PFT1, a transcriptional Mediator complex subunit, controls root hair differentiation through reactive oxygen species (ROS) distribution in Arabidopsis

Authors

  • Kalaipandian Sundaravelpandian,

    1. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
    2. Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, National Chung-Hsing University, Academia Sinica, Taipei, Taiwan
    3. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan
    Search for more papers by this author
  • Nulu Naga Prafulla Chandrika,

    1. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
    2. Molecular and Cellular Biology, Taiwan International Graduate Program, Academia Sinica, National Defense Medical Center, Taipei, Taiwan
    Search for more papers by this author
  • Wolfgang Schmidt

    Corresponding author
    1. Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, National Chung-Hsing University, Academia Sinica, Taipei, Taiwan
    2. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan
    3. Molecular and Cellular Biology, Taiwan International Graduate Program, Academia Sinica, National Defense Medical Center, Taipei, Taiwan
    4. Genome and Systems Biology Degree Program, College of Life Science, National Taiwan University, Taipei, Taiwan
    • Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
    Search for more papers by this author

Author for correspondence:

Wolfgang Schmidt

Tel: +886 2 2789 2997

Email: wosh@gate.sinica.edu.tw

Summary

  • Root hair morphogenesis is driven by an amalgam of interacting processes controlled by complex signaling events. Redox processes and transcriptional control are critical for root hair development. However, the molecular mechanisms that integrate redox state and transcription are largely unknown.
  • To elucidate a possible role of transcriptional Mediators in root hair formation, we analyzed the Arabidopsis root hair phenotype of T-DNA insertion lines that harbor homozygous mutations in genes encoding Mediator subunits.
  • Genetic evidence indicates that the Mediator subunits PFT1/MED25 and MED8 are critical for root hair differentiation, but act via separate mechanisms. Transcriptional profiling of pft1 roots revealed that PFT1 activates a subset of hydrogen peroxide (H2O2)-producing class III peroxidases. pft1 mutants showed perturbed H2O2 and superoxide (math formula) distribution, suggesting that PFT1 is essential to maintain redox homeostasis in the root. Chemical treatments rescued the pft1 mutant phenotype, indicating that correct reactive oxygen species (ROS) distribution is an essential prerequisite for root hair differentiation. In addition, PFT1 positively regulates cell wall remodeling genes that are essential for root hair formation.
  • Our results demonstrate that PFT1 maintains ROS distribution which, in turn, controls root hair differentiation. Thus, our findings reveal a novel mechanism in which the Mediator controls ROS homeostasis by regulating the transcriptional machinery.

Introduction

Root hairs, long tubular-shaped outgrowths from root epidermal cells, are an excellent model for the study of cell specification and differentiation. Their wide distribution among all major groups of vascular plants provides evidence for a long evolutionary history and their importance to the success of diverse plant groups in the adaptation to changing environmental conditions (Peterson & Farquhar, 1996). Root hairs increase the root surface area to aid plants in nutrient acquisition, anchorage and interactions with microbes (Grierson & Schiefelbein, 2002). In Arabidopsis, the specification of epidermal cells is biased by an as yet unidentified positional signal derived from the cortex that is perceived by the leucine-rich repeat (LRR) kinase SCRAMBLED (Kwak & Schiefelbein, 2008). The signal, which is supposedly stronger at epidermal cells that lie over the clefts of two underlying cortical cells, represses the expression of WEREWOLF (WER) and MYB23, which negatively regulate the root hair cell fate. A homeodomain protein, GLABRA2 (GL2), represses the hair cell fate by altering the cell developmental program in epidermal cells that occupy nonhair positions (i.e. that are located over tangential cell walls of cortical cells). After cell specification, root hair development is initiated by the formation of a cell wall bulge at the basal end of the cell. The third stage of root hair formation is characterized by the rapid elongation of the hair via tip growth. Tip growth is associated with the establishment of tip-focused Ca2+ gradients, extensive cytoskeleton reorganization, and the transport and deposition of cell wall materials. The restriction of growth at the hair tip is controlled by the reactive oxygen species (ROS)-generating NADPH oxidase ROOT HAIR DEFECTIVE2 (RHD2; AtrbohC) by activating Ca2+ channels (Foreman et al., 2003). The Rho GTPase GDP dissociation inhibitor SCN1/AtrhoGDI1 has been identified as a component that focuses the RHD2/AtrbohC-catalyzed generation of ROS to hair tips (Carol et al., 2005).

