Figure 1.
Schematic representation of BIA pathway.
Biosynthetic pathway and corresponding enzymes leading to synthesis of benzylisoquinolines (papaverine, reticuline, laudanine), morphinans (thebaine, oripavine, codeine, morphine), protoberberines (berberine), phthalideisoquinolines (narcotoline, noscapine), benzophenanthridines (sanguinarine) and papaverrubine. Abbreviations: TYDC tyrosine/dopa decarboxylase; TyrAT tyrosine aminotransferase; NCS norcoclaurine synthase; 6OMT (S)-norcoclaurine 6-O-methyltransferase; CNMT (S)-coclaurine Nmethyltransferase; NMCH (S)-N-methylcoclaurine 3-hydroxylase; 4-OMT (S)-30-hydroxy-N-methylcoclaurine 4-O-methyltransferase; N7OMT norreticuline 7-O-methyltransferase; 7OMT reticuline 7-Omethyltransferase; BBE berberine bridge enzyme; SOMT scoulerine 9-O-methyltransferase; CAS canadine synthase; STOX (S)-tetrahydroxyprotoberberine oxidase; CoOMT Columbamine O-methyltransferase; CheSyn cheilanthifoline synthase; StySyn stylopine synthase; TNMT tetrahydroprotoberberine N-methyltransferase; MSH methylstylopine hydroxylase; P6H protopine 6-hydroxylase; DBOX dihydrobenzophenanthridine oxidase; DRS 1,2-dehydroreticuline synthase; DRR 1,2-dehydroreticuline reductase; SalSyn salutaridine synthase; SalR salutaridine reductase; SalAT salutaridinol 7-O-acetyltransferase; T6ODM thebaine 6-O-demethylase; COR codeinone reductase; CODM codeine O-demethylase.
Figure 2.
Classification and expression analysis of ESTs.
(A) Functional classification of expressed sequence tags (ESTs) generated after 5 hours of wound treatment in Opium Poppy. (B) Semi-quantitative RT-PCR (reverse transcription PCR) analysis of selected ESTs, confirmed increased accumulation of transcripts after 3 and 5 hours of wounding (C). Wound induced transcripts were also monitored after 3 and 5 hours in response to exogenous application of 50 μM methyl jasmonate. Actin and rRNA were used as loading controls.
Table 1.
A List of total 80 ESTs obtained after repetitive round of subtractive cDNA library preparation in response to 5 hours of wounding whose fold expression was determined by nylon filter hybridization.
Figure 3.
Real Time expression analysis of selected BIAs biosynthetic pathway genes.
(A) The expression analysis of transcript level of BIAs biosynthetic pathway genes after 1, 3, and 5 hours of wounding. Intact seedlings without wounding were taken as control at respective time points. (B) Real time expression analysis of BIAs pathway genes after 3 hours of exogenous application of 50 μM methyl jasmonate. Intact seedlings treated with an equal volume of ethanol in 0.1% triton X-100 was taken as control. The data represented means of triplicate biological and experimental repeats, error bars represented SDs.
Figure 4.
Analysis of benzylisoquinoline alkaloids.
Morphine, narcotine, papaverine and thebaine were analyzed after 5 hours of wounding in different tissues (straw, capsule, and leaf) of Opium Poppy. The data represented in % content/gram of dry weight. Data was an average of two independent quantifications repeated in triplicate and error bars represented SDs.
Figure 5.
Phylogenetic analysis of PsWRKY.
phylogenetic tree of PsWRKY and selected WRKY family proteins from other plant species was constructed by Neighbor joining method using MEGA5 software. The statistical reliability assessed by bootstrap value provided along with the tree. Selected WRKY proteins, respective plant species and GenBank accession numbers are as follows: Nbdouble WRKY [Nicotiana benthamiana double WRKY, Accession no. BAI63296.1], NtWRKY2 [Nicotiana tabacum, Accession no. BAA77383.1], NaWRKY3 [Nicotiana attenuata, Accession no. AAS13439.1], CaWRKY [Capsicum annuum, Accession no. ABD65255.1], StWRKY [Solanum tuberosum, Accession no. BAI63294.1], SPF1 protein [Ipomoea batatas, Accession no. ABD65255.1], MtWRKY [Medicago trucatula, Accession no. XP_003615949.1], CrWRKY1 [Catharanthus roseus, Accession no. ADT82685.1].
Figure 6.
Real time expression analysis of PsWRKY under control and different stress conditions in Opium poppy.
Figure 7.
Transactivation of reporter genes in yeast by PsWRKY protein.
PsWRKY cDNA cloned in yeast expression vector (pGBKT7) fused with GAL4 DNA BD, was transformed into yeast strain carrying three reporter genes, HIS3, ADE2, and LacZ, under the control of the GAL4 promoter. (A) Growth of Yeast colonies carrying no vector (Con), vector only (Vec), and two transformants (PsWRKY-1 and PsWRKY-2) having WRKY on YPDA (B) and on synthetic medium lacking His and Ade (C). Activation of the third reporter gene as in (D) was shown by β-galactosidase assay of the transformants using ONPG.
Figure 8.
Gel-retardation assay demonstrating that PsWRKY protein binds to the W-box probe.
The probes (1 ng) used in all reactions were 32P-labeled dimers of the oligonucleotides shown in W-box1 and W-box2. The two consensus cis-motifs with sequence TGACT and TGACC predominantly identified in BIAs pathway gene promoters were used in the oligonucleotide. Recombinant PsWRKY protein expressed in E. coli Bl21 (DE3) in fusion with GST was purified with GST-agarose columns. PsWRKY cloned in antisense orientation was also purified and used as a negative control.
Figure 9.
Activation of TYDC promoter by PsWRKY protein in yeast S cerevisiae AH109 (HIS−LEU−) and tobacco BY2 Cells.
PsWRKY cDNA cloned with LEU marker under a constitutive alcohol dehydrogenase promoter was transformed. The resulting transformants were further transformed with TYDC promoter harboring HIS marker. (A) The transfrormants were grown at 30°C on YPD and (B) synthetic drop out medium lacking the histidine and leucine. Two individual transformants carrying PsWRKY are shown. (C) Activity of GUS reporter construct fused to tydc 5′-upstream activating sequence in tobacco BY2 cells harboring empty vector or PsWRKY. The promoter–GUS construct was introduced through Agrobacterium into the tobacco BY2 cells already transformed with control vector or PsWRKY. GUS activity level in the PsWRKY-expressing protoplasts relative to that in the control BY2 cells is presented. The data presented means of triplicate transformation repeats and error bars represented SDs.
Figure 10.
Biosynthetic pathways showing the expression of BIA transcripts in Papaver somniferum after wounding and methyl jasmonate treatment.
Enzymes shown in blue are commonly induced by both wounding as well as methyl jasmonate. Enzymes shown in red are induced by methyl jasmonate only and enzymes in green are induced by wounding only. Fold induction was represented in comparison to actin.