Identification & characterization of a rice spikelet-specific germin like protein : is superoxide dismutase activity its only role?

Reactive oxygen species (ROS) such as superoxide, peroxide, hydroxyl ion and singlet oxygen radical are by-products of specific metabolic reactions in plants. ROS damage cellular biomolecules and are thus actively scavenged. However, ROS also act as signal transducers during development and stress responses. Redox enzymes such as superoxide dismutase (SOD) contribute to achieving a balance between ROS being scavenged and being available as signal transducers. Spatio-temporally differential expression of SODs in various tissues plays a critical role in scavenging the superoxide radical. Using a nitroblue-tetrazolium-riboflavin-based in-gel SOD assay, up-regulation of two low mobility (~96 kDa and ~92 kDa) putative SODs was observed in rice spikelets. The two proteins were purified by gel electrophoresis and both were identified through mass spectrometry as homotetrameric versions of a single germin-like protein (GLP), encoded by the gene LaC _Os08g08960 (Rice Genome Annotation Project). Staining with a glycoprotein gel stain and enzymatic deglycosylation showed that the protein is post-translationally modified by N-glycosylation; differential modification probably accounts for_the difference between the two forms. Biochemical characterization- of this developmentally regulated spikelet-specific GLP (ssGLP) indicated that its SOD activity is Mn-SOD type and is highly stable in denaturing agents and at elevated temperatures. Tetrameric ssGLP lacks oxalate oxidase activity. The biological functions of GLPs have not been fully defined; among the different functions attributed to them their role in biotic stress tolerance is relatively well studied. Rice germins, especially some of those with genes on chromosome 8 in the same cluster as the LaC _Os08g08960 gene, although not this specific gene, have previously been found to contribute to the basal mechanism of resistance to pathogens causing rice blast and sheath blight disease. ssGLP was estimated to contribute nearly 11% of the total SOD activity in rice spikelets, indicating a crucial developmental role. Expression analysis with the in-gel SOD assay at the spatio-temporal level, as well as treatment with various hormones, chemicals and abiotic variables such as wounding and light indicated that the expression of ssGLP is affected by developmental rather than stress stimuli. Further, expression analysis at the spatio-temporallevel through RT-PCR confirmed the developmental patterns observed previously at the protein level. In situ mRNA hybridization studies provided evidence for ssGLP gene expression in pollen and endosperm and in localized regions of leaves, and for production of antisense RNA. Consistent with the up-regulation in spikelets, in silico comparison of the rice GLP promoters revealed more seed and pollen specific motifs in LOC_Os08g08960 than in other GLP genes. Additionally, presence of a GAF protein domain typical of cyclic nucleotide phosphodiesterases is unique to ssGLP. Preliminary enzyme assays and in silico results suggested that ssGLP may act as a nucleotide-sugar pyrophosphatase/phosphodiesterase (NSPPase) similarly to a previously-described barley GLP. As a first step to a fuller definition of the enzyme activity of ssGLP and in particular to testing the hypothesis that a different oligomeric configuration acts as NSPPase, a gene for recombinant ssGLP was constructed, cloned and expressed in Pichia pastoris.