Fig 1.
BIA biosynthetic pathways in BIA-producing plants.
Black and gray letters show biosynthetic enzymes in C. japonica and E. californica, respectively. Broken lines indicate uncharacterized enzyme reactions. NCS, (S)-norcoclaurine synthase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; CNMT, (S)-coclaurine-N-methyltransferase; CYP80B1, (S)-N-methylcoclaurine 3’-hydroxylase; 4’OMT, (S)-3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase; BBE, berberine bridge enzyme; SMT, (S)-scoulerine 9-O-methyltransferase of C. japonica; CYP719A1, (S)-canadine synthase; THBO, (S)-tetrahydroprotoberberine oxidase; CYP719A5, (S)-cheilanthifoline synthase; CYP719A2/3, (S)-stylopine synthase; TNMT, (S)-tetrahydroprotoberberine cis-N-methyltransferase, MSH, (S)-N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase; SR, sanguinarine reductase; G3OMT, (S)-scoulerine 9-O-methyltransferase of California poppy; G11OMT, putative 10-hydroxydihydrosanguinarine 10-O-methyltransferase. CYP1 and CYP2 are putative dihydrobenzophenanthridine 10-hydroxylases.
Fig 2.
CjWRKY1-overexpressing transgenic California poppy cells were established.
(A) Three transgenic cell lines were cultured for 5 days. (B) The medium from CjWRKY1-OX cell lines showed clear brown pigmentation. (C) Constitutive expression of CjWRKY1 in transgenic cell lines. The transcript level of CjWRKY1 was determined by quantitative RT-PCR. Nine biological replicates were used of each cell line.
Fig 3.
Effects of CjWRKY1-overexpression on genes encoding BIA biosynthetic enzymes in transgenic California poppy cell lines.
The transcript levels of Ec6OMT, EcCYP80B1, Ec4’OMT, EcBBE, EcCYP719A5, EcCYP719A2, EcCYP719A3, EcTNMT, EcMSH, EcP6H, EcDBOX, EcSR, EcG3OMT, and EcG11OMT were determined by quantitative RT-PCR. Nine biological replicates were used of each cell line. Asterisks indicate a significant difference compared with VC cell lines (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed paired Student’s t-test).
Fig 4.
Effects of overexpression of the CjWRKY1 gene on bHLH transcription factor genes in transgenic California poppy cell lines.
The expression levels of EcbHLH1-1 and EcbHLH1-2 were determined by quantitative RT-PCR. Nine biological replicates were used of each cell line. An asterisk indicates a significant difference compared with VC cell lines (*P < 0.05; two-tailed paired Student’s t-test).
Fig 5.
The accumulation of BIAs in the medium from transgenic California poppy cell lines.
The contents of sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, 10-hydroxychelerythrine, and reticuline were estimated based on the peak areas using the standard curve created from an authentic sanguinarine sample. For each cell line, nine biological replicates were used. Asterisks indicate significant differences from VC cell lines (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed paired Student’s t-test).
Fig 6.
The accumulation of representative BIAs in transgenic cells of California poppy.
The contents of sanguinarine, chelerythrine, chelirubine, protopine, and 10-hydroxychelerythrine in cell extract were calculated using the standard curve of sanguinarine. Nine biological replicates were used of cell line. Asterisks indicate significant differences from VC cells (**P < 0.01, two-tailed paired Student’s t-test).
Fig 7.
Correlation analysis of the expression of genes encoding biosynthetic enzymes and the accumulation of total alkaloids in flasks growing transgenic California poppy cells.
Asterisks indicate significant correlations (df = 18; *P < 0.05, **P < 0.01, ***P < 0.001).