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Sylvain Boutigny

A few observations about the role of copper ATPases HMA1 and PAA1 in the control of chloroplastic copper's homeostasis.

Published on 28 October 2009

Thesis presented October 28, 2009

The chloroplast is an organelle specific of the plant cell. It is surrounded by a double membrane or envelope which carries numerous transport systems for ions and metabolites, essential for the function of the chloroplast and the plant cell. At this point, only a few transporters associated with chloroplastic envelope had been identified: An iron transporter, a magnesium transporter and two copper P1B ATPases: HMA1 and PAA1. PAA1 is known to be the main pathway of copper import in the chloroplast, metal mainly used for photosynthesis. HMA1 is thought to be an additional pathway for copper import, essential to cope with an oxidative stress witch appears when the plant is grown under high light condition. In order to better understand the respective roles of these two ATPases in chloroplastic copper’s homeostasis, two complementary approaches had been developed:
- An in planta approach had been initiated to obtain some new lines affected in the expression of one (paa1 mutant overexpressing HMA1) or both ATPases (hma1/paa1 double knock out), then to identify conditions (light stress, saline stress, metal excess) revealing the essential role of these ATPases or inducing a different transcriptional response for genes encoding for theses ATPases or other actors of copper’s homeostasis… Results obtained show that the function of HMA1, known to be essential during a light stress, is also required when plants have to cope with a salt stress. These results are new evidences that HMA1 has a role in copper’s delivery to the chloroplastic copper/zinc superoxide dismutase. Moreover, we have shown that in photoautotrophic culture condition, copper can partially restore the photosensibility of a hma1 mutant thus validating that HMA1 in linked with copper homeostasis. We also have demonstrated that HMA1 and PAA1 function are not redundant. The copper imported by these two ATPases is very likely delivered to target proteins by distinct pathways. At last, we have shown that a third copper import pathway in the chloroplast exists, pathway still not characterized.
- An in vitro approach meant to produce HMA1 and PAA1 ATPases​ in the Lactococcus lactis heterologous expression system had been developed to determine their ionic specificities and biochemical characteristics. The prokaryotic expression system had been developed in the laboratory is perfectly suited for membrane protein production including HMA1 and PAA1. Yields obtained are appropriate for further biochemical analyses. We have determined the conditions of solubilisation and purification of these two proteins. So far, we had not been able to measure ATPase activity associated with these proteins but our data indicates that both of the proteins can bind one of their substrate: ATP. We also demonstrated that PAA1 can bind copper under its 1+ and 2+ form. Finally, these data suggests that the control of chloroplastic copper homeostasis requires several independent transport systems and a tight regulation of these copper import pathways in order to fill up the needs linked to photosynthesis and resistance mechanism to oxidative stress.

Chloroplast, envelope, membrane protein, P-type ATPase, copper, Arabidopsis thaliana, Lactococcus lactis

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