Maizel Alexis Maizel orcid.org/0000-0001-6843-1059 Center for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany Contribution: Investigation, Visualization Search for more papers by this author Stefanie Weidtkamp-Peters Stefanie Weidtkamp-Peters orcid.org/0000-0001-7734-3771 Center for Advanced Imaging, Heinrich-Heine University, Düsseldorf, Germany Contribution: Validation Search for more papers by this author Yvonne Stahl Corresponding Author Yvonne Stahl [email protected] orcid.org/0000-0002-7543-5186 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Rebecca C Burkart Rebecca C Burkart orcid.org/0000-0003-0015-5312 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Vivien I Strotmann Vivien I Strotmann orcid.org/0000-0003-4822-6706 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Gwendolyn K Kirschner Gwendolyn K Kirschner Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Abdullah Akinci Abdullah Akinci Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Laura Czempik Laura Czempik Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation Search for more papers by this author Anika Dolata Anika Dolata Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Investigation, Visualization Search for more papers by this author Alexis Maizel Alexis Maizel orcid.org/0000-0001-6843-1059 Center for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany Contribution: Investigation, Visualization Search for more papers by this author Stefanie Weidtkamp-Peters Stefanie Weidtkamp-Peters orcid.org/0000-0001-7734-3771 Center for Advanced Imaging, Heinrich-Heine University, Düsseldorf, Germany Contribution: Validation Search for more papers by this author Yvonne Stahl Corresponding Author Yvonne Stahl [email protected] orcid.org/0000-0002-7543-5186 Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Rebecca C Burkart1, Vivien I Strotmann1, Gwendolyn K Kirschner1,4, Abdullah Akinci1, Laura Czempik1,5, Anika Dolata1, Alexis Maizel2, Stefanie Weidtkamp-Peters3 and Yvonne Stahl *,1 1Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany 2Center for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany 3Center for Advanced Imaging, Heinrich-Heine University, Düsseldorf, Germany 4Present address: Biological and Environmental Sciences and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia 5Present address: Molecular Plant Science/Plant Biochemistry, University of Wuppertal, Wuppertal, Germany *Corresponding author. Tel: +49 211 81 15809; E-mail: [email protected] EMBO Reports (2022)23:e54105https://doi.org/10.15252/embr.202154105 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination. SYNOPSIS Stem cell maintenance in the Arabidopsis root is controlled by transcription factor complexes containing PLETHORAs and WOX5. Their localization in nuclear bodies (NBs), dependent on the differentiation status, controls cell fate determination. PLETHORA-WOX5 interaction and subnuclear localization regulate root stem cell fate. Transcription factors WOX5 and PLTs control QC quiescence and CSC fate interdependently. Intrinsically disordered prion-like domains (PrDs) in PLTs recruit PLT-WOX5 complexes to subnuclear microdomains. Partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination. Introduction The root system of higher plants is essential for plant life, as it provides anchorage in the soil and access to nutrients and water. It arises from a population of long-lasting stem cells residing in a structure called root apical meristem (RAM) at the tip of the root. Within the Arabidopsis thaliana RAM, the stem cell niche (SCN) consists of on average four to eight slowly dividing cells, the QC cells, which act as a long-term reservoir and signaling center by maintaining the surrounding shorter-lived, proliferating stem cells (also called initials) in a non-cell autonomous manner (van den Berg et al, 1997; Lu et al, 2021). These stem cells continuously divide asymmetrically, thereby generating new stem cells that are still in contact with the QC. The hereby-produced daughter cells frequently undergo cell divisions and are shifted further away from the QC to finally differentiate into distinct cell fates. By this mechanism, the position of the stem cells in the root remains the same throughout development and their precise orientation of division leads to the formation of concentrically organized clonal cell lineages representing a spatio-temporal developmental gradient (Dolan et al, 1993; van den Berg et al, 1997; Benfey & Scheres, 2000). From the inside to the outside, the following root cell tissues develop: vasculature, pericycle, endodermis, cortex, and epidermis plus columella and lateral root cap at the