Immunogold-labelling localization of chlorophyllase-2at different developmental stages of Pachira macrocarpa leaves

Abstract

Chlorophyllases (Chlases) are housekeeping proteins in plant cells. The dephytylating enzymes can catalyze chlorophyll (Chl) to form chlorophyllide, but the distribution of Chlases in plant cells is still an interesting debate. Previously, we showed that PmCLH2 was a nuclear-encoded gene, and PmCLH2 protein was located in cytosol and chloroplasts of Pachira macrocarpa (Pm). In this study, the antibody of PmCLH2 was made and used by the immunogold-labelling technique to detect the localization of Chlase of Pm leaves at four developmental stages (young, mature, yellowing, and senescent). The transmission electron microscopy results show that Chlases were comprehensively found in parts of the chloroplast, such as the inner membrane of the envelope, grana, and the thylakoid membrane as well as in cytosol, and vacuoles at young, mature, and yellowing stages of Pm leaves, but not in the cell wall, plasma membrane, mitochondria, and nucleus. In short, PmCLH2 was mainly detected in vacuoles at the senescent stage, but a few were found in the chloroplasts. A pathway is proposed to explain the birth and death of Chl, Chlase, and chloroplasts in higher plants.