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Cadmium (Cd) exposure can disturb the homeostasis of essential elements. In Arabidopsis thaliana, Cd induces a squamosa promoter binding protein-like 7 (SPL7)-dependent Cu deficiency response. We investigated how Cd induces a Cu deficiency response. The Cu deficiency response consists of the active SPL7 transcription factor binding to GTAC motifs in promoters of among others several Cu transporters, a Cu chaperone, and cupro-miRNAs to regulate Cu homeostasis. We demonstrated that the addition of supplemental Cu to Cd-exposed A.thaliana plants diminished the Cu deficiency response in roots, while it even disappeared in leaves. Exposure of plants to Cd in combination with extra Cu reduced Cd levels in both roots and leaves resulting in an improved cellular oxidative state. Furthermore, we demonstrated a role for phytochelatins (PCs) in the Cd-induced Cu deficiency response, because it was reduced in roots of cad1-3 mutant plants exposed to Cd. In conclusion, a working mechanism is provided in which it is suggested that Cd increases PC levels that can complex both Cd and Cu. This results in cellular Cu deficiency and subsequently the activation of SPL7 and hence the induction of the Cu deficiency response. Summary statement We demonstrated that addition of supplemental Cu to Cd-exposed Arabidopsis thaliana plants diminished the SPL7-dependent Cu deficiency response in roots, while it even disappeared in leaves. Moreover, in roots of phytochelatin (PC-) deficient cad1-3 mutants, this Cu deficiency response was reduced. Therefore, a working mechanism is provided in which it is suggested that Cd increases PC levels that can complex both Cd and Cu. This results in cellular Cu deficiency and subsequently the activation of SPL7 and hence the induction of the Cu deficiency response. This work was supported by Hasselt University by a PhD grant for Heidi Gielen and through the Methusalem project [08M03VGRJ].

Lipid peroxidation in leaves and roots of A. thaliana wild-type and spl7 knockout plants. Nineteen-days-old plants were exposed for 24 h to 2 μM CuSO4, 5 μM CdSO4 or grown under control conditions. Data are mean ± S.E. of 4 biological replicates. Significant treatment differences (P

Forward and reverse primers used to determine gene expression levels via quantitative real-time PCR. E-E-jn, exon-exon junction; E-I-b, exon-intron boundary; YLS, yellow-leaf-specific; UBQ10, ubiquitin; ACT, actin ; EF, elongation factor; APS, ATP sulfurylase; SULTR, sulfate transporter; LAC, laccase; CSD, Cu/Zn superoxide dismutase; FSD, Fe superoxide dismutase. *Pri-miRNA primer concentrations were increased to 900 nM. (DOCX 18 kb)

Verification of the T-DNA insertion in the spl7 mutant. Primers were designed using T-DNA primer design. (DOCX 32 kb)

Quantitative real-time PCR parameters according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines derived from Bustin et al. [51]. (DOCX 16 kb)

Plants exposed to excess metals are challenged by an increased generation of reactive oxygen species (ROS) such as superoxide (O-2(center dot-)), hydrogen peroxide (H2O2) and the hydroxyl radical ((OH)-O-center dot). The mechanisms underlying this oxidative challenge are often dependent on metal-specific properties and might play a role in stress perception, signaling and acclimation. Although ROS were initially considered as toxic compounds causing damage to various cellular structures, their role as signaling molecules became a topic of intense research over the last decade. Hydrogen peroxide in particular is important in signaling because of its relatively low toxicity, long lifespan and its ability to cross cellular membranes. The delicate balance between its production and scavenging by a plethora of enzymatic and metabolic antioxidants is crucial in the onset of diverse signaling cascades that finally lead to plant acclimation to metal stress. In this review, our current knowledge on the dual role of ROS in metal-exposed plants is presented. Evidence for a relationship between H2O2 and plant metal tolerance is provided. Furthermore, emphasis is put on recent advances in understanding cellular damage and downstream signaling responses as a result of metal-induced H2O2 production. Finally, special attention is paid to the interaction between H2O2 and other signaling components such as transcription factors, mitogen-activated protein kinases, phytohormones and regulating systems (e.g. microRNAs). These responses potentially underlie metal-induced senescence in plants. Elucidating the signaling network activated during metal stress is a pivotal step to make progress in applied technologies like phytoremediation of polluted soils. This work was supported by the Research Foundation Flanders (FWO) by a postdoctoral grant for EK and projects [G0D3414] and [G0D11141]. Additional funding came from Hasselt University (BOF12NI28, BOF14DOC04) and PhD grants from the Agency for Innovation by Science and Technology (IWT-Flanders).