distal root tip (Fig 1A). Figure 1. WOX5 positively regulates PLT3 expression A. Schematic representation of the Arabidopsis root meristem. The QC cells (red) maintain the surrounding stem cells (initials) outlined in black together building the root stem cell niche (SCN). The different cell types are color coded. QC = quiescent center (red); CSC = columella stem cells (yellow); CC = columella cells (green); LRC = lateral root cap (light purple); ep = epidermis (purple); c = cortex (light turquoise); en = endodermis (dark turquoise); bright turquoise = cortex/endodermis initials; dark purple = epidermis/lateral root cap initials; dark orange = stele initials; stele = light orange; grey dots = starch granules. B, C. Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D. Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100%. E, F. Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. G. Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. Data information: (D, G) Box = 25–75% of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, X = minimum/maximum. The data were statistically analyzed by one-way ANOVA and Holm–Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analyzed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. (B, C, E, F) Scale bars represent 10 µm. SCN = stem cell niche; PI = propidium iodide; YFP = yellow fluorescent protein; CFP = cyan fluorescent protein. Download figure Download PowerPoint The necessary longevity and continuous activity of the RAM can only be achieved if its stem cell pool is constantly replenished, since cells are frequently leaving the meristematic region due to continuous cell divisions. Therefore, complex regulatory mechanisms involving phytohormones and key TFs regulate stem cell maintenance and the necessary supply of differentiating descendants (Drisch & Stahl, 2015). Here, the APETALA2-type PLT TF family and the homeodomain TF WOX5 play important roles (Aida et al, 2004; Sarkar et al, 2007). WOX5 is expressed mainly in the QC, but maintains the surrounding stem cells non-cell-autonomously by repressing their differentiation (Sarkar et al, 2007; Pi et al, 2015). Loss of WOX5 causes the differentiation of the CSCs, also called distal stem cells, into starch-accumulating columella cells (CCs), while increased WOX5 expression causes CSC over-proliferation. Hence, WOX5 abundance is critical and necessary to suppress premature CSC differentiation (Sarkar et al, 2007; Pi et al, 2015). WOX5 also represses QC divisions, maintaining the quiescence of the QC by repressing CYCLIN D (CYCD) activity within the QC (Forzani et al, 2014). The auxin-induced PLTs form a clade of six TFs and act as master regulators of root development, as multiple plt mutants fail to develop functional RAMs (Aida et al, 2004; Galinha et al, 2007; Mähönen et al, 2014). PLT1, 2, 3, and 4 are expressed mainly in and around the QC and form an instructive gradient, which is required for maintaining the balance of stem cell fate and differentiation. This PLT gradient is also necessary for separating auxin responses in the SCN, for the correct positioning of the QC, and the expression of QC markers (Aida et al, 2004; Galinha et al, 2007; Mähönen et al, 2014). Genetically, WOX5 and PLT1 were shown to play an interconnected role in auxin-regulated CSC fate, whereas PLT1 and PLT3 were found to directly positively regulate WOX5 expression (Ding & Friml, 2010; Shimotohno et al, 2018). Although PLTs and WOX5 are known for controlling stem cell regulation and maintenance in the Arabidopsis RAM and genetic evidence for cross regulation exists, the underlying molecular mechanisms are until now largely elusive. Here, we show for the first time that the mutual regulation of expression, but importantly also the ability of PLTs to directly interact with and recruit WOX5 to NBs in CSCs controls stem cell homeostasis in the Arabidopsis RAM. NBs are membrane-less, self-assembling protein/RNA containing compartments thought to regulate a variety of physiological responses to differential environmental cues like light, temperature, or osmotic changes (Mao et al, 2011; Jung et al, 2020; Meyer, 2020). Therefore, we propose a model in which differential PLT/WOX5 complexes depending on their subnuclear localization in NBs or in the nucleoplasm regulate stem cell fate in the RAM, possibly by phase separation. Results WOX and PLTs regulate each other's expression in the root SCN WOX5 and PLTs are essential players in distal stem cell maintenance (Aida et al, 2004; Galinha et al, 2007; Sarkar et al, 2007; Pi et al, 2015). This, as well as their overlapping expression and protein localization domains in the root SCN raised the