The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression
2013; Springer Nature; Volume: 32; Issue: 21 Linguagem: Inglês
10.1038/emboj.2013.216
ISSN1460-2075
AutoresAdriana Garay‐Arroyo, Enrique Ortiz-Moreno, María de la Paz Sánchez, Angus Murphy, Berenice García‐Ponce, Nayelli Marsch‐Martínez, Stefan de Folter, Adriana Corvera-Poiré, Fabiola Jaimes‐Miranda, Mario A. Pacheco-Escobedo, Joseph Dubrovsky, Soraya Pelaz, Elena Álvarez‐Buylla,
Tópico(s)Plant Reproductive Biology
ResumoArticle11 October 2013free access Source Data The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression Adriana Garay-Arroyo Adriana Garay-Arroyo Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Enrique Ortiz-Moreno Enrique Ortiz-Moreno Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author María de la Paz Sánchez María de la Paz Sánchez Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Angus S Murphy Angus S Murphy Department Plant Science and Landscape Architecture. 2104 Plant Science Bldg. University of Maryland. College Park, USA Search for more papers by this author Berenice García-Ponce Berenice García-Ponce Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Nayelli Marsch-Martínez Nayelli Marsch-Martínez Departamento de Biotecnologia y Bioquimica, CINVESTAV-IPN Unidad Irapuato, Irapuato, México Search for more papers by this author Stefan de Folter Stefan de Folter Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Irapuato, México Search for more papers by this author Adriana Corvera-Poiré Adriana Corvera-Poiré Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Fabiola Jaimes-Miranda Fabiola Jaimes-Miranda Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Mario A Pacheco-Escobedo Mario A Pacheco-Escobedo Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Joseph G Dubrovsky Joseph G Dubrovsky Depto. de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, UNAM., Cuernavaca, México Search for more papers by this author Soraya Pelaz Soraya Pelaz Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB, Barcelona, Spain ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, Spain Search for more papers by this author Elena R Álvarez-Buylla Corresponding Author Elena R Álvarez-Buylla Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, MéxicoPresent address: 431 Koshland Hall, University of California, Berkeley, CA 94720, USA. Search for more papers by this author Adriana Garay-Arroyo Adriana Garay-Arroyo Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Enrique Ortiz-Moreno Enrique Ortiz-Moreno Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author María de la Paz Sánchez María de la Paz Sánchez Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Angus S Murphy Angus S Murphy Department Plant Science and Landscape Architecture. 2104 Plant Science Bldg. University of Maryland. College Park, USA Search for more papers by this author Berenice García-Ponce Berenice García-Ponce Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Nayelli Marsch-Martínez Nayelli Marsch-Martínez Departamento de Biotecnologia y Bioquimica, CINVESTAV-IPN Unidad Irapuato, Irapuato, México Search for more papers by this author Stefan de Folter Stefan de Folter Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Irapuato, México Search for more papers by this author Adriana Corvera-Poiré Adriana Corvera-Poiré Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Fabiola Jaimes-Miranda Fabiola Jaimes-Miranda Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Mario A Pacheco-Escobedo Mario A Pacheco-Escobedo Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México Search for more papers by this author Joseph G Dubrovsky Joseph G Dubrovsky Depto. de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, UNAM., Cuernavaca, México Search for more papers by this author Soraya Pelaz Soraya Pelaz Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB, Barcelona, Spain ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, Spain Search for more papers by this author Elena R Álvarez-Buylla Corresponding Author Elena R Álvarez-Buylla Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, MéxicoPresent address: 431 Koshland Hall, University of California, Berkeley, CA 94720, USA. Search for more papers by this author Author Information Adriana Garay-Arroyo1,‡, Enrique Ortiz-Moreno1,‡, María de la Paz Sánchez1, Angus S Murphy2, Berenice García-Ponce1, Nayelli Marsch-Martínez3, Stefan de Folter4, Adriana Corvera-Poiré1, Fabiola Jaimes-Miranda1, Mario A Pacheco-Escobedo1, Joseph G Dubrovsky5, Soraya Pelaz6,7 and Elena R Álvarez-Buylla 1 1Depto. de Ecología Funcional. Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Ext. Junto a J. Botánico, Ciudad Universitaria, UNAM, México DF, México 2Department Plant Science and Landscape Architecture. 