Microbial Sources Of Chlorinated Catechol 1,2 - Dioxygenase Genes In Arabidopsis Degradation Mechanism Of Aromatic Compounds

Transgenic exploitation of bacterial degradative genes in plants has been considered a favorable strategy for degrading organic pollutants in the environment. The aromatic ring characteristic of these pollutants is mainly responsible for their recalcitrance to degradation. In this study, a Plesiomonas-derived chlorocatechol 1, 2-dioxygenase (TfdC) gene (tfdC), capable of cleaving the aromatic ring, was introduced into Arabidopsis thaliana. Morphology and growth of transgenic plants are indistinguishable from those of wild-type plants. In contrast, they show significantly enhanced tolerances to catechol. Transgenic plants also exhibit strikingly higher capabilities of removing catechol from their media and high efficiencies of converting catechol to cis,cis-muconic acid. As far-less-than-calculated amounts of cis,cis-muconic acid were accumulated within the transgenic plants , existence of endogenous TfdD- and TfdE-like activities was postulated and subsequently putative orthologs of bacterial tfdD and tfdE were detected in Arabidopsis. However, no TfdC activity and no putative orthologs of either tfdC or tfdF were identified. This work indicates that the TfdC activity, conferred by tfdC in transgenic Arabidopsis, is a key requirement for phyto-removal and degradation of catechol, and also suggests that microbial degradative genes may be transgenically exploited in plants for bioremediation of aromatic pollutants in the environment. Chlorophyllase (EC 3.1.1.14) is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thaliana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here, we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly, 'activation' of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.