Abstract
Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.
Highlights
Which is labile in non-acidic environments due to the ionization of the hydroxyl group on the benzene ring[6], is stably stored in the acidic vacuolar compartment but the mechanism of its intracellular transport from the endoplasmic reticulum (ER) to the vacuole is unknown[7]
We report that the biosynthetic gene cluster for dhurrin includes a gene encoding a tonoplast localised multidrug and toxic compound extrusion (MATE) transporter for dhurrin uptake, demonstrating that the analysis of plant gene clusters can contribute to transporter identification
The genes showing the highest co-expression with CYP79A1, encoding the first enzyme of the dhurrin biosynthetic pathway, were CYP71E1, immediately followed by SbMATE2 (Fig. 1b, Supplementary Table 1)
Summary
Our results demonstrate the presence of a non-biosynthetic component, the SbMATE2 gene encoding a vacuolar transporter for dhurrin, in the gene cluster for a plant chemical defence pathway. Its inclusion in the dhurrin biosynthetic gene cluster is consistent with ideas that selection for reduced recombination between beneficially interacting alleles leads to gene cluster formation[15,16,41] Such selection is proposed to result from antagonistic selection pressures, such as the benefits maintaining a functional pathway provides in specific ecological context, e.g. the presence of non-adapted herbivores, against the trade-off costs associated with it[1,15]. Apart from the catabolic genes, the DAL cluster contains the DAL4 gene encoding an allantoin permease Given these examples from fungi, it can be expected that the future detailed analysis of genomic regions containing gene clusters for plant specialised metabolites will contribute to the identification of additional non-biosynthetic pathway components such as regulators or transporters
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