Cell wall expansion is a fundamental process in the morphogenesis of plant cells. Several cell wall-related proteins, such as CELLULOSE SYNTHASE LIKE D2 (CSLD2), CSLD3/RHD7, LEUCINE-RICH REPEAT EXTENSIN1 (LRX1) and PROLINE-RICH PROTEIN3 (PRP3), promote root hair formation (Bernhardt & Tierney, 2000; Baumberger et al., 2001; Favery et al., 2001; Bernal et al., 2008). CSLD3 is required for the organization of both cellulose and xyloglucans on root hair cell walls (Galway et al., 2011). Oxidative cross-linking of tyrosine (Tyr) residues in the extensin (EXT) domain is crucial for LRX1 function in the cell wall (Baumberger et al., 2001; Ringli, 2010). Peroxidases are known to mediate such cross-linking during plant defense reactions, and have also been predicted to form cross-links with EXTs during root hair formation (Almagro et al., 2009; Velasquez et al., 2011).

The redox state of the cell is crucial for cell cycle progression and is critical for the transition from cell proliferation to differentiation. In mammals, ROS levels have been found to dictate the fate and differentiation of cells (Owusu-Ansah & Banerjee, 2009). Decreased ROS levels in rhd2 root tip cells interfered with microtubule organization and induced macrotubule assembly in Arabidopsis (Livanos et al., 2012). In addition, disturbed ROS homeostasis caused atypical tubulin formation and affected mitosis in root tip cells of Triticum turgidum and Arabidopsis (Livanos et al., 2012). A molecular link between ROS distribution and transcription was revealed in a study on the UPBEAT1 (UPB1) transcription factor in Arabidopsis. UPB1 negatively regulates peroxidases that control the hydrogen peroxide (H2O2) and superoxide (math formula) balance, which are crucial for the transition from proliferation to differentiation in the Arabidopsis root (Tsukagoshi et al., 2010).

Transcriptional Mediators, central co-regulators of transcription, have been identified as large protein complexes in eukaryotes. They form a bridge between RNA polymerase II (RNA Pol II) and transcription factors to initiate transcription. The Mediator complex plays important roles in several developmental processes in yeast, Drosophila, mice and humans (Hentges, 2011). However, the function of Mediator subunits in plant development has only recently begun to be explored. Biochemical purification of plant Mediators identified 21 conserved and six Arabidopsis-specific Mediator subunits plus six paralogs (Backstrom et al., 2007). In Arabidopsis, Mediator subunits play diverse roles, including plant growth and development. For instance, STRUWWELPETER (SWP)/MED14 controls the cell number during primordia initiation and also regulates the duration of cell proliferation in aerial organs (Autran et al., 2002). The Mediator subunits MED12-MED13 have been shown to regulate developmental timing during embryo patterning (Gillmor et al., 2010). PHYTOCHROME AND FLOWERING TIME1 (PFT1)/MED25 has been shown to restrict cell growth, whereas MED8 acts independent of PFT1 to control organ growth (Xu & Li, 2011, 2012). Transcriptional Mediators and ROS regulate the transcription during growth and development in both plants and animals. However, no direct relationship has been implicated at the transcriptional level in either plants or animals.

In an attempt to uncover as yet unexplored roles of Mediators in plant developmental processes, we set out to explore a possible involvement of Mediator subunits in the morphogenesis of root hairs, a prime model for plant cell differentiation. We report that the transcriptional Mediator complex subunit PFT1 controls ROS balance in roots and is critical for root hair differentiation and elongation. From transcriptional profiling experiments, we conclude that PFT1 regulates peroxidase-mediated H2O2 formation to maintain the balance between H2O2 and math formula during root hair formation. Another Mediator subunit, MED8, is also involved in root hair formation, but acts independently of PFT1. Our results provide evidence for a control of root epidermal cell differentiation by the spatial distribution of ROS along the root.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana (L.) Heynh Columbia (Col-0) ecotype was used as the wild-type control. The mutant lines (Supporting Information Table S1) were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA). Homozygous lines were identified with the primer sequences provided by the Salk Institute (http://signal.salk.edu/tdnaprimers.2.html). The pft1-1 mutant and the pft1-1 complementation line (G1) have been described previously (Cerdan & Chory, 2003). The med8 pft1-2 double mutant was generated by genetic crossing. Homozygous double mutants were identified in the F2 generation by PCR using gene-specific primers. Plants were grown in a growth chamber on medium as described by Estelle & Somerville (1987). Seeds were surface sterilized and germinated in a medium containing KNO3 (5 mM), MgSO4 (2 mM), Ca(NO3)2 (2 mM), KH2PO4 (2.5 mM), H3BO3 (70 μM), MnCl2 (14 μM), ZnSO4 (1 μM), CuSO4 (0.5 μM), CoCl2 (0.01 μM), Na2MoO4 (0.2 μM) and FeEDTA (40 μM), solidified with 0.4% Gelrite pure (CP Kelco, Atlanta, Georgia, USA), 1.5% sucrose and 1 g l−1 MES hydrate. The pH was adjusted to 5.5. Seeds were sown on Petri plates and stratified for 1 d at 4°C in the dark before being transferred to a growth chamber and grown at 21°C under continuous illumination (50 μmol m−2 s−1). Plant roots were observed 7 d after germination for root hair phenotype.

