ROS mediated program cell death in rice endosperm development: in situ evidence through TUNEL [Terminal deoxynucleotidyl transferase dUTP Nick End Labeling]

Apoptosis or program cell death (PCD) is a crucial component of plant development. Cell death in cereal endosperm is characterized by an increase in nuclease activity and internucleosomal fragmentation of nuclear DNA. Endosperm development in rice, a highly metabolic activity is accompanied b the generation of reactive oxygen intermediates (R0I), mediating PCD. The authors demonstrated earlier that a germin-like protein (GLP, OsGLP8960) was an essential gene for endosperm formation. This gene was shown to be developmentally regulated and largely spikelet-specific. It codes for a bifunctional protein, which acts both as superoxide dismutase (SOD) and oxalate oxidase (OXO). The product of both these enzyme activities is hydrogen peroxide (H2O2). To determine the effect of GLP in program cell death during rice endosperm development, TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labelling) assay was conducted in a control genotype Nipponbare, as well as in plants homozygous and heterozygous for OsGLP8960 insertion mutation with the maize Spm transposon. This assay employs the labeling of fragmented double stranded DNA in the free 3'OH termini with a modified nucleotide. Fluorescein, a commonly used labeling fluorophore, is then detected using anti-flourescein antibody Fag fragments from sheep and viewed using epifluorescence microscopy. The assay was done in rice endosperm at 10, 16 and 22 days after fertilization (DAF). Results showed that more double stranded DNA breaks were detected in highest number at 22 DAF across genotype. This confirmed the gradual progression of PCD in rice endosperm as the grain matures. Results also showed that Nipponbare control has the most number of double stranded DNA breaks compared with plants homozygous for maize Spm transposon insertion mutation. This was reflected by the intensity of fluorophore detected at a particular given stage. This suggests that GLP-mediated release of H2O2 through SOD and/or OXO activity regulates the rate of PCD and hence influences proper grain maturation.