The Arabidopsis Copper Transporter COPT1 Functions in Root Elongation and Pollen Development

Vicente Sancenón,Sergi Puig,Isabel Mateu-Andrés,Eavan Dorcey,Dennis J Thiele,Lola Peñarrubia

doi:10.1074/jbc.m313321200

Vicente Sancenón, Sergi Puig + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m313321200

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Abstract

Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of (64)Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters.

Highlights

Plants use copper as a cofactor for a wide variety of physiological processes such as photosynthesis, mitochondrial respiration, superoxide scavenging, cell wall metabolism, and ethylene sensing
We focus on the function of copper transport in Arabidopsis by investigating both the COPT1 expression pattern and the phenotypes obtained in COPT1 antisense plants
The addition of 30 ␮M copper to the growth medium completely eliminated the observed differences in development and fresh weight (Fig. 6). These results demonstrate that the growth of COPT1 antisense plants is more sensitive than the wild type to copper limitation imposed by a copper-specific chelator

Summary

EXPERIMENTAL PROCEDURES

Plant Growth Conditions and Treatments—Seeds of A. thaliana, ecotype Columbia (Col 0), were grown in pots and covered with a clear plastic dome. To study the sensitivity of seedlings to copper availability, the seeds were germinated in either standard MS medium plates including 1% sucrose (except for the root length experiments) [28] or supplemented with the indicated concentrations of bathocuproine disulfonic acid (BCS), metal ions or both. To balance the great excess of 18 S rRNA with respect to the message of COPT2 and CCH, a series of PCRs were performed varying the ratio of competimers/(primers ϩ competimers) from 0 to 1. Copper Uptake and Accumulation Measurements—For the metal transport experiments, 10 mg of A. thaliana seeds ecotype Columbia (Col 0) and COPT1 antisense A 2–1, A 3– 4, and A 3– 6 transgenic lines were sterilized and incubated at 4 °C for 1–2 days to synchronize germination.

RESULTS

Copper Uptake in Arabidopsis

DISCUSSION