question if they could act together in distal stem cell regulation, where, in comparison to all the other PLTs, particularly PLT3 is highly expressed (Fig 1B) (Galinha et al, 2007). Furthermore, PLT3 was recently predicted as one of the central nodes regulating other QC-enriched TFs in the underlying gene regulatory network (GRN) within the Arabidopsis root SCN. In contrast, PLT1 and PLT2 were predicted as minor nodes only and PLT4 (BBM) was not predicted as a node (de Luis Balaguer et al, 2017). First, we tested if WOX5 influences PLT3 expression. Both a transcriptional and translational PLT3 fluorescent reporter line showed a reduced expression in the QC and CSC in a wox5 mutant background to around 57–70% compared to the Col-0 (Col) wild-type roots (Fig 1B–G, Appendix Table S3). Next, we addressed, if PLT3 expression is regulated directly or indirectly upon WOX5 induction by using the published Arabidopsis lines 35S::WOX5-GR (Sarkar et al, 2007) and 35S::WOX5-GFP-GR (Berckmans et al, 2020) in quantitative PCR experiments (qPCR) (Appendix Fig S1A, Appendix Table S1) and crosses with pPLT3::erCFP (Galinha et al, 2007) (Appendix Fig S1B–E, Appendix Table S2), respectively. In both independent experiments, we found no change of PLT3 expression 4 h after WOX5 induction. After 21 h of WOX5 induction, we found PLT3 expression significantly upregulated up to two-fold and therefore, we conclude that PLT3 expression is not directly regulated by WOX5 (Appendix Fig S1B–E, Appendix Table S2). This extends the previously reported regulation of PLT1 expression by WOX5 (Ding & Friml, 2010) and shows that WOX5 positively regulates expression of several PLTs, albeit in an indirect manner. To test if WOX5 expression also depends on PLTs, we produced a transcriptional reporter, which expresses a nuclear-localized mVenus under control of the WOX5 promoter. In agreement with previous reports, expression of WOX5 in our transcriptional reporter line is confined to the QC and is only weakly expressed in the stele initials (Sarkar et al, 2007; Pi et al, 2015) (Fig 2A). Figure 2. PLTs constrain the WOX5 expression domain A–F. Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3, and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. (E′, F′) Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. (E″, F″) Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G. Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H. Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. Data information: (G, H) Box = 25-75% of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, X = minimum/maximum. (G, H) Kruskal–Wallis ANOVA with subsequent Dunn's test (G) or one-way ANOVA and post-hoc Holm–Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analyzed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localization signal. Download figure Download PowerPoint PLTs are known for their redundant function in SCN maintenance, that can be very strong especially when PLT1 is mutated in combination with other PLTs (Aida et al, 2004; Galinha et al, 2007). Because we aimed to look at the rather subtle QC and distal SCN phenotypes, we therefore included only plt2 mutants for our analyses. In plt2 and plt3 single mutants, we observed additional mVenus-expressing cells in the QC region, which may derive from aberrant periclinal cell divisions of the QC (Fig 2B and C, Appendix Table S4). This effect is even stronger in the plt2, plt3 double mutant roots, where extra cells are found in all observed roots and often even form an additional cell layer of WOX5 expressing cells (Fig 2D). Previously, it was reported that the Arabidopsis wild-type QC is composed of four to eight cells with a low division rate (Truernit et al, 2008; Cruz-Ramírez et al, 2013; Stahl et al, 2013; Lu et al, 2021). We quantified the number of WOX5 expressing cells and the area of WOX5 expression per root by acquiring transverse optical sections through the roots. We observed four to ten WOX5 expressing cells in the Col wild type (Fig 2E and G, Appendix Table S4), whereas we found eight to 14 WOX5 expressing cells and a laterally expanded WOX5 expression domain in the plt2, plt3 double mutants (Fig 2F–H, Appendix Table S4). Taken together, our data show that WOX5 positively regulates PLT expression, here shown for PLT3, whereas PLT2 and PLT3 redundantly restrict WOX5 expression to a limited number of cells at QC position, possibly by negative feedback regulation. These observations are in agreement with a previous report, where a role for PLT1 and PLT2 in confining WOX5 expression was reported (Sarkar et al, 2007). A novel SCN staining method for simultaneous QC division and CSC differentiation