Root hair density and length measurement

A ZEISS DISCOVERY V.12 microscope equipped with a scale bar in the eye piece was used to measure root hair density and length. Root hair density was measured from 2 to 4 mm from the tip of the primary root. At least 300 root hairs from 10 roots were measured for root hair length data. A microscope equipped with a ZEISS AXIOCAM MRC CCD camera was used to capture root hair pictures. Statistically significant deviations from the wild-type were determined by Student's t-test.

RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from roots of 7-d-old plants with an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Nucleic acid quantity was analyzed with a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). One microgram of RNA was incubated in gDNA Wipeout Buffer to remove contaminating genomic DNA. Quantiscript Reverse Transcriptase and RT primer mix were used for cDNA synthesis (Qiagen).

Real-time quantitative RT-PCR (qRT-PCR) was performed using double-stranded DNA binding dye POWER SYBR GREEN PCR master MIX (Applied Biosystems, Life Technologies, Taipei, Taiwan) in an ABI 7500 real-time PCR system. Each reaction was run in triplicate. Melting curves were analyzed using Dissociation Curves Software (Applied Biosystems) to ensure that only a single product was amplified. EF1A was used as an endogenous reference. Primers were designed by the Primer Express software (Applied Biosystems). Root samples were collected 7 d after germination with or without treatment. For PFT1 expression in response to ROS, Col-0 plants were germinated for 6 d and treated with 500 μM H2O2 and 1 mM KI for 24 h before sample collection. A list of qRT-PCR primers is provided in Table S2.

Microarray data analyses

The Affymetrix gene chip Arabidopsis ATH1 Genome Array was used for microarray analysis. The total RNA sample was prepared as described above. All RNA samples were quality assessed using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Complementary RNA synthesis was performed using the GeneChip One-Cycle Target Labeling Kit (Affymetrix). Hybridization, washing, staining and scanning procedures were performed as described in the Affymetrix technical manual.

Gene expression data were imported directly into GeneSpring (version 11.5; Agilent). The software was used to normalize the data per chip to the 50th percentile and per gene to the control samples. Genes that were flagged as absent in two replicates were not considered in the analysis. P values for the Westfall and Young permutation method were calculated by Genespring 11.5 (Agilent). Genes that exhibited more than two-fold induced or repressed transcript levels between the genotypes in two independent experiments with P ≤ 0.05 were defined as being differentially expressed. Gene ontology (GO) enrichment analysis was conducted with the Gene Ontology Browsing Utility (GOBU) software (Lin et al., 2006; http://www.openfoundry.org/of/projects/192/download).

Nitroblue tetrazolium (NBT) staining for math formula

Ten to 20 roots of 7-d-old seedlings were stained for 15 min in a solution of 2 mM NBT (Sigma-Aldrich) in 20 mM phosphate buffer, pH 6.1 (Dunand et al., 2007). Roots were mounted on a glass slide with a drop of water and a coverslip, and observed with a ZEISS AXIO IMAGER microscope equipped with a ZEISS AXIOCAM MRC CCD camera that was used to capture photographs. A histogram function from Adobe Photoshop was used to assess the NBT staining intensity of the elongation zone (0, white; 255, black). Roots from 7-d-old seedlings with or without chemical treatment were used.

Hydroxyphenyl fluorescein (HPF) staining for H2O2

Ten to 20 roots of 7-d-old seedlings were incubated for 2 min in 0.1 M phosphate buffer, pH 7.4, containing 5 μM 3′-(p-hydroxyphenyl) fluorescein (HPF; Sigma) (Setsukinai et al., 2003; Dunand et al., 2007). Roots were then mounted on a glass slide in a drop of buffer, covered and observed with a laser scanning microscope (ZEISS LSM 510 META). Fluorescence images were immediately captured on the elongation zone. An argon ion laser of 488 nm was used for excitation; the emission fluorescence was collected by the 500–550-nm bandpass for HPF fluorescence. The photographs were captured with ×10 magnification. Roots from 7-d-old seedlings with or without chemical treatment were used.

Chemical treatments

Chemical treatments were performed at the following concentrations: H2O2 (500 μM), KCN (75 μM), diphenyleneiodonium (DPI) (2.5 μM). Seeds were germinated on medium containing the chemicals for 7 d and the roots were used for further experiments. For the analysis of PFT1 expression in response to ROS treatment, seeds were germinated for 6 d and transferred to medium containing H2O2 (500 μM) and KI (1 mM) for 24 h.

