Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7

Heidi Gielen, Tony Remans, Jaco Vangronsveld, Ann Cuypers

MicroRNAs are important posttranscriptional regulators of gene expression playing a role in developmental processes as well as in stress responses, including metal stress responses. Despite the identification of several metal-responsive miRNAs, the regulation and the role of these miRNAs and their targets remain to be explored. In this study, miRNAs involved in the response to Cd and Cu excess in Arabidopsis thaliana are identified. In addition, the involvement of the transcription factor SPL7, namely the key regulator of Cu homeostasis, in these metal stress responses is demonstrated by the use of an spl7 knockout mutant. Furthermore, more insight is given in the Cd-induced Cu deficiency response through determining the effects of adding supplemental Cu to Cd-exposed plants. Thirteen miRNAs were identified in response to Cu and Cd excess in A. thaliana. Several of these miRNAs (miR397a, miR398b/c and miR857) were oppositely affected under Cu and Cd exposure. The induced expression of these miRNAs after Cd exposure was totally abolished in the spl7 mutant (SQUAMOSA promoter binding protein like7), indicating a major role for SPL7 in the Cd response. Plants exposed to Cd showed a higher Cu content in the roots, whereas the Cu content in the leaves of the spl7 mutant was reduced. Furthermore, the Cd-induced Cu deficiency response disappeared when supplemental Cu was added. Copper- and Cd-responsive miRNAs were identified and several of them are SPL7-dependently regulated. SPL7 seems to be a shared component between both the Cu toxicity and the Cd toxicity response, yet oppositely regulated, that is inactivated after Cu exposure and activated after Cd exposure. Since SPL7 is the key regulator of Cu homeostasis, and Cd affects the Cu homeostasis, we hypothesize that SPL7 is activated in response to Cd possibly due to a Cd-induced Cu deficiency. Since adding additional Cu to Cd-exposed plants resulted in the disappearance of the Cu deficiency response, Cd possibly provokes Cu deficiency, thereby activating SPL7 and inducing subsequently the Cu deficiency response.