analyses QC cells rarely divide as they provide a long-term reservoir to maintain the surrounding stem cells (Cruz-Ramírez et al, 2013; Vilarrasa-Blasi et al, 2014). As WOX5 and PLTs control QC cell divisions and CSC maintenance (Aida et al, 2004; Galinha et al, 2007; Sarkar et al, 2007; Forzani et al, 2014; Mähönen et al, 2014; Pi et al, 2015), we asked if these two aspects are interdependent. Therefore, we analyzed the cell division rates in the QC and the CSC phenotypes in wild-type and mutant roots. To assess these two phenotypes and to probe for their interdependency, we needed to measure the number of dividing QC cells and CSC layers within the same root simultaneously. To enable this, we established a novel staining method, named SCN staining, by combining the 5-ethynyl-2′-deoxyuridine (EdU) and modified pseudo Schiff base propidium iodide (mPS-PI) stainings to simultaneously visualize cell divisions, starch granule distribution as well as cell walls within the same root (Truernit et al, 2008; Schiessl et al, 2012§; Cruz-Ramírez et al, 2013). Applying this new staining combination, potential correlations between QC-divisions and CSC cell fates can be uncovered. The EdU-staining is used to analyze QC-divisions by staining nuclei that have gone through the S-phase, detecting cells directly before, during, and after cell division (Cruz-Ramírez et al, 2013). However, cell layers and different cell types are hard to distinguish using only EdU staining due to the lack of cell wall staining. Therefore, we additionally applied the mPS-PI-method to stain cell walls and starch which is commonly used for CC and CSC cell fate determination (Truernit et al, 2008; Stahl et al, 2009, 2013). CCs are differentiated, starch granule-containing cells in the distal part of the root mediating gravity perception. They derive from the CSCs that form one or, directly after cell division, two cell layers distal to the QC. The CSCs lack big starch granules and can thereby easily be distinguished from the differentiated CCs by mPS-PI staining (Truernit et al, 2008; Stahl et al, 2009, 2013) (see Fig 3A, B and I, Appendix Table S5). Figure 3. plt and wox5 mutants show more CSC differentiation and QC divisions A. Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI (grey) and EdU (red) staining for 24 h (SCN staining) to analyze the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B–H. Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowheads. I. Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J. Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K–Q. Representative images of transversal sections with QC cells outlined in yellow. R. Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥ 4 dividing QC cells are plotted as bar graphs. Data information: Number of analyzed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU = 5-ethynyl-2′-deoxyuridine, scale bars represent 5 µm. Source data are available online for this figure. Source Data for Figure 3 [embr202154105-sup-0004-SDataFig3.xlsx] Download figure Download PowerPoint QC division rate and CSC differentiation correlate in the root SCN WOX5 was shown to be necessary for CSC maintenance, as loss of WOX5 causes their differentiation, while inducible overexpression of WOX5 leads to enhanced proliferation (Sarkar et al, 2007; Pi et al, 2015; Berckmans et al, 2020; Savina et al, 2020). In agreement with this, we found that the wox5 mutants lack a starch-free cell layer in 78% of analyzed roots, indicating differentiation of the CSCs, compared to 17% in Col (Fig 3A, B, F and I, Appendix Table S5). In the plt2 and plt3 single mutants, the frequency of roots lacking a CSC layer increases to above 30% (36 and 32%, respectively), and in the plt2, plt3 double mutant to 41% (see Fig 3C–E and I, Appendix Table S5). After overexpression of PLT3-mV by estradiol induction in wild-type Col-0 background, we observed the opposite effect, an increase from 29 to 50% of two CSC layers (see Appendix Fig S2A–F). Therefore, we argue, that the observed CSC phenotypes are due to PLT3 function and are not caused by potential early embryonic defects described previously for multiple plt mutants (Aida et al, 2004). Interestingly, the wox5, plt3 double mutant as well as the wox5, plt2, plt3 triple mutant show a frequency of differentiated CSCs comparable to the wox5 single mutant (71 and 77%, respectively) (Fig 3G–I, Appendix Table S5). This data suggests that PLTs and WOX5 may act together in the same pathway to maintain CSC homeostasis, as there is no additive effect observable in the multiple mutant roots. To analyze QC division phenotypes in detail, we quantified the number of EdU-stained cells in