The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy

Abstract

Noscapine is an antitumor alkaloid produced in opium poppy (Papaver somniferum) and some members of the Papaveraceae family. It has been primarily used for its antitussive effects; more recently, its anticancer properties were shown. Herein, we detected an SSR embedded in the promoter region of the CYP82Y1 gene, which was found to be the first committed-step enzyme in the noscapine biosynthesis pathway, using the MISA program. Some collected ecotypes of P. somniferum were investigated for understanding of SSRs role in the regulation of gene expression and metabolite content. Quantitative PCR showed that a variation in the motif repeat number (either a decrease or increase) down-regulated the expression of the CYP82Y1 gene. Furthermore, the analysis of noscapine content suggested that a variation in the promoter region influence noscapine amount. Moreover, P. bracteatum was analyzed in both transcript and metabolite levels, and illustrated much less expression and metabolite level in comparison to P. somniferum. By exploiting the transcriptome data from the eight genera of the Papaveraceae family, we found that noscapine biosynthesis genes are present in P. bracteatum and are not shared in other genera of the Papaveraceae family. This results may explain production of a confined metabolite within a genus.

Figure 1

Agarose gel electrophoresis of PCR amplified of detected SSR located in CYP82Y1 upstream region from different collected ecotypes of opium poppy from Iran; (Ps#1-Ps#16) P. somniferum L. ecotypes. M; 100 bp DNA ladder (a). Sequence alignment of CYP82Y1 promoter region (approximately 200 bp) of the four P. somniferum L ecotypes, ‘Ps#7’, ‘Ps#3’, ‘Ps#6’ and ‘Ps#12’. Variation in the number of AT motifs is distinctly observed. AT motifs are colored in red in three ecotypes, which is also considered as a putative TATA-box. The translation start codon (ATG) was also highlighted in yellow. The blue sequences indicates a part of CDS, first exon (b).

Figure 2

Agarose gel electrophoresis of PCR amplified of detected SSR embedded in upstream region of MT1 gene from different ecotypes of opium poppy; (‘Ps#1’-‘Ps#16’) P. somniferum L. ecotypes. M; 250 bp DNA ladder.

Figure 3

Relative abundance of CYP82Y1, MT1 and SDR1 transcripts in some geographical collected ecotypes of P.somniferum L. (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum from Iran in stem (a) and leaf (b) tissues.

Figure 4

Amount of noscapine in stem tissue from collected ecotypes of P.somniferum (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum.

Figure 5

Unrooted neighbor-joining phylogenetic tree for selected CYP82s, constructed using MEGA 7 software. Bootstrap frequencies for each clade were based on 1,000 iterations (a). Comparison of substrate recognition site across some CYP82s from Papaveraceae family. The red rectangle represents YPASXXXER conserved motif that is present in PsCYP82Y1 and PbCYP82Y1 (b). Abbreviated species names are given before gene identifiers for each protein related to CYP82Y1 are as follows: PbCYP82Y1, P. bracteatum CYP82Y1; PsCYP82Y1, P. somniferum (AFB74617); NtCYP82E4v2, Nicotiana tabacum nicotine demethylase (ABN42695.1); NtCYP82E3, N. tomentosiformis nicotine demethylase (ABM46919.1); PsCYP82N4, P. somniferum (L7X3S1.1); GmCYP82C1p, Glycine max cytochrome P450 (AAB94590.1); AlCYP82G1, Arabidopsis lyrata DMNT/TMTT homoterpene synthase (XP_002885694.1); PsCYP82X1, P. somniferum CYP82X1 (AFB74614); PbCYP82X1, P. bracteatum CYP82X1; AmCYP82Y1, Argemone Mexicana; CmCYP82Y1, Chelidonium majus; CcCYP82Y1, Corydalis cheilanthifolia; EcCYP82Y1, Eschscholzia californica; GfCYP82Y1, Glaucium flavum; ScCYP82Y1, Sanguinaria canadensis; SdCYP82Y1, Stylophorum diphyllum.