Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

New Breeding Techniques (NBTs) represent the newest technology for introduction of new variation into crop plants for plant breeding, in particular the methods that aim to make targeted mutagenesis at specific sites in the plant genome (NBT mutagenesis). In a decision from 2018, the EU Court of Justice deemed plants modified by these techniques to be GMO. The decision was based in a context of conventional plant breeding using mutagenesis of crop plants by physical or chemical treatments. These plants are explicitly exempted from the EU GMO regulation, based on the long-termed use of mutagenesis. In its decision, the EU Court claims that the NBTs operate “at a rate out of all proportion to those resulting from the application of conventional methods of mutagenesis”. In this paper, we argue that in fact this is not the case anymore; instead, a convergence has taken place between conventional mutagenesis and NBTs, in particular due to the possibilities of TILLING methods that allow the fast detection of mutations in any gene of a genome. Thus, by both strategies mutations in any gene across the genome can be obtained at a rather high speed. However, the differences between the strategies are 1) the precision of the exact site of mutation in a target gene, and 2) the number of off-target mutations affecting other genes than the target gene. Both aspects favour the NBT methods, which provide more precision and fewer off-target mutations. This is in stark contrast to the different status of the two technologies with respect to EU GMO regulation. In the future, this situation is not sustainable for the European plant breeding industry, since it is expected that restrictions on the use of NBTs will be weaker outside Europe. This calls for reconsiderations of the EU regulation of plants generated via NBTs.

Heterotrimeric G proteins are intracellular membrane-attached signal transducers involved in various cellular processes in both plants and animals. They consist of three subunits denoted as α, β and γ. The γ-subunits of the so-called AGG3 type, which comprise a transmembrane domain, are exclusively found in plants. In model species, these proteins have been shown to participate in the control of plant height, branching and seed size and could therefore impact the harvestable yield of various crop plants. Whether AGG3-type γ-subunits influence yield in temperate cereals like barley and wheat remains unknown. Using a transgenic complementation approach, we show here that the Scottish malting barley cultivar (cv.) Golden Promise carries a loss-of-function mutation in HvDep1, an AGG3-type subunit encoding gene that positively regulates culm elongation and seed size in barley. Somewhat intriguingly, agronomic field data collected over a 12-year period reveals that the HvDep1 loss-of-function mutation in cv. Golden Promise has the potential to confer either a significant increase or decrease in harvestable yield depending on the environment. Our results confirm the role of AGG3-type subunit-encoding genes in shaping plant architecture, but interestingly also indicate that the impact HvDep1 has on yield in barley is both genotypically and environmentally sensitive. This may explain why widespread exploitation of variation in AGG3-type subunit-encoding genes has not occurred in temperate cereals while in rice the DEP1 locus is widely exploited to improve harvestable yield.