Results

The Mediator subunits PFT1 and MED8 play critical roles in root hair morphogenesis

To elucidate whether Mediator subunits are essential for root hair development, we screened T-DNA lines harboring mutations in Mediator subunits for their root hair phenotypes. We obtained 52 mutant lines corresponding to 28 genes from the Arabidopsis Biological Resource Center (ABRC). Homozygous insertion lines were identified by PCR using specific primers (http://signal.salk.edu/tdnaprimers). Three mutant lines comprising two Mediator subunits, MED8 (SALK_092406) and MED25 (SALK_129555 and SALK_059316), showed a marked reduction in root hair number and length relative to the wild-type (Fig. 1a–c). MED25 has been described previously as PHYTOCHROME AND FLOWERING TIME1 (PFT1; Cerdan & Chory, 2003). The mutant lines SALK_129555 and SALK_059316 were named pft1-2 and pft1-3 by Kidd et al. (2009). PFT1 encodes a protein containing a von Willebrand factor type A (vWF-A) domain in the N-terminus and a glutamine-rich (Gln-rich) region in the C-terminus. The mutant lines pft1-2 and pft1-3 contain T-DNA insertions in the fifth and 14th exons, respectively, corresponding to the vWF-A and Gln-rich domains (Kidd et al., 2009).

Figure 1.

Mutations in Mediator subunits induce root hair-defective phenotypes. (a) Root hair zone of 7-d-old Arabidopsis seedlings. Bar, 100 μm. (b) Root hair density per millimeter; data are averages of 10 roots. (c) Root hair length; data are averages of 300 hairs; Error bars represent means ± SE.

To further confirm that the root hair defective phenotype was caused by a loss of function of PFT1, pft1-1 mutants that were transformed with a genomic copy of PFT1 (G1 complementation line; Cerdan & Chory, 2003) were included in the analysis. The severe root hair defects of pft1-1 plants were fully rescued in the G1 line, supporting a critical function of PFT1 in root hair differentiation (Fig. 1a–c). Similar to the pft1 mutants, the MED8 mutant line SALK_092406 (med8) showed severe defects in root hair development (Fig. 1a–c). Double mutants resulting from the crossing of med8 and pft1-2 showed an additive root hair defective phenotype, indicating independent effects of the mutations (Fig. 1a). The med8 pft1-2 double mutant developed only a few bulges, suggesting that root hair formation is inhibited at an early stage. Together, these results indicate that the Mediator subunits MED25 and MED8 are important for root hair morphogenesis in Arabidopsis.

PFT1 regulates the transcription of a suite of genes including class III peroxidases and NADPH oxidases

To catalog genes that are affected by PFT1, we conducted microarray experiments using roots of pft1-2 mutant plants and the wild-type. A subset of 304 genes was differentially expressed between pft1-2 and the wild-type (two-fold change or more, P < 0.05; Supporting Information Tables S3, S4). As anticipated from the function of Mediator in transcriptional activation, the vast majority of differentially expressed genes (261) in pft1-2 plants were down-regulated (Fig. 2a). Using more stringent criteria (three-fold change or more, P < 0.05), we found that 131 genes were down-regulated, whereas only seven genes showed higher expression levels in the mutants (Fig. 2b). Interestingly, several redox-related genes, including a group of class III peroxidases, were affected in the pft1-2 background (Table 1). In total, 13 class III peroxidase genes were down-regulated in pft1-2 mutants when compared with the wild-type, six of which were strongly (< 0.5-fold) repressed by the mutation in the microarray and confirmed by qRT-PCR (Fig. 2c). A similar trend was observed in the pft1-3 allele, albeit less pronounced (Fig. 2c). Notably, PRX33, PRX34 and PRX71, which have been shown to generate H2O2 in previous studies (Rouet et al., 2006; Daudi et al., 2012), were particularly affected in both mutant alleles. Four class III peroxidases and three NADPH oxidases (AtrbohI, AtrbohA and AtrbohG) showed higher transcript levels in pft1-2 roots when compared with the wild-type. NADPH oxidases are the major source of math formula generation in plants. According to publicly available expression data (https://www.genevestigator.com/gv/), the PFT1-regulated class III peroxidases and NADPH oxidases were predominantly expressed in the root hair zone (Table 1), supporting a possible role in root hair formation. Taken together, these results indicate that PFT1 regulates the transcription of genes involved in ROS signaling.

Figure 2.

Differentially expressed genes in pft1-2 roots. (a) Two-fold or more up- and down-regulated genes. (b) Genes with three-fold or more change in expression level (P < 0.05). (c) Validation of PHYTOCHROME AND FLOWERING TIME1 (PFT1)-regulated gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Bars represent relative fold changes relative to the wild-type (light gray bars, pft1-2; dark gray bars, pft1-3). EF1A was used as endogenous control. Gene-specific primers are listed in Supporting Information Table S2. Error bars represent SD from two independent runs. Seven-day-old Arabidopsis plants were used for the experiments.

