Transgenic tobacco and Arabidopsis plants expressing the two multifunctional sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesis

Abstract

Novel cyanogenic plants have been generated by the simultaneous expression of the two multifunctional sorghum (Sorghum bicolor [L.] Moench) cytochrome P450 enzymes CYP79A1 and CYP71E1 in tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis under the regulation of the constitutive 35S promoter. CYP79A1 and CYP71E1 catalyze the conversion of the parent amino acid tyrosine to p-hydroxymandelonitrile, the aglycone of the cyanogenic glucoside dhurrin. CYP79A1 catalyzes the conversion of tyrosine to p-hydroxyphenylacetaldoxime and CYP71E1, the subsequent conversion to p-hydroxymandelonitrile. p-Hydroxymandelonitrile is labile and dissociates into p-hydroxybenzaldehyde and hydrogen cyanide, the same products released from dhurrin upon cell disruption as a result of pest or herbivore attack. In transgenic plants expressing CYP79A1 as well as CYP71E1, the activity of CYP79A1 is higher than that of CYP71E1, resulting in the accumulation of several p-hydroxyphenylacetaldoxime-derived products in the addition to those derived from p-hydroxymandelonitrile. Transgenic tobacco and Arabidopsis plants expressing only CYP79A1 accumulate the same p-hydroxyphenylacetaldoxime-derived products as transgenic plants expressing both sorghum cytochrome P450 enzymes. In addition, the transgenic CYP79A1 Arabidopsis plants accumulate large amounts of p-hydroxybenzylglucosinolate. In transgenic Arabidopsis expressing CYP71E1, this enzyme and the enzymes of the pre-existing glucosinolate pathway compete for the p-hydroxyphenylacetaldoxime as substrate, resulting in the formation of small amounts of p-hydroxybenzylglucosinolate. Cyanogenic glucosides are phytoanticipins, and the present study demonstrates the feasibility of expressing cyanogenic compounds in new plant species by gene transfer technology to improve pest and disease resistance.

Figure 1

The biosynthetic pathway of the cyanogenic glucoside dhurrin. In sorghum, the pathway is catalyzed by CYP79A1 and CYP71E1, two multifunctional membrane-bound P450s, and by a soluble UDPG-glucosyltransferase. The glucosylated intermediates and metabolites that are detected in transgenic Arabidopsis and tobacco expressing one or both P450s are indicated. The retention time (rt) of the TMS derivatives observed upon GC analysis is shown below each structure.

Figure 2

Expression of CYP79A1 and CYP71E1 in transgenic Arabidopsis and tobacco. Microsomes isolated from selected transgenic lines were incubated with either radiolabeled Tyr (Y) or radiolabeled p-hydroxyphenylacetaldoxime (Ox). After incubation, the reaction mixtures were extracted with ethyl acetate and the ethyl acetate extracts analyzed by TLC. −, Purity of the radiolabeled p-hydroxy-phenylacetaldoxime precursor used. Plants expressing CYP79A1 (79) catalyze the conversion of Tyr to p-hydroxyphenylacetaldoxime (Oxime). Plants expressing both CYP79A1 and CYP71E1 (2×) catalyzethe conversion of Tyr to p-hydroxyphenylacetaldoxime and further conversion of p-hydroxyphenylacetaldoxime to p-hydroxymande-lonitrile. p-Hydroxymandelonitrile is detected as its decomposition product p-hydroxybenzaldehyde (Aldehyde).

Figure 3

Tyr-derived glycosylated metabolites in transgenic tobacco plants expressing CYP79A1 and CYP71E1 (2×) as analyzed after deglucosylation, using either β-glucosidase (B) or Viscozym L (V). Aglucones were extracted into ethyl acetate and separated by TLC.

Figure 4

GC-CIMS analysis of Tyr-derived glucosides in transgenic Arabidopsis and tobacco lines. A and B, Comparison of the total ion trace versus that of m/z 361 using methanol extract prepared from an Arabidopsis plant expressing CYP79A1 and CYP71E1 (2×). C to E, m/z 361 ion trace of wild-type Arabidopsis (C) and transgenic Arabidopsis expressing CYP79A1 (79) (D) or CYP79A1 + CYP71E1 (2×) (E). F to H, m/z 361 ion trace of wild-type tobacco (F) and transgenic tobacco expressing CYP79A1 (79) (G) or CYP79A1 + CYP71E1 (2×) (H). Suc 21.7 min. p-Hydroxybenzylglucosinolate, 25.7 min (1); p-glucosyloxy-phenylethanol, 23.7 min (2); p-glucosyloxy-phenylacetonitrile, 23.9 min (3); p-hydroxyphenyl-(acetaldoxime glucoside), 24.1 min (4); p-glucosyloxy-benzaldehyde, 21.9 min (5); p-glucosyloxy-phenylmethanol, 22.6 min (6); glucosyl p-hydroxybenzoate, 23.3 min (7); and p-glucosyloxy-benzoic acid, 24.4 min (8).

Figure 5

Intermediates in dhurrin biosynthesis accumulate in transgenic tobacco. Radiolabeled Tyr was administered to detached tobacco leaves from the highest expresser of CYP79A1 (79), from the highest and two additional expressers of both CYP79A1 and CYP71E1 (2×), and from a control line (wt). After 18 h of incubation, intermediates were extracted and analyzed by TLC.

Figure 6

Cyanide potential of leaves and methanol extracts of transgenic and wild-type tobacco. Cyanide released was measured colorimetrically. 2×, Tobacco expressing both CYP79A1 and CYP71E1; 79, tobacco expressing CYP79A1; wt, tobacco control plant transformed with pPZP111.