Cytochemical localization of H2O2 in pigment glands of cotton (Gossypium hirsutum L.)

Programmed cell death (PCD) plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species (ROS) produced by the aerobic metabolism of cells, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands of Gossypium hirsutum L. with 3,3-diaminobenzidine (DAB) staining, 2',7'-dichlorodihydrofluorescein diacetate (DCFH2)-DA fluorescent labeling and CeCl3 cytochemical localization techniques. The results showed that the pigment glands of G. hirsutum could generate H2O2, and the amount and localization of H2O2 varied at different developmental stages. At the early developmental stage, a small amount of H2O2 accumulated in the vacuole membrane of pigment gland cells. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cell walls started to accumulate a small amount of H2O2. When pigment gland cell degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cells. With the degradation of the sheath cells, H2O2 was detected in cell wall and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2 are consistent with the process of PCD of individual gland cells, which started from the degradation of intracellular membrane and ended with the degradation of cell walls. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.