2104 Plant Science Bldg. University of Maryland. College Park, USA 3Departamento de Biotecnologia y Bioquimica, CINVESTAV-IPN Unidad Irapuato, Irapuato, México 4Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Irapuato, México 5Depto. de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, UNAM., Cuernavaca, México 6Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB, Barcelona, Spain 7ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, Spain ‡These authors contributed equally to this work. *Corresponding author. ER Alvarez-Buylla, Instituto de Ecología, Universidad Nacional Autónoma de México, 3er Circuito Exterior, Junto a Jardín Botánico, CU, Coyoacán, México DF 04510, México. Tel.:+52 55 5622 9013; Fax:+52 55 5622 9013; E-mail: [email protected] The EMBO Journal (2013)32:2884-2895https://doi.org/10.1038/emboj.2013.216 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 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. Introduction Plant and animal development is guided by transcriptional networks that control cell-fate decisions in conjunction with dynamic patterning modules, such as those that regulate the differential distribution (gradients) of hormones (Davidson and Erwin, 2006; Newman et al, 2009). Establishing the molecular links between such networks and hormone or nutrient distribution is a key for understanding cell patterning. Our current comprehension of how these two types of processes are linked spatially and temporally in vivo is limited (Newman et al, 2009). The root of Arabidopsis thaliana has become an exemplar for in vivo studies of molecular developmental mechanisms, particularly of the molecular basis of stem-cell niche patterning and dynamics (van den Berg et al, 1997; Aida et al, 2004; Sarkar et al, 2007; Fulcher and Sablowski, 2009). The root stem-cell niche is a part of the cell proliferation domain (Ivanov and Dubrovsky, 2013) and, like that of other multicellular organisms, has in the centre an organizer, called the Quiescent Centre (QC) in roots. Tissue-specific progenitor cells, multipotent stem cells or initial cells, surround the QC. The derivatives of the initial cells exhibit a brief period of rapid cell proliferation shootward of the root apical region. After 4–6 rounds of division, cells begin elongation and ultimately differentiation (Dolan et al, 1993; van den Berg et al, 1998). Expression of GRAS family SCARECROW and SHORTROOT (SCR/SHR) and AP2 family PLETHORA (PLT) transcription factors is necessary for the formation and maintenance of the stem-cell niche in Arabidopsis roots (Sabatini et al, 1999, 2003; Helariutta et al, 2000; Aida et al, 2004). Auxins are fundamental plant hormones in embryonic development (Möller and Weijers, 2009), organogenesis (Vanneste and Friml, 2009), and root cell patterning (Friml et al, 2003). An auxin gradient with a maximum level at the QC is required for correct specification of the stem-cell niche. Moreover, the cellular levels of auxin define root cell fate: intermediate auxin concentrations are found in the cell proliferation zone and low auxin levels characterize the zones of elongation and differentiation along the root longitudinal axis (Burstrom, 1957; Friml and Palme, 2002; Petersson et al, 2009; Jurado et al, 2010). Directional auxin transport between cells, partially mediated by PIN auxin-efflux carriers, is crucial for generating these auxin gradients (Blilou et al, 2005; Vieten et al, 2005; Wisniewska et al, 2006). Differential PIN expression has been explored extensively (Aida et al, 2004; Vieten et al, 2005; Ruzicka et al, 2009), but little is known about the factors that directly regulate PIN gene transcription or PIN expression in response to auxins, as well as how this regulation impacts PIN-dependent developmental processes. Initial studies suggested that the PLT and SHR/SCR genes constituted the main components of root stem-cell niche patterning; based on the recent gene discoveries and a new gene regulatory network (GRN) model it is hypothesized that additional components remain undiscovered (Sarkar et al, 2007; Welch et al, 2007; Azpeitia et al, 2010). Plant MADS-box genes have been extensively characterized as regulators of reproductive development, flower transition, and organ identity (Coen and Meyerowitz, 1991; Álvarez-Buylla et al, 2000a; Álvarez-Buylla et al, 2000b; Burgeff et al, 2002), and recent studies suggest that auxin-regulated MADS-box genes, such as XAANTAL1/AGL12 (XAANTAL is the Mayan word for ‘go slower’ in recognition of the retarded root growth phenotypes of xaantal mutants), also regulate root development (Gan et al, 2005; Tapia-López et al, 2008). We report here that the Arabidopsis MADS-box gene, AGL14/XAANTAL2, closely related to the flowering gene, SUPPRESOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (Martinez-Castilla and Alvarez-Buylla, 2003) is required for root stem-cell niche and meristem patterning. Furthermore, AGL14/XAL2 regulates auxin transport and