Rice bran oil is good quality edible oil, rich in antioxidants and comprised typically of oleic-linoleic type fatty acids. However, presence of a highly lipolytic enzyme Phospholipase D alpha1 (OsPLDα1) increases free fatty acid content in the oil which further leads to stale flavor and rancidity of the oil, making it unfit for human consumption. In this study, we compared the upstream regions of OsPLDα1 orthologs across 34 accessions representing 5 wild Oryza species and 8 cultivars, to uncover sequence variations and identify cis-elements involved in differential transcription of orthologs. Alignment of the upstream sequences to the Nipponbare OsPLDα1 reference sequence revealed the presence of 39 SNPs. Phylogenetic analysis showed that all the selected cultivars and wild species accessions are closely related to the reference except for three accessions of O. rufipogon (IRGC89224, IRGC104425, and IRGC105902). Furthermore, using exon-specific qRT-PCR, OsPLDα1 expression patterns in immature grains indicated significant differences in transcript abundance between the wild species accessions. In comparison to the control, lowest gene expression was observed in IRGC89224 accession (0.20-fold) followed by IRGC105902 (0.26-fold) and IRGC104425 (0.41-fold) accessions. In-silico analysis of the OsPLDα1 promoter revealed that the copy number variations of CGCGBOXAT, GT1CONSENSUS, IBOXCORE, NODCON2GM, OSE2ROOTNODULE, SURECOREATSULTR11, and SORLIP1AT cis-elements play an important role in the transcriptional activities of orthologous genes. Owing to the presence of ARFAT and SEBF elements only in the IRGC89224 accession, which had the lowest gene expression as well, these putative upstream regulatory sequences have been identified as novel cis-elements which may act as repressors in regulating the OsPLDα1 gene expression. The accessions identified with low OsPLDα1 expressions could be further deployed as potential donors of ideal OsPLDα1 allele for transfer of the desired trait into elite rice cultivars.
Keywords: Oryza; Phospholipase D; Promoter mining; cis-Elements; qRT-PCR.