Altering the expression of the chlorophyllase gene ATHCOR1 in transgenic Arabidopsis caused changes in the chlorophyll-to-chlorophyllide ratio.

The Arabidopsis gene ATHCOR1, which encodes the CORI1 (coronatine-induced) protein, was expressed in bacterial cells. Soluble recombinant CORI1 was purified and shown to possess chlorophyllase (Chlase) activity in vitro. To determine its activity in vivo, wild-type Arabidopsis and coi1 mutant, which lacks ATHCOR1 transcripts, were transformed with sense and antisense forms of the gene. Wild-type and coi1 plants overexpressing ATHCOR1 showed increased contents of chlorophyllide (Chlide) without a substantial change in the total amount of the extractable chlorophyll (Chl). These plants presented high Chlide to Chl ratios in leaves, whereas antisense plants and nontransformed coi1 mutant showed undetectable ATHCOR1 mRNA and significantly lower Chlide to Chl ratios, relative to wild-type control. Overexpression of ATHCOR1 caused an increased breakdown of Chl a, as revealed by the Chlide a to b ratio, which was significantly higher in sense than wild-type, coi1 mutant, and antisense plants. This preferential activity of CORI1 toward Chl a was further supported by in vitro analyses using the purified protein. Increased Chlase activity was detected in developing flowers, which correlated to the constitutive expression of ATHCOR1 in this organ. Flowers of the antisense plant showed reduced Chlide to Chl ratio, suggesting a role of CORI1 in Chl breakdown during flower senescence. The results show that ATHCOR1 has Chlase activity in vivo, however, because coi1 flowers have no detectable ATHCOR1 mRNA and present Chlide to Chl ratios comparable with the wild type, an additional Chlase is likely to be active in Arabidopsis. In accordance, transcripts of a second Arabidopsis Chlase gene, AtCLH2, were detected in both normal and mutant flowers.