Two P-Type ATPases Are Required for Copper Delivery in Arabidopsis thaliana Chloroplasts

Abstract

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.

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

“…S3, ESI †), as previously reported. 22 Furthermore, the expression of transporters such as ZIP2 (ZRT, IRT-like protein 2) 32 and COPT2 (ref. 13) was lower under high Cu conditions as compared to low Cu conditions, while that of HMA5, a P-type ATPase that plays a role in Cu efflux, 33 was higher.…”

Transition metal transport

“…Not surprisingly, Arabidopsis mutants with defects in the transport of essential transition metals have severe phenotypes [4][5][6]. The dynamic range of optimal internal concentrations, within which plants are unaffected by deficiency or toxicity, is particularly narrow for transition metals [3].…”

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7

“…To allow the dismutation of superoxide an FeSOD is expressed in the stroma under Cu limitation. Interestingly, in the paa2 mutant, defective in Cu transport to the thylakoids, an increased expression and activity of Cu/ZnSOD in the stroma was observed [9]. These observations prompted us to investigate Cu delivery to stromal Cu/ZnSOD and its regulation.…”

“…It was recently shown that Cu feeding dramatically affects the activity of FeS-OD and Cu/ZnSOD isoforms, whereas the activity of MnSOD is not affected by Cu availability [9]. Arabidopsis plants grown on media in which Cu is not limiting express Cu/ZnSOD in the cytosol and stroma.…”

“…Copper is imported into the chloroplast stroma by the P-type ATPase PAA1 and mutations in PAA1 affect both stromal Cu/ZnSOD activity and PC [8]. The Arabidopsis genome encodes a protein with high sequence similarity to PAA1, called PAA2, which functions to move Cu into the thylakoid lumen for delivery to plastocyanin [9].…”