Triplin, a small molecule, reveals copper ion transport in ethylene signaling from ATX1 to RAN1

Abstract

Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human.

OsATX1 Interacts with Heavy Metal P1B-Type ATPases and Affects Copper Transport and Distribution

“…S4A). It has been shown that AtATX1-mCherry is localized to the cytoplasm and nucleus and adheres to endoplasmic reticulum membranes (Puig et al, 2007;Li et al, 2017).…”

“…In Arabidopsis, AtATX1 was proposed to deliver Cu to AtHMA5 for Cu detoxification in roots Huang et al, 2016). Using triplin as a Cu ion chelator, AtATX1 was shown to act upstream of RAN1 for Cu + delivery (Li et al, 2017). In rice, the expression of OsHMA4, OsHMA5, OsHMA6, and OsHMA9 was The wild-type yeast strain BY4741 and its mutant strain Δycf1 were transformed with the empty vector pYES2 or OsATX1.…”

“…In plant shoot tissues, a Cu concentration of 5 mg kg −1 dry weight is considered to be sufficient for plant growth and development, whereas more than 20 mg kg −1 can induce toxicity (Marschner, 1995;Burkhead et al, 2009), and plants have developed a complex Cu homeostatic network to facilitate its use and avoid toxicity in shoots. Cu homeostasis in plants depends on the control of root uptake (Sancenón et al, 2004), Cu trafficking via P-type ATPases and Cu chaperones (Himelblau et al, 1998;Hirayama et al, 1999;Abdel-Ghany et al, 2005a;Puig et al, 2007;Li et al, 2017), and regulation of the mRNAs of Cu proteins by microRNAs in response to Cu availability (Yamasaki et al, 2007).…”

“…PAA1 (AtHMA6) transports Cu across the plastid envelope, whereas PAA2 (AtHMA8) functions in Cu transport through the thylakoid membrane (Shikanai et al, 2003;Abdel-Ghany et al, 2005b). The Responsive to Antagonist (RAN1)/AtHMA7 transporter transports Cu to the ethylene receptors (Hirayama et al, 1999;Woeste and Kieber, 2000;Li et al, 2017). The AtHMA5 transporter, the closest homolog of RAN1 in the P 1B -type ATPase subfamily, is involved in Cu efflux and functions in loading Cu into the xylem for root-to-shoot translocation or Cu detoxification in Arabidopsis roots (Andrés-Colás et al, 2006;Kobayashi et al, 2008;Huang et al, 2016).…”

“…Cu + ions do not access Cu + -ATPases in a free (hydrated) form but are bound to a chaperone protein (González-Guerrero and Argüello, 2008). Genetic evidence shows that AtATX1 delivers Cu + to RAN1 for ethylene receptor biogenesis and signaling (Li et al, 2017). In Arabidopsis, AtATX1 but not AtCCH chaperone contributes to the tolerance of both excess and slightly deficient Cu (Woeste and Kieber, 2000;.…”