Fig. 3: Silencing of DpCYP87A106 by RNAi in D. purpurea confirms its role in cardenolide biosynthesis.
From: Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants
a,b, De novo production of pregnenolone in transgenic Arabidopsis leaves overexpressing DpCYP87A106 (red) (a) or CpCYP87A103 (blue) (b) compared with wild-type leaves (black). Lines 2 and 3 are independent DpCYP87A106-Ox transgenic Arabidopsis lines, whereas lines 1 and 3 are independent CpCYP87A103-Ox transgenic Arabidopsis lines. Extracted ion chromatograms are shown. GC–MS was used for sterol profiling. c, Levels of pregnenolone in leaves of DpCYP87A106-RNAi transgenic D. purpurea lines compared with wild type. Pregnenolone was not detected in the DpCYP87A106-RNAi lines. d, Knockdown of DpCYP87A106 resulted in almost complete loss of cardenolides in transgenic D. purpurea leaves. The relative cardenolide levels shown are the sum of the peak areas obtained from six cardenolides (verdoxin, digitoxigenin fucoside, digitoxin, purpurea glycoside A, purpurea glycoside B and glucogitaloxin) that typically accumulate in D. purpurea10. Four independent DpCYP87A106-RNAi transgenic D. purpurea lines (lines 1, 2, 3 and 4) were generated using a stable transformation approach and analysed in the T0 generation. Values in c and d indicate means ± s.e.m. of biological replicates (n = 4 for wild type and n ≥ 2 for individual transgenic lines). Biological replicates (for example, for line 1, n = 2) refer to the individual plants that emerged from the same callus explant. Asterisks indicate significant changes compared with wild-type samples, as calculated by two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001. e, Phylogenetic context of CYP87A proteins obtained from cardenolide-producing and cardenolide-free plants. The percentage identity at the amino acid level for each CYP87A protein compared with DpCYP87A106 is presented. The blue squares (with the + sign) indicate presence of chemotype (that is, cardenolide) in a particular plant species. In planta pregnenolone-producing activity of CYP87A clade proteins seen in N. benthamiana transient expression experiments is shown by red squares (with the + sign). Increased pregnenolone accumulation by three CYP87A proteins (marked in red), EcCYP87A126, DpCYP87A106 and CpCYP87A103, correlated well with the presence of cardenolides in these corresponding plant species. The asterisk represents negligible pregnenolone-formation activity in transient experiments by CYP87A enzymes (for example, tomato) from non-cardenolide-producing plants. CYP87A sequences from the following species were used in phylogenetic analysis: S. lycopersicum (Sl; tomato), N. benthamiana (Nb), O. sativa (Os; rice), A. thaliana, S. indicum (Si; sesame), O. europaea (Oe; olive), E. cheiranthoides, D. purpurea, C. procera and H. sapiens (humans). The amino acid sequences used in the phylogenetic analysis are provided in Supplementary Data 1. Percentage bootstrap values are shown at the nodes of each branch. Scale bar represents branch lengths measured in the number of amino acid substitutions per site.