gradients in the root via direct regulation of PIN transcription. Results XAANTAL2 (XAL2/AGL14) controls root stem-cell niche delimitation and cell proliferation We sought to test the role of MADS-box gene XAL2 in Arabidopsis root development. We obtained lines with loss-of-function mutations in XAL2, a type II MADS-box gene that belongs to the SOC1 clade (Figure 1A) and is highly expressed in roots (Figure 1B). ‘In situ’ data of RNA expression derived from both ‘in situ’ PCR and dig-labelled ‘in situ’ experiments demonstrated that mRNA is found in lateral root cap, epidermis, endodermis, and columella of the root meristematic region, as well as in the vascular cylinder in differentiated zones of the primary root and in emerged lateral root primordia (Figures 1C–H; Supplementary Figures S1B–H and S2). In addition, we generated a 1-kb XAL2 promoter that also recovers the expression of the gene promoter in the vascular tissue and in the lateral roots. This line recovers only a scanty and light expression in the root meristematic tissues after a few hours of GUS staining, maybe because the cloned promoter fraction misses some important enhancer sequences. This genetic marker is strongly expressed in the root meristem only after several days of staining (data not shown). Nonetheless, both this and the ‘in situ’ data show clear and strong expression of XAL2 in the central cylinder and the emerging lateral roots. These data parallel the reports by Birnbaum et al (2003) and Nawy et al (2005) for XAL2; who report expression in the meristematic tissues as our ‘in situ’ assays, and in the QC (see Supplementary Figure S1A). We isolated two loss-of-function alleles for this XAL2 (called hereafter xaantal2-1 and xaantal2-2). Both mutants develop shorter wild-type roots in the Columbia background, similar in length to XAL1 mutants (Figure 1I; Tapia-López et al, 2008). The xal2-2 allele exhibited the most pronounced retarded root growth and altered cellular structure at the stem-cell niche, and concordantly lacked detectable levels of XAL2 mRNA, whereas the xal2-1 allele exhibited intermediate phenotypes between wild type and xal2-2, and exhibited residual XAL2 expression (Figures 1I–K). The root growth and stem-cell niche phenotypes, as well as the correlated levels of expression of XAL2 in these two independent alleles demonstrate that the observed phenotype can be attributed to the loss of XAL2 function (Figure 2A). For the rest of the functional analyses, we therefore focused on the strong allele xal2-2. Figure 1.XAL2, a MADS-box gene, is highly expressed in roots and affects root growth. (A) Bayesian reconstruction of the phylogenetic relationships among selected type II A. thaliana MADS-box genes, with XAL2 position indicated by an arrow. (B) RT–PCR used to visualize XAL2 expression in different tissues from 7 days post sowing (d.p.s.) in wild-type. Ubiquitin (UBI) was used as an internal control. (C–H) Whole-mount ‘in situ’ RT–PCR hybridization (see Materials and methods for details of procedures used) of XAL2 in columella cells and lateral root cap (C), in the vascular bundle in differentiated cells (E) and in the primordia of lateral roots (G). (D, F, and H) Negative sense controls (Bar=50 μm). (I) XAL2 gene structure schematic model with the sites of transposon insertions. Squares correspond to exons while lines represent introns. Root length phenotypes of seedlings at 7 d.p.s. of Col-0 wild type, xal2-1, and xal2-2 (n=60). Tubulin (TUB) was used as an internal control. (J) RT–PCR of XAL2 at 7 d.p.s. in wild type, xal2-1, and xal2-2. (K) Root growth curves of wild-type, xal2-1, and xal2-2 plants grown in medium supplemented with 2% sucrose for 10 days (n=30). Average and s.d. are shown. Download figure Download PowerPoint Figure 2.XAL2 controls meristem size and patterning of the root apical meristem. (A) Organization of the quiescent center and initial cells in the stem-cell niche from representative phenotypes for wild-type and XAL2 mutants in longitudinal root sections (100%; n=30) (Bar=50 μm). (B) Meristem length of wild-type and xal2-2 plants at 7 d.p.s.; the white line indicates the size of the meristematic region (Bar=50 μm). (C) Cell length of fully elongated cells (see red arrowheads) at 7 d.p.s. (n=30) in cleared roots observed with Nomarski optics (Bar=50 μm). (D) Cell production rate and length of completely elongated cortical differentiated cells; quantifications are described in detail in Materials and methods. All data were analysed with the JMP 5.1.1 version statistical package. Measurements were obtained directly by measuring 10 cortical cells from 10 plants. Average and s.d. are shown. (E) Nomarski photographs of wild-type and xal2-2 plants. The position of the QC is indicated (see white arrowhead) and white arrows indicate position of columella initial tiers. Lugol staining marks starch