Table 1. Regulation of class III peroxidases and NADPH oxidases by PHYTOCHROME AND FLOWERING TIME1 (PFT1)
GeneRegulationFold changeTissue-specific expression
  1. Tissue-specific expression data were obtained from Genevestigator (Hruz et al., 2008).

PRX59Down8.2Root, root hair zone, hypocotyl
PRX33Down7.1Root, senescing leaf, sepal
PRX34Down7.1Root, senescing leaf, sepal
PRX14Down2.1Root, hypocotyl
PRX15Down2.1Root, hypocotyl
PRX71Down2Root, stele
PRX35Down1.6Root, root hair zone
PRX60Down1.5Root, root hair zone
PRX52Down1.5Anther, ovary
PRX8Down1.5Root, root hair zone
PRX39Down1.5Root, root hair zone, stigma
PRX49Down1.4Root, hypocotyl
PRX53Down1.4Anther, stigma
PRX25Up1.8Root, stele
PRX4Up1.7Root, root hair zone
PRX27Up1.4Root, root hair and elongation zone
PRX61Up1.4Root, root apical meristem
AtrbohIUp1.6Root, root hair zone, pollen
AtrbohAUp1.5Root, root hair zone, radicle
AtrbohGUp1.4Root, root hair zone

PFT1 maintains ROS distribution in Arabidopsis roots

As class III peroxidases were strongly repressed in pft1-2 and pft1-3 roots, we reasoned that ROS homeostasis might be compromised in roots of the mutants. To test this assumption, we analyzed the level and distribution of the two major plant ROS, H2O2 and math formula, in roots of the wild-type, pft1-2 and pft1-3 mutant plants. math formula accumulates primarily in the meristematic zone, whereas H2O2 was observed preferentially in the elongation zone and at the junction of the differentiation zone in previous studies (Dunand et al., 2007; Tsukagoshi et al., 2010). HPF was used to detect H2O2 in the roots, which yields the strongly fluorescent fluorescein when reacting with H2O2 (Setsukinai et al., 2003; Dunand et al., 2007). Interestingly, HPF fluorescence was lower in pft1-2 and pft1-3 roots relative to the wild-type, indicative of a decreased H2O2 level (Fig. 3a,b). math formula was probed by NBT, which reacts with math formula to form an insoluble formazan that precipitates in contact with math formula (Bielski et al., 1980). Formazan production was higher in roots of pft1-2 and pft1-3 plants, indicating elevated math formula levels when compared with the wild-type (Fig. 3c,d). These findings suggest that PFT1 maintains the ROS balance in Arabidopsis roots.

Figure 3.

Reactive oxygen species (ROS) distribution along the roots. (a) Roots of 7-d-old Arabidopsis plants were stained with 3′-(p-hydroxyphenyl) fluorescein (HPF) for hydrogen peroxide (H2O2). (b) Quantification of HPF fluorescence intensity. (c) Roots stained with nitroblue tetrazolium (NBT) for superoxide. (d) Quantification of NBT staining intensity. Quantification was conducted in the elongation zone. Error bars represent SE, n = 20. Bars: (a, c) 50 μm.

PFT1 controls root hair growth through redox processes

To determine whether PFT1-regulated ROS distribution is critical for root hair differentiation, several chemicals that react with ROS were used to treat the mutants. To demonstrate whether a reduced accumulation of H2O2 causes the root hair phenotype, H2O2 was supplied to the mutants under investigation. Exogenous H2O2 fully rescued the phenotypes of both mutant alleles (Fig. 4b). The addition of peroxidase inhibitors can increase the H2O2 content by inhibiting the action of H2O2-scavenging peroxidases. Treatment of the mutants with the peroxidase inhibitor KCN (Tsukagoshi et al., 2010) suppressed the mutant phenotypes, suggesting that the H2O2 level, or its distribution, is critical for root hair growth (Fig. 4c). Next, we treated mutant roots with the NADPH oxidase inhibitor DPI which reduces the level of math formula (Foreman et al., 2003). This treatment also suppressed the mutant phenotype of both alleles, indicating that a reduction in math formula radicals is sufficient to confer the wild-type phenotype (Fig. 4d). A root hair initiation mutant, rhd6, was used as a negative control for the H2O2, KCN and DPI treatments. None of these treatments rescued the hair-less phenotype of rhd6 plants, indicating a specific effect of the chemicals in the pft1 backgrounds (Fig. 4b–d). We then assessed the level of H2O2 and math formula in the mutant lines and wild-type after H2O2, KCN and DPI treatments. Consistent with the suppression of the mutant phenotype (Fig. S1), H2O2 and math formula levels in the mutants did not differ significantly from those of the wild-type after the treatments (Fig. S2), indicating the recalibration of ROS homeostasis. These findings further support the assumption that PFT1-controlled ROS distribution is critical for root hair differentiation and elongation.

