To a considerable degree, research on metal hyperaccumulation was based on the implicit assumption that metal hyperaccumulation or hypertolerance mechanisms must be inducible by exposure of a hyperaccumulator plant to elevated concentrations of the cognate metal. By contrast, it came as a surprise that all candidate genes—including HMA4, Metal Tolerance Protein1 (MTP1) and Nicotianamine Synthase2 (NAS2) that are presently known to be of central functional importance—are constitutively highly expressed in A. halleri by comparison to closely related non-accumulators (Becher et al. 2004; Weber et al. 2004). A subgroup of highly expressed candidate genes of A. halleri consists of Zn deficiency response genes (Talke et al. 2006). Their transcript levels are generally high in A. halleri as a consequence of HMA4-mediated Zn depletion in roots (Hanikenne et al. 2008), but they also retain their responsiveness to Zn-mediated repression of transcript levels known for their homologues in A. thaliana. In agreement with this, root-shoot Zn partitioning in A. halleri depends on external Zn supply (Talke et al. 2006).
As shown here, both herbivory and mechanical leaf wounding enhanced leaf Cd accumulation in A. halleri (Fig. 1). This must involve altered activities of processes in roots, and thus systemic signaling. Wounding-triggered leaf-to-leaf systemic signaling was recently shown to involve the movement of electrical surface potential changes dependent on ionotropic glutamate receptor-related plant proteins and to occur very fast (Mousavi et al. 2013). It is surprising that in A. halleri, wounding-activated processes act preferentially or even specifically to enhance shoot Cd accumulation. Cd2+ is not an essential nutrient in higher plants so that it generally accumulates in plants along pathways of chemically related nutrient metal cations such as Fe2+ or Zn2+ (Clemens et al. 2013). Our data suggested a comparably large variation of leaf Cd accumulation (see Fig. 1). We attribute this to the difficulty of both administering reproducible degrees of wounding and preventing accidental wounding in control plants. Moreover, both insect herbivory and pathogens can trigger overlapping signaling pathways, for example those involving the oxylipin family of plant hormones including jasmonates. A. halleri is particularly prone to pathogen and insect pests, and thus it is technically difficult to entirely exclude the presence of all biotic stress in experiments (Maja Schellenberg, Ina Talke, Ricardo Stein, Enrico Martinoia and Ute Krämer, unpublished observations). We expect that, in addition to leaf wounding, other biotic factors also act to enhance leaf Cd accumulation. For example, it has been reported that the natural root microbiome of A. halleri had a modestly enhancing effect on leaf Cd accumulation (Farinati et al. 2009; Muehe et al. 2015), but also this system remains challenging to control (Farinati et al. 2011). Rich microbiomes are known to colonize both above- and below-ground organs of plants, adding to the complexity of these experiments (Bulgarelli et al. 2012; Horton et al. 2014; Lundberg et al. 2012).
Leaf wounding is known to activate oxylipin-based signaling (Nemhauser et al. 2006; Reymond et al. 2004; Taki et al. 2005). This was observed here in the systemic transcriptional response of roots of A. thaliana and—to a lesser degree—A. halleri, with the activation of transcriptional methyl jasmonate, 12-oxophytodienoic acid (OPDA) and herbivory responses (Fig. 3a). Especially among the responses of A. halleri, we observed a striking overrepresentation of Fe deficiency responses and metal homeostasis genes. Upon closer investigation, this response consisted to a large extent of the transcriptional repression of Fe deficiency response genes, in particular genes of the FIT1 regulon (Supplementary Tables 1 and 2). Future studies will address whether or not this transcriptional response contributes to the wounding-induced leaf Cd accumulation response in A. halleri.
In A. thaliana, there was no increase in leaf Cd accumulation in response to wounding (data not shown). In agreement with this, despite a strong activation of herbivory responses in A. thaliana (see Fig. 3a), the transcriptional repression of Fe deficiency response genes was quantitatively far less pronounced in A. thaliana, with fewer genes detected to respond in our microarray analysis. Our data are consistent with a report that jasmonate treatment of A. thaliana resulted in decreased transcript levels of Fe deficiency response genes (Maurer et al. 2011).
Our transcript profiling identified some candidate genes for contributions to wounding-enhanced leaf Cd accumulation in A. halleri. MTP3 was the most strongly repressed transcript in response to leaf wounding. In A. thaliana, transcription of the FIT1 target MTP3 is activated when root Zn2+ uptake rates are enhanced under Fe deficiency and excess Zn. Under these conditions, the vacuolar-membrane localized MTP3 protein acts to sequester Zn2+ in vacuoles of root epidermal and cortex cells, thus decreasing shoot Zn accumulation (Arrivault et al. 2006). A decrease in MTP3 expression would thus be predicted to enhance root-to-shoot Zn transport. A. thaliana MTP3 was found not to transport Cd2+, but the specificity of A. halleri MTP3 remains to be investigated. Another interesting candidate gene is the FIT1 target HMA3. Similar to MTP3, this P1B-type ATPase can also mediate the vacuolar sequestration of Zn2+, as well as of Cd2+ and Pb2+, in the root (Morel et al. 2009). The transcriptional repression of HMA3 in roots of wounded A. halleri plants could thus decrease Cd immobilization inside roots and enhance the translocation of Cd into the shoots.
In A. thaliana, the systemic transcriptional repression of other FIT1 targets, for example FRO2 (Fig. 3b) and IRT1 (Supplementary Fig. 1), in the root is predicted to decrease the reduction of FeIII chelates to Fe2+ and root uptake rates of Fe2+. In addition, root uptake of Cd2+ is expected to decrease because this heavy metal cation is primarily taken up through the high-affinity Fe2+ uptake system IRT1 in A. thaliana. The same effect is predicted in A. halleri unless this species possesses another, yet unidentified, root uptake system for Cd2+. If, indeed, A. halleri possessed such a root uptake system for Cd2+, the decreased expression of FIT1 regulon genes upon leaf wounding would have entirely different consequences for overall metal homeostasis: For example, the decreased expression of FRO2, in particular, would be expected to lower the concentration of extracellular Fe2+ competing with Cd2+ for uptake into root cells and to enhance plant Cd accumulation, and the latter was observed here in A. halleri. Future work will address each of these hypotheses.
In conclusion, our data suggest that in the metal hyperaccumulator A. halleri wounding induces signals that act systemically in the root to trigger enhanced leaf Cd accumulation, which in turn functions as a defense against attack by herbivores (Fig. 2), and possibly also pathogens (Boyd 2007). The existence of inducible metal hyperaccumulation in A. halleri provides strong circumstantial support for the elemental defense hypothesis (Boyd 2007). Furthermore, this observation requires an analysis of the underlying molecular mechanisms, and it will guide the design of future experiments addressing metal hyperaccumulation.