Detection and analysis of Clp protease mRNA in rice using RT-PCT and Northern hybridization

Towards a better understanding of leaf senescence in rice, leaves of seedlings were analyzed for the presence of Clp protease mRNA. This ATP-dependent protease has been found to be highly conserved in various eukaryotes and prokaryotes. Their presence and high degree of conservation among various prokaryotes and eukaryotes implies that they have an important role to play. High degree of homology was found between the wheat Orf216, encoding the ClpPgene, and rice chloroplast genome gene. The ClpP gene encodes the small catalytic subunit of the Clp protease. Further homology search revealed the presence of a sequence in rice which was homologous to the tomato CD4A and CD4B genes encoding ATP-dependent proteases. This sequence from rice was found to be highly conserved in various eukaryotic plants encoding the ClpC subunit of the Clp protease. Primer pairs for the two subunits were based on these homologous, highly conserved sequences. RT-PCR with total RNA from the rice leaves revealed the absence of intronds in the ClpP gene and also the presence of a family of genes encoding ClpC-like proteases in rice. Primers based on the Arabidopsis thaliana ERD1 sequence, which encodes a ClpC-like protease, amplified two bands of sizes smaller than expected. The levels of these subunits, detected by Northern hybridization, was found to be more in light-grown than in etiolated seedlings. The amount of the ClpC transcripts was seen to increase once the etiolated seedling were transferred into light, similar to what was observed for Rubisco large subunit levels. ClpP, the smaller subunit could be detected in the light-grown seedlings, but was not so detectable in etiolated seedlings, although it was present. Western blot analysis cone with anti-tobacco ClpP and anti-pea ClpC- antibodies was negative for the presence of both subunits in seedlings