Figure 4.

Suppression of the mutant phenotype of pft1-2 and pft1-3 by various chemical treatments. (a) Untreated Arabidopsis roots. (b) Hydrogen peroxide (H2O2) (500 μM)-treated roots. (c) KCN (75 μM)-treated roots. (d) Diphenyleneiodonium (DPI) (2.5 μM)-treated roots. Bars, 100 μm.

Regulation of cell wall-related genes and other targets by PFT1

To extract further information with regard to the function of the genes that were differentially expressed between pft1-2 and the wild-type, we performed a GO enrichment analysis of the microarray data using GOBU (Lin et al., 2006). Among the down-regulated genes, the GO categories, ‘light-harvesting complex’, ‘photosynthesis’ and ‘response to light’ were markedly over-represented, which confirms the role of PFT1 in light signaling (Fig. 5). The GO terms ‘apoplast’, ‘peroxidase activity’, ‘response to H2O2’, ‘response to jasmonic acid (JA)’ and ‘defense response’ are indicative of an apoplastic localization of H2O2-generating peroxidases, probably involved in defense and development. In addition, several GO categories comprising genes with cell wall-related functions were over-represented, suggesting an activation of cell wall remodeling genes by PFT1. In particular, a group of genes consisting of five EXTs, four xyloglucan endo-transglycosylase/hydrolases (XTHs), an expansin (EXP), a pectate lyase-like (PLL) gene and a proline-rich protein (PRP) were repressed in pft1-2. Validation of the differential expression of these genes by qRT-PCR in pft1-2 and pft1-3 mutants confirmed the microarray data (Fig. 6). The expression of the majority of these genes was induced on H2O2 treatment in pft1-2 mutant roots (Fig. 6). Thus, it appears that PFT1 controls this group of genes through the production of H2O2.

Figure 5.

Functional categories of PHYTOCHROME AND FLOWERING TIME1 (PFT1)-regulated genes. Enriched gene ontology (GO) categories of genes that are positively regulated by PFT1. x-axis shows the log values (log10 1/P value) of over-represented GO categories calculated by the Gene Ontology Browsing Utility (GOBU) software package.

Figure 6.

Analysis of PHYTOCHROME AND FLOWERING TIME1 (PFT1)-regulated gene expressions by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The light-gray bars (pft1-2) and mid-gray bars (pft1-3) represent gene expression levels without treatment and fold changes relative to untreated wild-type Arabidopsis plants. The dark-gray bar denotes hydrogen peroxide (H2O2)-treated pft1-2 mutant plants (fold change relative to H2O2-treated wild-type plants). EF1A was used as endogenous control. Gene-specific primers are listed in Supporting Information Table S2. Error bars represent SD from two independent runs. Seven-day-old plants were used for the experiments.

Among the up-regulated genes, the GO categories ‘methionine gamma-lyase activity’ and ‘methionine catabolic process’ were over-represented, indicating that PFT1 might be involved in the regulation of methionine synthesis. Other over-represented GO categories included ‘sterol-related processes’, ‘phospholipid transfer to membrane’, ‘extracellular transport’, ‘cell wall’, ‘spindle assembly’, ‘apoplast’ and ‘indole butyric acid-related processes’, which collectively indicate that PFT1 participates in the regulation of growth and cell cycle processes (Fig. S3). Overall, this analysis shows that PFT1 is critical for the regulation of several groups of genes with various functions.

Discussion

Collectively, the results from this study revealed critical, but independent, roles of PFT1 and MED8 in root hair morphogenesis. med8 pft1-2 double mutants showed a severe root hair defective phenotype with an apparently additive effect. PFT1 controls organ growth by restricting cell proliferation in Arabidopsis (Xu & Li, 2011). Consistent with our findings, MED8 has been reported to act independently of PFT1 in regulating cell expansion and organ growth (Xu & Li, 2012). In addition, PFT1 and MED8 are known to exert additive genetic effects on flowering time and the pathogen defense response (Kidd et al., 2009). It appears that PFT1 exerts its effect on root hairs through ROS distribution along the roots by regulating redox-related genes.

