Few factors have been identified to date that help plants tolerate toxic conditions of excess Zn2+, which can occur, for instance, as a consequence of mining, metal smelting, or application of sewage sludge to agricultural soil. Understanding metal tolerance is a prerequisite for biotechnological approaches to phytoremediation (Sinclair and Krämer, 2012). Perhaps even more importantly, basal metal tolerance is a consequence of mechanisms of metal homeostasis. Exploration and dissection of mechanisms of Zn2+ tolerance can therefore advance the understanding of the interplay of Zn transport and Zn-binding activities that result in the safe trafficking of Zn to the large number of target sites in tissues, cell types and cellular compartments of a plant. Also, it can help uncover novel aspects of the biological roles played by Zn. In spite of this potential, Zn2+ tolerance is as yet genetically underexplored (Richard et al., 2011).
The specificities of the isolated ozs mutants can help unravel metal homeostasis and metal toxicity
Known Zn2+ tolerance mechanisms differ in their metal specificity. While vacuolar sequestration in A. thaliana is mediated by the Zn-specific transporters MTP1 and MTP3 (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005; Arrivault et al., 2006), efflux is dependent on Zn- and Cd-transporting P1b-ATPases such as HMA2 and HMA4 (Hussain et al., 2004; Mills et al., 2005). Similarly, cytosolic Zn buffering is partly dependent on phytochelatins, which efficiently bind Cd as well (Tennstedt et al., 2009). The results of our genetic screen revealed different sensitivity patterns too. Mutants ozs1, ozs2 and ozs3 are hypersensitive specifically to excess Zn2+, while ozs4, ozs5 and ozs6 also show growth inhibition in the presence of a varying set of other metal cations. The observed combinations do not immediately suggest the involvement of any known metal tolerance pathways. All three mutants share Cu2+ hypersensitivity. Two of them show Cd2+ hypersensitivity as well. Taken together, these findings suggest the existence of both a variety of Zn toxicity targets and Zn tolerance mechanisms that can now be elucidated through the molecular characterization of the ozs mutants.
Furthermore, the Zn2+-specific hypersensitivity of ozs1 and ozs2 had already demonstrated that the mutant phenotypes are not the result of a general higher susceptibility to oxidative stress, which is often a consequence of exposure to toxic metals (Clemens, 2006). This was further supported by the wild-type growth of ozs2 when exposed to other abiotic stresses (Figure S4). Thus, the ozs mutants are likely to be affected in processes that are directly linked to metal biology.
ozs1 represents a loss-of-function allele of AtMTP1
Mapping of the causal mutation in ozs1 led to the identification of a new atmtp1 allele. A change of amino acid 293 from aspartic acid to asparagine renders the protein non-functional, as demonstrated by expression in S. cerevisiae zrc1cot1 mutant cells (Figure S3). D293 is highly conserved in cation diffusion facilitator (CDF) proteins from bacteria to humans and was recently found to be essential for Zn2+ transport activity when expressed in zrc1cot1 (Kawachi et al., 2012). Our data provide in planta support for this finding. Furthermore, the strength of the ozs1 Zn-hypersensitivity phenotype confirms the major role of AtMTP1 in Zn2+ tolerance at the seedling stage (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005).
The ozs2 mutant carries a mutation in AtPME3
Specific Zn2+ hypersensitivity was also found for ozs2. In contrast to ozs1 as well as the mutants ozs3-6, the mutation in ozs2 is semi-dominant. It was mapped to an amino acid change at position 497 (glycine to valine) in AtPME3. While in accordance with the semi-dominant character no complementation with wild-type AtPME3 was achieved and two T-DNA insertion lines showed wild-type Zn2+ tolerance, the available evidence strongly suggests that the change is causal for the ozs2 phenotype. First, no other mutation was found in the mapped interval. Second, and most importantly, down-regulation of PME3 transcript levels by RNAi fully restored wild-type Zn2+ tolerance in ozs2. Third, overexpression of the mutated PME3 version in wild-type plants led to severe and specific Zn2+ hypersensitivity. Surprisingly, overexpression of the Col-0 version was equally detrimental (Figure 6a). For both AtPME3 versions we found a remarkably strong correlation between transcript abundance and degree of growth inhibition by excess Zn2+ (Figure 6c,d).
