Copper Chaperone Antioxidant Protein1 Is Essential for Copper Homeostasis    

Abstract

Copper (Cu) is essential for plant growth but toxic in excess. Specific molecular mechanisms maintain Cu homeostasis to facilitate its use and avoid the toxicity. Cu chaperones, proteins containing a Cu-binding domain(s), are thought to assist Cu intracellular homeostasis by their Cu-chelating ability. In Arabidopsis (Arabidopsis thaliana), two Cu chaperones, Antioxidant Protein1 (ATX1) and ATX1-Like Copper Chaperone (CCH), share high sequence homology. Previously, their Cu-binding capabilities were demonstrated and interacting molecules were identified. To understand the physiological functions of these two chaperones, we characterized the phenotype of atx1 and cch mutants and the cchatx1 double mutant in Arabidopsis. The shoot and root growth of atx1 and cchatx1 but not cch was specifically hypersensitive to excess Cu but not excess iron, zinc, or cadmium. The activities of antioxidant enzymes in atx1 and cchatx1 were markedly regulated in response to excess Cu, which confirms the phenotype of Cu hypersensitivity. Interestingly, atx1 and cchatx1 were sensitive to Cu deficiency. Overexpression of ATX1 not only enhanced Cu tolerance and accumulation in excess Cu conditions but also tolerance to Cu deficiency. In addition, the Cu-binding motif MXCXXC of ATX1 was required for these physiological functions. ATX1 was previously proposed to be involved in Cu homeostasis by its Cu-binding activity and interaction with the Cu transporter Heavy metal-transporting P-type ATPase5. In this study, we demonstrate that ATX1 plays an essential role in Cu homeostasis in conferring tolerance to excess Cu and Cu deficiency. The possible mechanism is discussed.

Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings

“…However, most gene expression analyses under Cu excess conditions have been carried out at high Cu concentrations (25-50 mM), where reduced growth and other physiological symptoms are already evident. [25][26][27][28][29] The main objectives of the current work are to define Cu sufficiency limits at the molecular level and to provide new molecular markers for mild Cu deficiency and excess conditions. For these purposes, global changes in the gene expression under mild Cu deficiency and Cu excess conditions were compared.…”

IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis

“…Plant growth procedures were modified from a previous study (Shin et al, 2012). Arabidopsis thaliana ecotype Columbia-0 and the idf1 T-DNA insertion lines (SALK_038445, idf1-1; and SALK_129958, idf1-2) seeds were obtained from the ABRC.…”

“…The procedure was described previously (Shin et al, 2012). Frozen root tissues (0.5 g) were ground in liquid nitrogen using a tissue homogenizer (SH-48; J&H Technology).…”

“…The protein extraction procedure was as described previously (Shin et al, 2012). Plant or Xenopus laevis oocyte samples were extracted with extraction buffer (23 SDS sample buffer containing 20 mM N-ethylmaleimide, 100 mM Na 2 S 2 O 5 , and one tablet of protease inhibitor cocktail [Roche Applied Science] per 50 mL).…”

“…Elemental analysis was as previously described (Shin et al, 2012). Harvested plant samples were washed with CaCl 2 and water and dried for 3 d before digestion.…”