Positive Selection in the Chloroplastic ATP-Synthase ß-Subunit and Its Relation to Virulence Factors.

The most paradigmatic examples of molecular evolution under positive selection involve genes related to the immune system. Recently, different chloroplastic factors have been shown to be important for plant defenses, among them, the α- and ß-subunits of the ATP synthase. The ß-subunit has been reported to interact with several viral proteins while both proteins have been implicated with sensitivity to tentoxin, a phytotoxin produced by the widespread fungus Alternaria alternata. Given the relation of both protein to virulence factors, we studied whether these proteins are evolving under positive selection. To this end, we used the dN/dS ratio to examine possible sites under positive selection in several Angiosperm clades. After examining 79 plant genera and 1232 species, we found three times more sites under pervasive diversifying selection in the N-terminal region of the ß-subunit compared to the α-subunit, supporting previous results which identified this region as responsible for interacting with viral proteins. Moreover, we found the site 83 of ß-subunit under positive selection in several plant genera, a site clearly related to the sensitivity to tentoxin according to biochemistry assays, which possibly reflects the selective pressure of the non-host specific tentoxin across various Angiosperm clades.

Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system.

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).

Coding of insertion-deletion events of the chloroplastic intergene atp beta-rbcL for the phylogeny of the Valerianeae tribe (Valerianaceae).

A preliminary analysis of the sequence alignment of the chloroplast intergene atp beta-rbcL in tribe Valerianeae reveals that insertion-deletion evolutionary events ('indels'), combined with nucleotide substitutions, have occurred in large zones in some of the studied taxa. Due to the frequent occurrence and large size of indels within this tribe, intergene length varies from 531 to 788 base pairs within the studied species. This situation poses gap coding problems that we had to tackle before phylogenetic analysis. Four methods of gap coding were used: elimination of gapped sites ('complete omission'), 'missing data', 'fifth base' and Barriel's coding method, which translates indels into new multistate characters in the data matrix. After application of these four methods of data treatment, phylogenetic analyses (maximum parsimony) did not lead to very different results. Three robust clades emerged in each case, corresponding to the Centranthinae subtribe (genus Centranthus), the Fediinae subtribe (genera Fedia and Valerianella), and the American species of Valeriana. The theoretical basis and biological significance of these four methods are discussed in order to apply the best ones in future studies.