Novel allelic variation in the Phospholipase D alpha1 gene (OsPLDα1) of wild Oryza species implies to its low expression in rice bran

Abstract

Rice bran, a by-product after milling, is a rich source of phytonutrients like oryzanols, tocopherols, tocotrienols, phytosterols, and dietary fibers. Moreover, exceptional properties of the rice bran oil make it unparalleled to other vegetable oils. However, a lipolytic enzyme Phospholipase D alpha1 (OsPLDα1) causes rancidity and 'stale flavor' in the oil, and thus limits the rice bran usage for human consumption. To improve the rice bran quality, sequence based allele mining at OsPLDα1 locus (3.6 Kb) was performed across 48 accessions representing 11 wild Oryza species, 8 accessions of African cultivated rice, and 7 Oryza sativa cultivars. From comparative sequence analysis, 216 SNPs and 30 InDels were detected at the OsPLDα1 locus. Phylogenetic analysis revealed 20 OsPLDα1 cDNA variants which further translated into 12 protein variants. The O. officinalis protein variant, when compared to Nipponbare, showed maximum variability comprising 22 amino acid substitutions and absence of two peptides and two β-sheets. Further, expression profiling indicated significant differences in transcript abundance within as well as between the OsPLDα1 variants. Also, a new OsPLDα1 transcript variant having third exon missing in it, Os01t0172400-06, has been revealed. An O. officinalis accession (IRGC101152) had lowest gene expression which suggests the presence of novel allele, named as OsPLDα1-1a (GenBank accession no. MF966931). The identified novel allele could be further deployed in the breeding programs to overcome rice bran rancidity in elite cultivars.

Figure 1

Evolutionary relationship across different wild species accessions and cultivars based on the nucleotide sequence of OsPLDα1 exons (a) first exon, (b) second exon, (c) third exon using a neighbor –joining algorithm calculated by boot-strap value of 1000 replicate.

Figure 2

Evolutionary relationship across different wild species accessions and cultivars based on the nucleotide sequence of OsPLDα1 introns (a) first intron, (b) second intron, (c) third intron, using a neighbor –joining algorithm calculated by boot-strap value of 1000 replicate.

Figure 3

Phylogenetic relationship of OsPLDα1 across Nipponbare, wild species accessions, and cultivars of rice based on the nucleotide sequence data of cDNA. Phylogenetic tree was generated using a neighbor - joining algorithm calculated by boot-strap value of 1000 replicate. The number 1–20 indicates 20 OsPLDα1 variants based on the nucleotide sequences of cDNA while the numbers I to XII indicate protein variants. ‘Ref’ denotes the nucleotide sequences variants which translates into the amino acid sequence same as that of the reference OsPLDα1 protein sequence of Nipponbare.

Figure 4

Three-dimensional structures of OsPLDα1 protein in (a) Nipponbare and (b) IR101152 accession of O. officinalis. (c) Superimposition of OsPLDα1 protein from Nipponbare and IR101152 accession of O. officinalis. Two β-strands (shown with arrows) were found missing in the IR101152 (depicted in pink color) upon superimposition with Nipponbare (depicted in blue color).

Figure 5

OsPLDα1 transcript levels in immature seeds from wild Oryza species accessions. Mean values for OsPLDα1 transcripts and standard deviation (S.D.) measured relative to Actin expression. Relative transcript levels of OsPLDα1, in accessions of wild Oryza species, for four qRT-PCR primers namely PLDE1 (designed from first exon of the gene), PLDE2.1 (designed from 5′end of second exon), PLDE2.2 (designed from 3′end of second exon), and PLDE3 (designed from third exon of the gene) are shown. Among all the wild species accessions, IR101152 accession of O. officinalis was found to have lowest transcript levels using all the four qRT-PCR primers.

Figure 6

Heatmap showing differential expression of OsPLDα1 transcripts between as well as within the accessions of wild Oryza species. PLDE1, PLDE2.1, PLDE2.2 and PLDE3 denotes the qRT-primers designed from exons of OsPLDα1 gene. Wild species accessions (horizontal) were hierarchially clustered (Pearson sorrelation, average linkage). Color patterns from green to red indicate low to high transcript levels, thus IRGC101152 have the lowest expression for all the four exon specific qRT primers.

Figure 7

Graphical representation of newly identified OsPLDα1 transcript variant, Os01t0172400-06. A new transcript form having only two exons was detected in the wild Oryza species accessions viz. O. barthii (IR104102 and IR106239), O. nivara (CR100126), O. glaumaepatula (IR104387), O. meridionalis (IR101146), and O. punctata (IR101434 and IR105158); and an accession of O. glaberrima (IR102489). Expression profiling in these accessions, using exon specific qRT-PCR primers, showed the low abundance of transcripts having third exon.