Double homozygous c3c4 plants exhibit severe patterning alterations, including a complete absence of roots (Figure 1b), which are strongly suggestive of defects in polar auxin transport (Liu et al., 1993; Shevell et al., 1994; Geldner et al., 2004). This would be consistent with the proposed role of PP2As in PIN dephosphorylation and trafficking (Michniewicz et al., 2007; Kleine-Vehn et al., 2009; Sukumar et al., 2009; Rahman et al., 2010). To gain further evidence for this hypothesis, we first analysed in roots of the different mutants the activity of the DR5 promoter as a proxy for in vivo auxin distribution (Sabatini et al., 1999; Friml et al., 2003; reviewed in Tanaka et al., 2006). As previously described (Xu et al., 2006), a strong DR5pro:GFP signal was observed in the quiescent centre (QC), in columella initials and in columella cells of wild-type roots (Figure 2a, g). Interestingly, DR5pro:GFP fluorescence distribution in both c4 and c3/+ c4 plants was reduced to a narrower domain, with fluorescence generally being weaker, and in the columella it was restricted to the two central files (Figure 2c, e, i, k). This reduced expression domain is indicative of a depletion of auxin from the root tip. The DR5pro:GFP fluorescence was also altered in c3 and c3 c4/+ plants but, remarkably, in an opposite way to c4 and c3/+ c4 mutants. There was an increase in the overall DR5pro:GFP fluorescence levels in c3 and c3 c4/+ root tips, suggesting a higher accumulation of auxin. Moreover, the domain of DR5pro:GFP expression was expanded, appearing in vasculature and cortex/endodermis initials, and in lateral root cap cells (Figures 2b, d, h, j). This expanded domain of DR5 promoter activity is identical to that observed at short times after stimulating auxin production in roots, before the internal capacitor re-establishes the correct auxin distribution (Grieneisen et al., 2007), supporting the hypothesis that it may result from increased auxin accumulation at the tip due to impairment of auxin transport. Importantly, in the single c3 and c4 mutants these changes in DR5pro:GFP fluorescence distribution are uncoupled from obvious morphological alterations, indicating that they are a direct consequence of the absence of PP2A-C3 and -C4 activity. In addition, DR5pro:GFP fluorescence in c3c4 seedlings is observed at irregular spots in the malformed cotyledons, whereas no fluorescence is detected at the position of the putative root primordium (Figure 2f, l), further supporting the absence of a proper root meristem in c3c4 seedlings. These results suggest that PP2A-C3 and PP2A-C4 play distinct, specific functions in the regulation of auxin distribution in the roots.
Figure 2. DR5pro:GFP fluorescence in single c3 and c4 mutants, c3 c4/+ and c3/+ c4 roots, and c3c4 seedlings.
Longitudinal sections of the root tip from 5-day-old plants expressing the auxin reporter DR5pro:GFP construct. The GFP fluorescence was visualized by confocal microscopy in the roots of wild-type (a, g), c3 (b, h), c4 (c, i), c3 c4/+ (d, j), and c3/+ c4 (e, k) mutant plants, and in c3c4 seedlings (f, l). Red signal: (a)–(e) propidium iodide stained cell walls, (f) propidium iodide staining and chlorophyll fluorescence in the cotyledons. Green signal: (a)–(l) GFP fluorescence. The brackets in (f) and (l) mark the presumptive root pole in c3c4 seedlings. Scale bar = 50 μm.
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Recent results suggest that PP2A is responsible for dephosphorylating and controlling the localization of the PIN family of auxin efflux transporters (Michniewicz et al., 2007; Kleine-Vehn et al., 2009; Rahman et al., 2010), which are essential for establishing the auxin gradients that guide many aspects of plant development (Benkova et al., 2003). As PIN1 is expressed in the stele and is responsible for the bulk of basipetal auxin transport into the root tip (Friml et al., 2003), we introduced the PIN1pro:PIN1-GFP marker in the mutant plants and analysed its expression and localization, along with potential root patterning defects similar to those described for mutants with compromised polar auxin transport (Geldner et al., 2004; Michniewicz et al., 2007). Since the double homozygous c3c4 mutant does not generate a root meristem, our analyses focused on the c3 and c4 single and c3 c4/+ or c3/+ c4 enhanced mutants. For all tested parameters, we found a general pattern of increasing phenotypic severity c3 = c4 < c3 c4/+ < c3/+ c4, reminiscent of what is observed at the whole plant level (Figure 1a). Average PIN1-GFP signal strength was diminished most markedly in the c3/+ c4 genotype (Figure 3a, topmost panel). Patterning defects and abnormalities in PIN1-GFP polarity were analysed and sorted into phenotypical classes (none, slight, severe; Figure 3a, b). Moreover, the percentage of cells in the meristem area with a clearly basal PIN1-GFP signal was determined (Figure 3a). Defects in patterning were most frequently observed around the QC, with ectopic cell divisions and aberrant division planes often leading to an increased number of cells in the QC, and a less clearly defined niche of initial cells, with aberrations in both their numbers and positions (Figure 3b). Both single c3 and c4 mutant plants exhibited a higher percentage of aberrations than the wild type. However, defects were never severe. In contrast, c3 c4/+ roots showed increasingly severe phenotypes, and c3/+ c4 roots were almost exclusively aberrant of the slight or severe classes (Figure 3a middle panel). Importantly, the increased frequency and severity of patterning defects were accompanied by a failure to polarly localize PIN1-GFP to the basal plasma membrane (Figure 3a, b, arrowheads). In severe cases, PIN1-GFP was basically absent from the basal plasma membrane, consistent with the results reported in knockout plants for the PP2A-A regulatory subunits (Michniewicz et al., 2007). Notably, complete reversion of PIN1-GFP polarity from the basal to the apical cell side, which has been described as an occasional occurrence in RNA interference (RNAi)-induced knockdown of all three regulatory PP2A-A subunit genes (Michniewicz et al., 2007), was not observed in our experiments, indicating that presumably one copy of either C3 or C4 still suffices to counter AGC3 kinase-dependent PIN phosphorylation for targeting PINs to the apical cell side (Dhonukshe et al., 2010).
Figure 3. PIN1 localization in roots.
(a) Topmost graph: box plot of relative PIN1-GFP signal strength (arbitrary units) in wild-type, c3, c4, c3 c4/+ and c3/+c4 seedling roots. Second graph: frequency of the three patterning defect classes (none, slight, severe) as exemplified in (b). Third graph: frequency of the three PIN1-GFP polarity aberration classes (none, mild, severe) as exemplified in (b). Lowermost graph: box plot of percentage of cells clearly exhibiting basally polarized PIN1-GFP signal.
(b) Example micrographs of the phenotypic classes of patterning defects revealed by propidium iodide staining (red channel) of cell boundaries, and of PIN1-GFP (green channel) polarity aberrations (see 'Experimental Procedures' for detailed description). Scale bar: 20 μM. Note the reduction in the number of cells exhibiting strongly polar PIN1-GFP signal at the basal plasma membranes in the ‘slight’ and ‘severe’ classes (arrowheads mark some of the cells with polar PIN1-GFP localization). In the box plots, whiskers indicate minimum and maximum of population, box indicates the lower and upper quartile, bars the median.
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Taken together, our results indicate that the subfamily II of the catalytic PP2A subunits is involved in regulating plant development, presumably by modulating PIN polarity to establish auxin pattern-guiding gradients.