granules observed in mature wild-type and xal2-2 columella-differentiated cells (Bar=50 μm). (F) Percentage of one or two tiers of columella initials (CI) in wild-type plants and xal2-2 mutants (n=56 for wild type; n=69 for xal2-2). Download figure Download PowerPoint Quantitative growth analyses of xal2-2 roots (Table I) showed that the mutant displays a significantly lower number of meristematic cells and lower rate of cell production, fewer cells in the elongation zone and shorter fully elongated cells compared with wild-type roots (Figures 2B–D; Table I) and the xal2-2 line exhibits normal cell-cycle duration and pCYCB1;1DB::GUS expression (Supplementary Figures S3A and B). Consequently, decreased root growth in plants with this strong loss-of-function xal2-2 allele results from a combination of decreased cell production and shorter cell length in the differentiation zone. The diminished size of the completely elongated cells observed in xal2-2 accounts for up to 86% of the decrease in root length in the mutant, while only 14% of such length decrease could be attributed to changes in a number of cells in the root apical meristem (RAM) proliferation domain (Table I). In addition, we documented that the diameter of the stele (provascular tissues) of wild-type plants is not larger (see Supplementary Figures S4A–E), in the root meristem compared to that of xal2-2 plants. In wild type, a greater number of pericycle cells are present in the transverse plane of the wild-type roots in comparison to the mutant ones (14.8 cells in wild type and 12.0 cells in xal2-2, Supplementary Figures S4C, D, and F). It is important to note, that wild-type and xal2-2 mutant lines had roots and steles with the same width also in the differentiation zone (see Supplementary Figure S4G). We can conclude that xal2-2 loss-of-function mutation significantly affects the proliferation of the pericycle cells, particularly the radial anticlinal divisions at the fifth cortical cell. Table 1. Quantitative cellular analysis of root development for wild-type and xal2-2 seedlings (7 d.p.s.) Root growth rate (μm h−1) Proliferation domain (PD) length (μm) Cortical cell number within PD Cortical cell length within PD (μm) Combined length of the transition domain and the elongation zone (μm) ****** * **** ** WT 265±11 173±12 33.3±2.14 5.2±0.1 510±32 xal2-2 163±3 142±5 26.5±1.43 5.4±0.1 385±22 Length of the growing part of the root (μm) Length of completely elongated cortical cells (μm) Cell-cycle duration (h) Cell production rate (1/h) Cortical cell number in the elongation zone **** ***** ***** ** WT 683±32 156±6 12.3±0.7 1.9±0.1 20.1±1.0 xal2-2 528±22 113±4 12.7±1.03 1.5±0.6 16.5±1.12 Average (marked with bold letters)±s.d. with α<0.1, α<0.05, α<0.02, α<0.005, α<0.002, and α<0.001 indicated as *, **, ***, ****, *****, and ******, respectively; n=30. To determine whether XAL2 is necessary for QC identity and stem-cell niche patterning, we analysed whole mount optical and thin plastic sections of roots for each allele. We identified an altered stem-cell niche phenotype characterized by altered shapes and distributions of the QC, initial, and columella cells. XAL2 does not seem to be necessary for QC identity, as WOX5, SCR, and PLT1 (COL148 (plt1-1-GUS); Aida et al, 2004) were properly expressed in the xal2-2 mutants (Supplementary Figure S5) and xal2-2 roots continued growth until day 10, although at a lower rate than wild-type roots (Figure 1K). The domains of expression of the QC-specific markers QC25 and QC46 were abnormally expanded in xal2-2 mutants towards the columella cells and provascular initials, respectively (Figure 3A; Supplementary Figure S5), suggesting that XAL2 is necessary for restriction of the QC to its normal spatial domain. Expression of the columella initial marker J2341 was expanded as well in 25% of the xal2-2 mutant roots (Figure 3A), and in these plants a supernumerary columella layer lacking starch granules was observed (Figures 2E and F); the rest of the mutant roots did not show this phenotype. We interpret this as indicating a delayed transition to columellar cell differentiation. The above results suggest that XAL2 seems to be required for normal spatial organization and patterning of the root stem-cell niche, as well as for maintaining meristem homeostasis. Figure 3.XAL2 is necessary to restrict QC and auxin distribution to its normal spatial domain in Arabidopsis root tip. (A) Two different markers in wild-type and xal2-2 mutant plants: QC46 expression (95%; n=50) and specific enhancer trap J2341 expression (25%; n=30). In the latter marker, two tiers of columella initials were observed in xal2-2 (white arrows) and the position of the QC is indicated (white arrowhead). For this line, red signal is emitted by propidium iodide that was used as a counterstain. 