Bibliografisk note

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We previously showed that SbGSTL1 and SbGSTL2 have stereospecific catalytic activity towards the glutathione conjugate in the dhurrin recycling pathway (Fig. 1) (Bjarnholt et al., 2018). They are 98% identical at the amino acid level and formed a specific subclade in a phylogenetic tree of GSTLs. Sorghum harbors two additional GSTLs, that have respectively no (GSTL3) or low and non-stereospecific (GSTL4) activity against the dhurrin-derived glutathione conjugate. In addition, SbGSTL3 belongs to a subclade, from which TaGSTL1 (Triticum aestivum; wheat) likewise has no activity against the dhurrin derived conjugate, while TaGSTLs and AtGSTLs (Arabidopsis thaliana; Arabidopsis) that group in other subclades have the same low and unspecific activity as SbGSTL4 (Bjarnholt et al., 2018). From barley, Rezaei et al. (2013) previously identified two GSTLs, namely HvGSTL1 (HORVU4Hr1G057890.5) and HvGSTL2 (HORVU4Hr1G057910.1). Using all GSTL sequences from the phylogenetic tree of Bjarnholt et al. (2018) as bait, we conducted a BLAST search of the 2016 version of the barley genome in the Barlex database (https://apex.ipk-gatersleben.de/apex/f?p=284:49) and identified two additional GSTLs, (HORVU4Hr1G057740.1 and HORVU4Hr1G057930.3). The latter was not expressed in the germinating barley grain (Betts et al. Supplementary Table S2A. 2020), nor in any of 14 different tissues for which expression data is available in the Barlex database, and it was therefore disregarded in the further analysis. However, HORVU4Hr1G057740.1 was expressed in both datasets and is therefore named HvGSTL3. An updated phylogenetic analysis adding these new sequences and others identified by BLAST of different genomes to the list from Bjarnholt et al. (2018), revealed that HvGSTL3 belongs to the same monocot specific conserved subclade as SbGSTL3, whereas no HvGSTLs clustered with SbGSTL1 and SbGSTL2 (Fig. 7a). In the updated tree, monocot GSTLs grouped into four different subclades supported by high bootstrap values. While the SbGSTL1/SbGSTL2 subclade contained only these two sequences, each of the five cereals in the tree was represented in the SbGSTL3/HvGSTL3 subclade, by only one sequence, although it should be noted that the tree only contains the previously confirmed sequences for wheat GSTLs (Dixon and Edwards, 2010) and there are likely to be more members in each subclade from this species, due to its hexaploidity. Each member contains the CXXC motif characteristic of thioredoxins (Quan et al., 2007), which is absent from the remaining cereal GSTLs. Furthermore, both HvGSTL3 and SbGSTL3 were highly expressed in the aleurone at the very early stages of germination, further supporting the conserved function of proteins from this subclade. All three barley GSTLs showed low levels of expression in the embryonic axis where the hydroxynitrile glucoside biosynthetic genes were expressed (Fig. 7b; Fig. S5a), in contrast to SbGSTL1 and SbGSTL2 which were somewhat highly expressed in this tissue in sorghum (Fig. 5a). It should be noted that Betts et al. (2020) removed the roots prior to RNA extraction at the time points where they had protruded. The results for embryonic expression levels are therefore not strictly comparable between species at the latest time points, but the correlation, or lack thereof, between hydroxynitrile biosynthetic genes and genes encoding GSTLs is.This research was funded by The VILLUM Foundation, Denmark, and Independent Research Fund Denmark by grants awarded to N.B. (respectively grant numbers 19151 and 9126-00003 B). We thank Dr. Tomas Laursen and Dr. Rita Del Giudice for help and guidance with the Western blot, Dr. Kirsten Jørgensen for advice on germination set-up and dissection and Dr. Søren Bak for help with the phylogenetic tree.

Funding Information:
This research was funded by The VILLUM Foundation, Denmark , and Independent Research Fund Denmark by grants awarded to N.B. (respectively grant numbers 19151 and 9126-00003 B ). We thank Dr. Tomas Laursen and Dr. Rita Del Giudice for help and guidance with the Western blot, Dr. Kirsten Jørgensen for advice on germination set-up and dissection and Dr. Søren Bak for help with the phylogenetic tree.

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