PFT1 controls the expression of redox-related genes

In mammalian cells, ROS distribution can switch cell proliferation to differentiation (Sarsour et al., 2008; Owusu-Ansah & Banerjee, 2009). A recent study in Arabidopsis has revealed that UPB1 controls ROS distribution, which, in turn, regulates the transition from proliferation to cell differentiation in roots. In upb1-1 mutants, the accumulation of H2O2 was reduced, whereas math formula levels were increased as a result of an up-regulation of peroxidases (Tsukagoshi et al., 2010). These results resemble the H2O2 and math formula distribution in pft1-2 and pft1-3 mutants. However, the ROS balance in the mutants was altered by the deregulated expression of peroxidases. In Arabidopsis, 73 class III peroxidase genes have been identified through database screening (Tognolli et al., 2002). Class III peroxidases have contrasting functions, that is they can either scavenge or generate H2O2. As Mediators activate transcription directly by interacting with transcription factors and RNA Pol II, it can be assumed that PFT1 induces the expression of a subset of class III peroxidases. Among the class III peroxidases that were repressed in the pft1-2 mutant, PRX33 and PRX34 were shown to generate H2O2 during the defense response, thereby conferring resistance to a wide range of pathogens (Bindschedler et al., 2006). Recently, PRX33- and PRX34-dependent oxidative bursts have been shown to play crucial roles in basal resistance mediated by the recognition of microbe-associated molecular patterns (MAMPs) and in the orchestration of pattern-triggered immunity in tissue culture cells (Daudi et al., 2012; O'Brien et al., 2012). Another class III peroxidase, PRX71, has been reported to produce active oxygen in the presence of cofactors in Arabidopsis cell suspension cultures, which can be prevented by peroxidase inhibitors (Rouet et al., 2006). It can thus be assumed that the reduction in the level of H2O2 in pft1-2 and pft1-3 mutants is caused by a compromised control of the expression of peroxidase genes.

NADPH oxidases are the main source of math formula production in Arabidopsis. The NADPH oxidase RHD2/AtrbohC has been shown to control root hair development by producing ROS that regulate cell expansion through the activation of Ca2+ channels (Foreman et al., 2003). In pft1-2 mutants, the increased math formula level was consistent with the up-regulation of the NADPH oxidases AtrbohI, AtrbohA and AtrbohG. It has been shown that the increased production of math formula in the meristematic zone is a mechanism to maintain ROS homeostasis when the H2O2 level is reduced in plant roots (Tsukagoshi et al., 2010). A similar mechanism might apply to pft1-2. Treatment of pft1-2 and pft1-3 plants with H2O2, KCN or DPI rescued the mutant phenotype and restored ROS to wild-type levels. These experiments provide evidence that PFT1 maintains ROS distribution, which is critical for root hair differentiation.

ROS distribution determines the differentiation of root epidermal cells

Based on the results outlined above, a working model on the role of PFT1 in root hair morphogenesis can be proposed (Fig. 7). By interacting with activators and RNA Pol II, PFT1 induces the transcription of a subset of genes that include class III peroxidases. This generates H2O2 that preferentially accumulates in the elongation zone and math formula that preferentially accumulates in the meristematic zone. A threshold level of ROS acts as a signal which is critical for root hair differentiation. As H2O2 can function as a secondary messenger in cell proliferation and differentiation, the signal that is essential for root hair differentiation may be H2O2. It is interesting to note that ROS do not seem to affect the expression of PFT1, indicating that no feedback loop exists between ROS and PFT1 (Fig. S4). As high levels of ROS can damage the cells, a feedback control might exist with the activator which interacts with PFT1.

Figure 7.

Working model of PHYTOCHROME AND FLOWERING TIME1 (PFT1)-regulated root hair differentiation. Mediator complex subunit PFT1 (blue) interacts with activators and RNA polymerase II (RNA Pol II) to initiate transcription. Possibly, PFT1 positively regulates class III peroxidases which produce hydrogen peroxide (H2O2) in the elongation zone (red). Superoxide (math formula) is produced by NADPH oxidases in the meristematic zone (black). The distribution of both H2O2 and math formula reaches a threshold concentration, which acts as a signal that determines the differentiation of root hair cells. This signal could be H2O2. GTFs, general transcription factors.

To investigate the cause of the short root hair phenotype of pft1-2, we mined the microarray data and found that a group of EXTs was down-regulated. EXTs are hydroxyproline-rich glycoproteins (HRGPs), which contain the Tyr-X-Tyr motif that forms intermolecular cross-links catalyzed by peroxidases (Held et al., 2004). The insoluble EXT network formation in the cell wall is a well-characterized peroxidase-mediated and H2O2-dependent process (Almagro et al., 2009). Once EXTs are secreted into the cell wall, mature EXTs form a network by the oxidative cross-linking of several Tyr residues (Lamport et al., 2011). The mutants ext6, ext7 and ext10 have been reported to form short root hairs (Velasquez et al., 2011); all three genes were significantly down-regulated in pft1-2 and pft1-3 mutant alleles. These EXTs have been predicted to form cross-links with PRX13 and PRX73 during root hair formation in root hair cell walls (Velasquez et al., 2011). In addition, PRX33 and PRX34 have been shown to be involved in H2O2-mediated cell wall remodeling (O'Brien et al., 2012). Both PRX33 and PRX34 are localized in the apoplast and promote cell elongation in Arabidopsis root, which is in line with their affinity for the Ca2+-pectate structure (Passardi et al., 2006). We found the cell wall loosening gene PLL to be strongly down-regulated in the pft1-2 and pft1-3 mutant alleles, which suggests the concerted action of peroxidases, EXTs and pectin in the formation of the cell wall. In pft1-2 mutants, H2O2 treatment normalized the expression of EXTs and PLL, and rescued the phenotype, supporting the assumption that these genes are regulated by ROS. These findings raise the possibility that PFT1 controls root hair growth by regulating H2O2-dependent and peroxidase-mediated cross-linking of EXTs.