QC position in transversal optical sections. QC cells were identified by their position within the root SCN, as they are located directly distal to the stele initials and surrounded by the CEIs in a circular arrangement (Fig 3A and J). In Col, 27% of the analyzed roots show at least one cell division in the QC within the 24 h staining window (Fig 3J, K and R, Appendix Table S5), which is consistent with already published frequencies (Cruz-Ramírez et al, 2013). This frequency almost doubles to 45–50% in the plt2 and plt3 single mutants and is even higher in the plt2, plt3 double mutant (57%) (Fig 3L–N and R, Appendix Table S5). Additionally, the plt double mutant roots often show disordered QC regions with a disruption of the circular arrangement of cells surrounding the QC (Fig 3N) which could be a result of uncontrolled divisions. wox5 mutants show a disordered SCN accompanied by a high overall QC cell division frequency of at least one dividing QC cell in 92% of roots (Fig 3O and R) and on average more dividing QC cells per root (Appendix Table S5). The number of dividing QC cells per root increases further in the wox5, plt3 double mutant and is even higher in the wox5, plt2, plt3 triple mutant; here, in one third of the roots all QC cells undergo cell division (Fig 3P–R, Appendix Table S5). Taken together, this data implies an additive effect of PLT2, PLT3, and WOX5 regarding the QC-division phenotype, suggesting that WOX5 and PLTs act in parallel pathways to maintain the quiescence of the QC. Additionally, we quantified roots showing at least one aberrant periclinal cell division in the QC in longitudinal optical sections (Fig EV1). Whereas the occurrence of these aberrant periclinal divisions in Col wild-type roots is very rare (3%) (Fig EV1A), it increases in the plt-single mutants to 21% and in wox5 and wox5, plt3 mutants to around 40% (Fig EV1B and C). We found the most severe phenotypes in the plt2, plt3 double and wox5, plt2, plt3 triple mutants with an occurrence of periclinal QC-cell divisions in 53% of the observed roots, indicating a synergistic regulatory role of PLTs in periclinal QC cell divisions (Fig EV1B and C, Appendix Table S6). Click here to expand this figure. Figure EV1. plt and wox5 mutants show more periclinal cell divisions in the QC A. Representative figure of an Arabidopsis wild-type root SCN staining. QC cells are outlined in yellow. Scale bars represent 10 µm. B. Representative figure of an Arabidopsis plt2, plt3 double mutant root SCN staining showing a periclinal cell division (PCD) in the QC (arrow). QC cells are outlined in yellow. Scale bars represent 10 µm. C. Analysis of the PCD phenotype. The frequency of roots (in percent) showing at least one PCD in the QC is plotted as a bar graph. Number of analyzed roots (n) (biological replicates) is indicated for each genotype and results from 2 to 5 technical replicates. PCD = periclinal cell division. Download figure Download PowerPoint 2D plots of SCN staining facilitate assessment of root phenotypes To visualize correlations of QC division and CSC differentiation, we combined the acquired data in 2D-plots in which the frequencies of the two phenotypes are color-coded (Fig 4). This visualization reveals a regular pattern for Col wild-type roots, which peaks at one CSC layer and no QC divisions (Fig 4A). The pattern of the plt single mutants is more irregular with a shift to less CSC layers (indicating more differentiation) and more EdU-stained QC cells (indicating more QC divisions) compared to the wild-type Col roots (Fig 4B and C). The plt2, plt3 double mutants have an additional maximum at a position showing no CSC layer and one divided QC cell, resulting in two phenotypic populations, one at a wild-type-like position, the other showing a strong mutant phenotype (Fig 4D). The 2D-pattern for the wox5 mutant shifts to less CSC-layers and more QC-divisions with a maximum at no CSC-layers and two QC-divisions (Fig 4E). The QC phenotype is more severe in the wox5, plt3 double mutant towards more cell divisions and is even stronger in the wox5, plt2, plt3 triple mutant which peaks at zero CSC layers and three QC-divisions (Fig 4F and G). In summary, our data acquired by applying the novel SCN staining demonstrates that higher CSC differentiation correlates with a higher division rate in the QC, possibly to replenish missing stem cells by increased QC divisions. Figure 4. QC divisions correlate negatively with the number of CSC layers A–G. The combined results of the SCN staining in Fig 3 are shown as 2D plots to visualize the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on
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