Plant PMEs are encoded by large gene families. The A. thaliana genome contains 66 PME genes, the poplar genome 89 (Pelloux et al., 2007). Pectin methylesterases catalyze the demethylesterification of homogalacturonan pectin subsequent to its secretion into the cell wall in a highly methylesterified state. This process plays a crucial role in the regulation of cell elongation as it greatly influences cell wall architecture and extensibility (Peaucelle et al., 2011a). Accordingly, important functions in vegetative and reproductive development have in recent years been assigned to the regulated modulation of methylesterification of homogalacturonan by PMEs (Wolf et al., 2012). For instance, the tip growth of pollen tubes is dependent on spatially controlled PME activity (Bosch and Hepler, 2005). Regulated pectin demethylesterification is an early and necessary event in phyllotaxis and organ initiation (Peaucelle et al., 2008, 2011a). In addition, PMEs have been studied in the context of plant–pathogen interactions (Lionetti et al., 2012). An increase in the number of free carboxylic groups renders pectin more susceptible to degradation through polygalacturonases secreted by pathogens.
However, few individual PMEs have been functionally characterized to date. Most mutants do not show phenotypes, probably because of overlapping activities of PMEs as well as compensatory responses (Wolf and Greiner, 2012). Phyllotaxis requires the differential regulation of AtPME5 in the shoot apical meristem and the elongating stem by the transcription factor BELLRINGER (Peaucelle et al., 2011b). Recently, a contribution of AtPME35 to mechanical strength of the stem was demonstrated (Hongo et al., 2012). AtPME3 was identified as a protein interacting with and possibly targeted by a cellulose-binding protein from the parasitic nematode Heterodera schachtii (Hewezi et al., 2008). Later, a role of AtPME3 in controlling adventitious root formation was described (Guénin et al., 2011).
AtPME3 is strongly expressed throughout the plant (Louvet et al., 2006). According to publicly available microarray data transcript levels are highest in the vascular tissue of roots and shoots (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). This is consistent with promoter::GUS data (Guénin et al., 2011). Mature AtPME3 protein, i.e. without the pro-region, has been detected in the cell wall (Boudart et al., 2005; Guénin et al., 2011).
Two major questions arise from the observations reported for the ozs2 mutant: (i) what is the effect of the mutation on the biological activity of AtPME3; and (ii) why does the ozs2 mutation as well as the ectopic expression of AtPME3 cause severe and specific Zn2+ hypersensitivity? The ozs2 mutation changes a highly conserved stretch of amino acids. The mutated glycine is nearly 100% conserved in PMEs across kingdoms (Markovic and Janecek, 2004). It is located next to an arginine that is part of the active center (Johansson et al., 2002), suggesting a change in enzyme activity of ozs2 AtPME3. It was not possible to directly test this hypothesis because no purified recombinant protein representing the PME domain could be obtained, a common problem with type-I PMEs (De-la-Peña et al., 2008). Instead, we tested whether the deleterious effect of ozs2 AtPME3 is dependent on enzyme activity by mutating two residues that, according to structural mechanistic knowledge and experimental validation (Dorokhov et al., 2006), lead to an enzymatically dead protein. For overexpressing plants we found at least equally pronounced growth inhibition by excess Zn2+ as for the ozs2 mutant version (Figure 6b), arguing strongly against an effect on activity as the underlying cause of the ozs2 phenotype. Consistent with this hypothesis, we did not detect any differences in total PME activity between any of the studied plant lines, regardless of pH, assay type or substrate.
A detectable biochemical consequence of the ozs2 mutation, however, concerned the proteolytic processing that type-I PMEs undergo before they reach the apoplast. The fraction of mature protein was consistently smaller for the ozs2 version compared with the wild-type AtPME3 (Figure 7). This difference was independent of Zn2+ exposure. While the ozs2 mutation is not directly located in one of the two basic cleavage motifs of the pro-protein (Wolf et al., 2009), the results still suggest that proteolytic processing is impaired in the mutant. It is widely assumed that PMEs are processed intracellularly, most likely before exit from the Golgi (Wolf et al., 2009). Possibly the processing of PMEs occurs in Golgi-localized protein complexes similar to those postulated recently for cell wall biosynthesis enzymes (Burton et al., 2010; Oikawa et al., 2013). We speculate that the semi-dominant ozs2 mutation affects the interaction of AtPME3 with proteases or other proteins along the secretory pathway. Accordingly, we hypothesize that strong overexpression of AtPME3 interferes with the function of proteins trafficking through the Golgi in a similar fashion. Both impaired processing and protein overdose would then indirectly affect synthesis and/or modification of the cell wall in a way that renders seedlings hypersensitive to Zn2+.