Transcriptional profiling of pft1-2 roots indicates that PFT1 regulates genes in the GO category ‘response to JA stimulus’. H2O2 has been shown to initiate the octadecanoid pathway leading to the biosynthesis of JA and JA-related compounds (Thomma et al., 2001). It is produced in response to a wide variety of abiotic and biotic stress signals, indicating that H2O2 mediates the cross-talk between signaling pathways and acts as a signaling molecule in contributing to cross-tolerance (Almagro et al., 2009). JA and methyl jasmonate (MeJA) have been reported to regulate peroxidase gene expression, and PFT1 has been shown to play a key role in JA-dependent defense and signaling (Kidd et al., 2009; Chen et al., 2012). These results indicate that JA might also be involved in the PFT1-mediated root hair formation.

In addition to EXTs and JA, a group of XTHs and an EXP were also down-regulated in pft1-2 roots. XTHs cross-link among cellulose microfibrils in cell walls. XTH exhibits the most prominent xyloglucan endo-transglycosylase (XET) activity in epidermal cells in the elongation zone and in trichoblasts in the differentiation zone of the primary root (Vissenberg et al., 2001, 2003). XTH18 and XTH21 play crucial roles in primary root development by altering the deposition of cellulose and the elongation of cell walls (Osato et al., 2006; Liu et al., 2007). XTH8 and XTH31 may be responsible for reduced leaf cell expansion (Miura et al., 2010), and the latter gene has been shown to be positively regulated by PFT1. XTHs appear to be critical in promoting cell wall expansion, and are therefore essential for cell expansion, and are also required for the construction of cell walls in cells that have completed the expansion process (Van Sandt et al., 2007). EXP1 has been shown to regulate guard cell expansion in the control of stomatal movement (Zhang et al., 2011), and this gene is also positively regulated by PFT1. Similar to EXTs, the expression of XTHs was also increased after H2O2 treatment, indicating that PFT1 may act through ROS to activate several cell wall remodeling genes.

In conclusion, our results show that PFT1 regulates an array of genes to control root hair formation through ROS distribution. PFT1 is known to play an essential role in light signaling and flowering time (Cerdan & Chory, 2003; Wollenberg et al., 2008; Inigo et al., 2012; Klose et al., 2012), JA-mediated pathogen defense (Kidd et al., 2009), organ growth (Xu & Li, 2011), abiotic stress (Elfving et al., 2011), and JA and abscisic acid (ABA) signaling (Cevik et al., 2012; Chen et al., 2012). Interestingly, numerous reports have indicated that ROS is critical in these traits. Thus, these results lead us to propose a hypothesis that PFT1 might control multiple traits through ROS homeostasis. However, future studies will be necessary to elucidate our hypothesis. PFT1 is a single-copy gene in Arabidopsis with homologs across plant species (Hecht et al., 2005; Backstrom et al., 2007; Mathur et al., 2011). TaPFT1, a PFT1 homolog from wheat (Triticum aestivum), complemented the defense and developmental defective phenotype of pft1-2, indicating that PFT1 function is highly conserved in diverse plant species (Kidd et al., 2009). The Mediator complex is conserved across eukaryotes, and the role of Mediator in maintaining ROS homeostasis may represent a common mechanism among plants and animals.

Acknowledgements

This work was supported by grants from Academia Sinica. We thank the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA) for providing the T-DNA insertion mutant and wild-type seeds used in this study, and Pablo Cerdan (Universidad de Buenos Aires, Argentina) for kindly providing the seeds of pft1-1 and its complementation line (G1). We also thank Drs Yue-Ie Hsing, Thomas Yang, Saminathan Thangasamy, Liang-Chi Chang (Institute of Plant and Microbial Biology, Academia Sinica, Taiwan), Shanmugam Varanavasiappan (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan) and Thomas J. Buckhout (Humboldt University, Germany) for valuable suggestions and critical comments on the manuscript. Affymetrix Gene chip assays were performed by the Affymetrix Gene Expression Service Laboratory (http://ipmb.sinica.edu.tw/affy/), supported by Academia Sinica.

Ancillary