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 levels were increased as a result of an up-regulation of peroxidases (Tsukagoshi et al., 2010). These results resemble the H2O2 and 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.
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 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 () is produced by NADPH oxidases in the meristematic zone (black). The distribution of both H2O2 and 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.
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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.