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Key Message: MMDH2 gene negatively regulates Cd tolerance by modulating reactive oxygen species (ROS) levels and the ROS-mediated signaling, thus, affecting the expression of PDR8. The molecular mechanism by which plants respond to stress caused by cadmium (Cd), one of the most toxic heavy metals to plants, is not well understood. Here, we show that MMDH2, a gene encoding mitochondrial malate dehydrogenase, is involved in Cd stress tolerance in Arabidopsis. The expression of MMDH2 was repressed by Cd stress. The mmdh2 knockdown mutants showed enhanced Cd tolerance, while the MMDH2-overexpressing lines were sensitive to Cd. Under normal and Cd stress conditions, lower H 2 O 2 levels were detected in mmdh2 mutant plants than in wild-type plants. In contrast, higher H 2 O 2 levels were found in MMDH2-overexpressing lines, and they were negatively correlated with malondialdehyde levels. In addition, the expression of the PDR8, a gene encoding a Cd efflux pump, increased and decreased in the mmdh2 mutant and MMDH2-overexpressing lines, in association with lower and higher Cd concentrations, respectively. These results suggest that the MMDH2 gene negatively regulates Cd tolerance by modulating reactive oxygen species (ROS) levels and the ROS-mediated signaling, thus, affecting the expression of PDR8.

Cadmium (Cd) extrusion is an important mechanism conferring Cd tolerance by decreasing its accumulation in plants. Previous studies have identified an Arabidopsis ABC transporter, PDR8, as a Cd extrusion pump conferring Cd tolerance. However, the regulation of PDR8 in response to Cd stress is still largely unknown. In this study, we identified an Arabidopsis cadmium-tolerant dominant mutant, designated xcd3-D, from the XVE-tagging T-DNA insertion lines by a gain-of-function genetic screen. The corresponding gene was cloned and shown to encode a nuclear WRKY transcription factor WRKY13. Expression of WRKY13 was induced by Cd stress. Overexpression of WRKY13 resulted in decreased Cd accumulation and enhanced Cd tolerance, whereas loss-of-function of WRKY13 led to increased Cd accumulation and sensitivity. Further analysis showed that WRKY13 activates the transcription of PDR8 by directly binding to its promoter. Genetic analysis indicated that WRKY13 acts upstream of PDR8 to positively regulate Cd tolerance. Our results provide evidence that WRKY13 directly targets PDR8 to positively regulate Cd tolerance in Arabidopsis., (© 2018 John Wiley & Sons Ltd.)

Key Message: The WRKY transcription factor WRKY12 negatively regulates Cd tolerance in Arabidopsis via the glutathione-dependent phytochelatin synthesis pathway by directly targeting GSH1 and indirectly repressing phytochelatin synthesis-related gene expression. Cadmium (Cd) is a widespread pollutant toxic to plants. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway plays key roles in Cd detoxification. However, its regulatory mechanism remains largely unknown. Here, we showed a previously unknown function of the WRKY transcription factor WRKY12 in the regulation of Cd tolerance by repressing the expression of PC synthesis-related genes. The expression of WRKY12 was inhibited by Cd stress. Enhanced Cd tolerance was observed in the WRKY12 loss-of-function mutants, whereas increased Cd sensitivity was found in the WRKY12-overexpressing plants. Overexpression and loss-of-function of WRKY12 were associated respectively with increased and decreased Cd accumulation by repressing or releasing the expression of the genes involved in the PC synthesis pathway. Transient expression assay showed that WRKY12 repressed the expression of GSH1, GSH2, PCS1, and PCS2. Further analysis indicated that WRKY12 could directly bind to the W-box of the promoter in GSH1 but not in GSH2, PCS1, and PCS2 in vivo. Together, our results suggest that WRKY12 directly targets GSH1 and indirectly represses PC synthesis-related gene expression to negatively regulate Cd accumulation and tolerance in Arabidopsis.