Cloning And Functional Analysis Of Programmed Cell Death-Related Genes In Rice (Orysa Sativa L.)

Programmed cell death (PCD) is a highly orchestrated process that plays a major role for plants during development, differentiation and stress survival. Many genes inducing or inhibiting PCD have been found in animals, but their homologues have rarely been isolated in plants.OsPDCD5, an ortholog to mammalian programmed cell death 5, was previously cloned from rice, and its overexpression can induce PCD in transgenic rice. In the present study, we will further study its characterization. The OsPDCD5 cDNA is a 416 bp at length, harboring a complete open reading frame of 128 aa. OsPDCD5 was highly expressed in old root, old leaf and stem near the tassel but weakly expressed in young root, young leaf and stem near the root, suggesting that OsPDCD5 was related to the senescence of leaf and root tissues as well as the development of stem tissues. Distribution of OsPDCD5 protein in various rice organs were analyzed by western blotting using anti-OsPDCD5 polyclonal antibody. The result revealed that OsPDCD5 protein was widely expressed in the tassel, leaf, leaf sheath, and different parts of the stem but not in the anther. Furthermore, OsPDCD5 was up-regulated by UV-B irradiation. Calcineurin B-like interacting protein kinase 23 (OsCIPK23), which is involved in the calcineurin B-like proteins (CLBs)/CBL-interacting protein kinases (CIPKs) signaling network, was identified as interacting with OsPDCD5 by yeast two-hybrid screening and subsequently confirmed by pull-down assay in vitro.Two main categories of PCD have been recognized in animals: apoptosis and autophagy. During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to vacuole for breakdown. Two conjugation systems are recruited in double-membrane autophagosome formation. One of systems is Atg8 lipid conjugation. The carboxyl-terminal arginine (R) residue of Atg8 is proteolytically removed by a cysteineprotease, Atg4. The exposed glycine (G) residue of Atg8 is activated by the El-like enzyme Atg7, and subsequently transferred to an E2 enzyme, Atg3. Atg8 is finally conjugated to PE by the formation of an amide bond between PE and Atg7. The recent studies revealed Atg genes have been isolated in Arabidopsis. Up till now, however, there is no report about Atg genes and autophagy pathway in rice. Here we cloned two autophagy genes, OsAtg8 and 0sAtg4, from rice. Express pattern analysis revealed that OsAtg8 and OsAtg4 were expressed in young leaf, mature leaf, young root, mature root, leaf sheath and spike. To determine whether the genes have any characterization similar to the yeast orthologs, OsAtg8 was cloned into a pGBKT7 vector and expressed in yeast. Western blotting analysis revealed that the C terminus of OsAtg8 was cleared off. Mutation analysis revealed that the C terminus of OsAtg8-G117A wasn' t removed, impling that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacts with 0sAtg4, and this interaction was not affected by the conserved Gly117 mutation. These data implied that the autophagy pathway is evolutionarily conserved in rice.