Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome

Abstract

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.

Fig. 1.

Biosynthesis of the sorghum-derived cyanogenic glucoside dhurrin and glucosinolates in transgenic A. thaliana plants. CYP79A1, CYP71E1, and UGT85B1 are enzymes encoded by the introduced sorghum transgenes. The dashed arrow illustrates metabolic crosstalk resulting from interaction of sorghum CYP79A1 with preexisting endogenous post-oxime-metabolizing enzymes (CYP83B1, SUR1, a sulfotransferase, and UGT74B1), enabling use of p-hydroxyphenylacetaldoxime produced by CYP79A1 by the post-oxime-metabolizing enzymes in the preexisting glucosinolate biosynthetic pathway. The presence of CYP71E1 prevents this interaction.

Fig. 2.

Comparison of plant morphology and metabolite composition in wild-type A. thaliana and transgenic lines. Plants expressing sorghum CYP79A1 are designated 1x, plants expressing sorghum CYP79A1 and CYP71E1 are designated 2x, and plants expressing CYP79A1, CYP71E1, and UGT85B1 are designated 3x. Morphological phenotype (A) and metabolite profile as monitored as total ion trace (TIC) and extracted ion chromatographs (EIC) in the three transgenic lines are shown (B). (C) Close-ups of the extracted ion chromatograms to facilitate visualization of minor components. Compound numbers and absolute and relative changes are tabulated in Table 1.

Fig. 3.

Transcriptome analyses of wild-type versus transgenic A. thaliana lines. Scatterplots of log2-transformed signal intensities from Cy3- and Cy5-labeled mRNA isolated from wild-type (WT) or transgenic A. thaliana lines. (A) 1x lines. (B) 2x lines. (C) 3x lines. The focused array (A–C) contains probes for 453 selected genes, and the global array (D) contains 27,216 probes. Signals below two times the average local background are boxed. Phenylpropanoid marker genes are light blue and represented by circles. PAL, phenylalanine ammonia-lyase; CYP73A5, cinnamate 4-hydroxylase; CYP98A3, coumarate 3-hydroxylase; CYP84A1, ferulate 5-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase. Glucosinolate marker genes (CYP79s, CYP83A1, CYP83B1, SUR1, and UGT74B1) are pink and represented by triangles. The Sorghum transgenes are yellow and represented by squares. Genes scored by the sam analysis as up-regulated are red and genes scored as down-regulated are green (Table 2). Blue lines indicate 2- or 4-fold up- or down-regulated expression levels.

Fig. 4.

Number of differentially expressed genes (log₂-transformed) as a function of the threshold for selection of differentially expressed genes (δ value) in the three transgenic lines (1x,2x, and 3x) as analyzed by sam analysis of the focused oligonucleotide array. The vertical line indicates a δ value of 0.16. A δ value of 1 is equivalent to a 2-fold change in ratio.