7 d.p.s. plants were used for the experiment with QC46 transgenic lines and 5 d.p.s. plants for the J2341 marker (Bar=50 μm). (B) pDR5-GUS (90%; n=80) expression of wild-type and mutant xal2-2 plants with or without an auxin treatment (IAA 1 μM, 4 h) (Bar=50 μm). (C) Quantification of free IAA levels in the root of wild-type and xal2-2 plants (P<0.01, n=50). (D) Direct measurements of 3H-IAA transport in shootward transport capacity from the root apex to the first 2 mm section (P=0.052, n=10). (E) Direct measurements of 3H-IAA transport in rootward transport capacity in the hypocotyl to root–shoot transition zone (P<0.001, n=10). In (C–E), bars represent standard errors (s.e.) and the * indicates significant differences (P<0.01, n=50). Download figure Download PowerPoint XAL2 controls auxin transport and concentration Auxin is required for normal cell proliferation and elongation: intermediate auxin levels are associated with highest cell proliferation, while lower levels with cell elongation and differentiation along the root (Burstrom, 1957; Grieneisen et al, 2007; Jurado et al, 2010). Given the growth defects of xal2-2 mutant roots, we hypothesized that XAL2 could be important for maintaining auxin gradients along the root longitudinal axis. Visualization of the auxin-responsive DR5 promoter (Sabatini et al, 1999; Friml et al, 2003) reporter suggests that auxin levels or responses are elevated in the QC, provascular initials, and columella cells of xal2-2 mutants compared with normal siblings (Figure 3B). Moreover, exogeneous indole acetic acid (IAA) treatment of xal2-2 roots resulted in increased pDR5:GUS expression throughout the root (Figure 3B; Supplementary Figure S6A) compared with wild-type treated roots. We performed several experiments to distinguish whether these effects reflected alterations in downstream signalling, free auxin levels, or auxin transport. First, we tested several auxin responsive markers in the wild-type and xal2-2 plants with or without auxin treatment and found that all these markers were similarly induced by auxin treatment in both genotypes (Supplementary Figure S6B), implying that auxin response is not altered in the mutant. Next, direct quantification of free IAA levels in xal2-2 seedlings confirmed that auxin levels are significantly increased in the xal2-2 root compared with wild type (Figure 3C). A reduction in rootward 3H-IAA transport, similar to levels seen in pin1 mutants (Blakeslee et al, 2007), was also observed (Figure 3E). Diminished shootward transport from the root apex (Figure 3D) is consistent with the increased pDR5::GUS signal observed in xal2-2. These results indicated that XAL2 is a positive regulator of auxin transport towards and within the root. We then addressed whether such alterations were a consequence, at least in part, of the misregulation of the transcription of PIN auxin transporters, in the xal2-2 background. XAL2 regulates the transcript accumulation of several PIN genes Despite functional redundancy, each PIN protein has been implicated in particular developmental processes (Gälweiler et al, 1998; Luschnig et al, 1998; Friml et al, 2002a, 2002b, 2003; Mravec et al, 2009). For example, PIN4 has been associated with root meristem activity and patterning (Friml et al, 2002b). Interestingly, roots with pin4 loss-of-function alleles are strikingly similar to xal2-2 roots, with altered stem-cell niches, two files of columella initials, expanded expression domains of QC markers, and altered auxin gradients (Friml et al, 2002b). Hence, we addressed whether XAL2 is involved in regulation of PIN4 expression. We crossed xal2-2 with a pPIN4:GUS line to assay an impact on RNA accumulation and a pPIN4:PIN4-GFP translational fusion line. The progeny of these crosses displayed clearly diminished GUS and GFP signals, respectively (Figures 4A and B). Consistent with this result, PIN4 transcript abundance in response to auxin was also decreased by this visual assay in xal2-2 mutants compared to wild type (Figures 4A and D). Figure 4.XAL2 upregulates PIN1 and PIN4 gene expression and controls PIN4 auxin response. (A) pPIN4-GUS (100%; n=60) expression of wild-type and mutant xal2-2 plants with or without an auxin treatment (IAA 1 μM, 4 h) (Bar=50 μm). (B) pPIN4:PIN4-GFP (30%; n=65) expression in wild-type and xal2-2 mutant plants. In these lines, confocal image of longitudinal optical sections of root meristem and red signal is emitted by propidium iodide that was used as a counterstain (Bar=50 μm). (C) pPIN1-GFP (50%: n=80) expression in wild-type and xal2-2 mutant plants (Bar=50 μm). Quantification of the fluorescent signal of the pPIN1-GFP reporter (shown above) for wild-type and xal2-2 plants (n=57 for wild type and n=52 for xal2-2 plants) Average and s.d. are shown. (D) qRT-PCR analysis of PIN1, PIN2, PIN4, and ABCB19 normalized to actin expression in
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