Two observations indicated alterations in the cell walls of ozs2 seedlings. First, upon exposure to Zn2+ ozs2 showed more extensive root hair outgrowth (Figure 3). This is consistent with the role of the pectin network in controlling cell expansion. Cell wall extensibility is the key parameter, and pectins as the most complex matrix components play a major role in the modulation of the physical properties of the cell wall (Wolf and Greiner, 2012). Accumulation of Zn in roots, however, was not affected by the change in root hair growth, arguing against an effect on Zn uptake.
Second, an excess of Ca2+ was able to suppress the Zn2+ hypersensitivity of ozs2 but not of ozs1 seedlings (Figure 8). This finding pointed towards alterations in cation-binding sites in the apoplast of ozs2 as the cause for the phenotype. Free carboxylic groups of demethylesterified pectin could potentially bind metal cations and thereby lower the uptake into the symplast. As an example, 30% of the total Zn in roots of the metal hyperaccumulator Noccaea caerulescens was estimated to be localized in cell walls (Salt et al., 1999). Also, it is documented in vitro that pectin with lower degree of methylation has a higher Zn-binding capacity (Khotimchenko et al., 2008). Increases in low-methylesterified pectin have been implicated in metal tolerance (Krzeslowska, 2011). Thus, it is suggestive to associate a loss of metal tolerance with a defect in demethylesterification of pectin. However, the specificity of the metal hypersensitivity of ozs2 strongly argues against a simple change in the metal-binding capacity of the cell wall. Other metal cations such as Cd2+ or Cu2+ should also bind to pectins and therefore potentially affect growth of ozs2. In fact, Cu2+ is known to bind more strongly to pectins than Zn2+. Accordingly, in a recent synchrotron-based x-ray fluorescence microscopy/x-ray absorption spectroscopy study with metal-exposed roots of cowpea, Cu was–in contrast to Zn–found to be mainly associated with polygalacturonic acids (Kopittke et al., 2011).
Moreover, careful analysis of Col-0 and ozs2 roots via immunostaining of pectins and electron microscopy did not reveal any consistently noticeable differences between the cell walls of the two genotypes, regardless of Zn2+ status. Taken together, our results do not agree with a modification of cell wall-binding strength as the reason for the ozs2 Zn2+ hypersensitivity.
Instead, we hypothesize that due to subtle changes caused by the ozs2 mutation the interference of Zn2+ ions with the complex dynamics of cell wall architecture is aggravated. Our observations suggesting changes in cell wall properties of ozs2 were restricted to seedlings exposed to Zn2+, indicating that Zn2+ can act as a trigger for cell wall remodeling. This adds to recent evidence for the effects of Zn on root development and morphology that could be due to effects on cell wall modulation. An analysis of natural variation in Zn tolerance in A. thaliana found indications that Zn is required for the initiation of lateral roots (Richard et al., 2011). Some A. thaliana accessions did not produce lateral roots under conditions of Zn2+ deficiency. A study on responses to Zn2+ excess on the other hand found effects on the shape of A. thaliana root hairs (Fukao et al., 2011). The ozs2 mutant might therefore help identify primary Zn2+-toxicity targets which are currently unknown (Clemens, 2010).
Furthermore, the ozs2 mutation provides opportunities to better understand cell wall architecture and remodeling. The cell wall in general is biochemically poorly understood. Up to 10% of all A. thaliana genes are estimated to be involved in cell wall biosynthesis, modification, degradation, and the regulation of these processes (Liepman et al., 2010). Structural analysis of pectins as the most complex polysaccharides in cell walls is particularly challenging, and pectin biosynthesis is the least understood (Mohnen, 2008). Complexity is potentiated through the existence of a large number of modifying enzymes including the PMEs. Their activity may promote a multitude of apparently contradictory changes including wall stiffening, wall loosening, wall degradation and wall signaling (Wolf et al., 2012). An important aspect of PME regulation is the proteolytic cleavage of the pro-domain (Wolf et al., 2009). The processing defect in the ozs2 AtPME3 can be used to unravel this aspect of control of PME activity. Moreover, the amazingly tight correlation between AtPME3 overexpression levels and the degree of Zn2+ hypersensitivity (Figure 6c,d) could be exploited to trigger and analyze changes in cell wall structure varying in magnitude over a wide range.