Zn2+-Dependent Nuclease Is Involved in Nuclear Degradation during the Programmed Cell Death of Secretory Cavity Formation in Citrus grandis ‘Tomentosa’ Fruits

Zn2+- and Ca2+-dependent nucleases exhibit activity toward dsDNA in the four classes of cation-dependent nucleases in plants. Programmed cell death (PCD) is involved in the degradation of cells during schizolysigenous secretory cavity formation in Citrus fruits. Recently, the Ca2+-dependent DNase CgCAN was proven to play a key role in nuclear DNA degradation during the PCD of secretory cavity formation in Citrus grandis ‘Tomentosa’ fruits. However, whether Zn2+-dependent nuclease plays a role in the PCD of secretory cells remains poorly understood. Here, we identified a Zn2+-dependent nuclease gene, CgENDO1, from Citrus grandis ‘Tomentosa’, the function of which was studied using Zn2+ ions cytochemical localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The full-length cDNA of CgENDO1 contains an open reading frame of 906 bp that encodes a protein 301 amino acids in length with a S1/P1-like functional domain. CgENDO1 degrades linear double-stranded DNA at acidic and neutral pH. CgENDO1 is mainly expressed in the late stage of nuclear degradation of secretory cells. Further spatiotemporal expression patterns of CgENDO1 showed that CgENDO1 is initially located on the endoplasmic reticulum and then moves into intracellular vesicles and nuclei. During the late stage of nuclear degradation, it was concentrated in the area of nuclear degradation involved in nuclear DNA degradation. Our results suggest that the Zn2+-dependent nuclease CgENDO1 plays a direct role in the late degradation stage of the nuclear DNA in the PCD of secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits.

Ultrastructural cytochemistry as a tool for studying transcriptional mechanisms

Ultrastructural cytochemistry is a powerful tool for investigating the biology of the cell nucleus. Thanks to its very high resolution, it has been possible to localize with extreme accuracy the sites of transcription, splicing and maturation of both mRNA and rRNA, their precise location as well as their movements. By means of immunocytochemical techniques, many nuclear proteins have been given a specific role in the transcriptional mechanisms, with the possibility of precisely mapping their location on a single RNA fibril. Starting from the 70s, the techniques have evolved from the resolution of autoradiography to that of gold-coupled antibodies, reaching a resolution of few nanometers. The use of correlative microscopy techniques as well as of electron tomography has also allowed the 3D imaging of ribonucleoprotein-containing structures in situ, in the nucleus. The association of a biological mechanism with the cytochemical localization of a specific molecule has been crucial in defining the functional organization of the cell nucleus, and has been invaluable for the understanding of many biological processes.

Cytochemical localization of peroxidase activity in early developmental stages of the moss Ceratodon purpureus. Light-microscopic observations

Peroxidase activity was localized in the cell walls and minute cytoplasm granules of swollen and germinating spores and in the several-celled protonema of the moss <i>Ceratodon purpureus</i> kept in darkness. Kinetin in a concentration of 100 μM inhibited the protonema development and also depressed the activity of this enzyme.

Cytochemical localization of certain hydrolytic enzymes at various stages of development of Achlya flagellata mycelium

The standard coupling azo dyes techniques were used to reveal the activities of acid phosphatase, alkaline phosphatase, esterase and β-galactoidase in the vegetative and reproductive cycle of <i>Achlya flagellata</i>. The end-products of the enzymic reactions, with the exception of E 600 sentisive esterese, which is localized in cytoplasm, occured in cytoplasmic granules. These granules are expected to be spherosomes. Acid phosphatase activity is high in differentiating sporangia, in antheridial hyphae and in degenerating oospheres where hydrolytic processes occur. β-galactosidase is the least active enzyme in the mycelium of <i>Achlya</i>.