Poaceae (R.Br.) Barnhart, 1895

de Bruijn, Wouter J. C., Gruppen, Harry & Vincken, Jean-Paul, 2018, Structure and biosynthesis of benzoxazinoids: Plant defence metabolites with potential as antimicrobial scaffolds, Phytochemistry 155, pp. 233-243 : 234-236

3. Benzoxazinone biosynthesis and distribution in Poaceae

3.1. General biosynthesis of benzoxazinones

The basic biosynthetic pathway of benzoxazinones (up to DIBOAGlc) was first studied and elucidated in maize ( Frey et al., 1997), and later also in wheat ( Nomura et al., 2002, 2003) and rye ( Bakera et al., 2015; Rakoczy-Trojanowska et al., 2017). The biosynthesis starts from indole-3-glycerolphosphate (IGP), which is consecutively converted to HBOA, the first benzoxazinoid, in 4 steps by the enzymes BX1 to BX4 ( Fig. 3 ). A subsequent glucosylation and hydroxylation leads to DIBOAGlc, which serves as the starting point for hydroxamic acid biosynthesis. All further downstream benzoxazinoids are synthesized as glucosides and most of the enzymes involved are unable to use the aglycones as substrates ( Jonczyk et al., 2008; Oikawa et al., 2002). The formation of these downstream metabolites has mainly been studied in maize and to a lesser extent in wheat and rye ( Dutartre et al., 2012; Handrick et al., 2016; Jonczyk et al., 2008; Makowska et al., 2015; Meihls et al., 2013; Tanwir et al., 2017). A summary of the current state of knowledge on the biosynthetic pathways of benzoxazinones is shown in Fig. 3 . As shown on the right-hand side of this figure, benzoxazinones can be transformed to benzoxazolinones, which is further discussed in Section 4.2. Next to the Poaceae , benzoxazinoids have been reported in dicots of the families Acanthaceae , Calceolariaceae , Lamiaceae , Plantaginaceae , and Ranunculaceae ( Table 1 ). The biosynthesis in these families might be similar to what has been reported in Poaceae , as indicated by analogues of the benzoxazinoid biosynthetic enzymes that have been found in dicots ( Dick et al., 2012; Schullehner et al., 2008). The overview in Fig. 3 indicates that several benzoxazinoid biosynthetic pathways require further investigation (dashed arrows). These aspects are discussed in the next section.

3.2. Tentative pathways in benzoxazinone biosynthesis

According to the generally accepted pathway, as shown in Fig. 3 , HBOA is converted to DIBOA by BX5, which is then glucosylated by BX8 or BX9. Whereas HBOA aglycons are relatively stable, DIBOA aglycons are reactive (Section 4) and phytotoxic (Section 5). Therefore, the formation of free DIBOA within the plant cell is likely omitted. One possibility could be the stabilisation and rapid glycosylation of DIBOA within a metabolon (i.e. a complex of sequential metabolic enzymes) by metabolic channelling. This would be similar to what has been demonstrated for toxic or labile intermediates in other secondary metabolic pathways (e.g. dhurrin biosynthesis in sorghum) ( Jørgensen et al., 2005). As an alternative, we propose a pathway in which HBOA is glycosylated prior to oxidation to form DIBOA-Glc. It was previously proposed that HBOA-Glc and DIBOA-Glc are in fact a redox pair ( Hofman and Hofmanová, 1969), which might present a mechanism for the possible interconversion of these compounds. The existence of an enzyme which catalyses the oxidation of HBOA-Glc to DIBOA-Glc has not been thoroughly explored in literature.

Maize BX8 and BX9 were both shown to be able to glycosylate HBOA albeit at a much lower conversion rate than DIBOA and DIMBOA ( von Rad et al., 2001). The glycosylation of DIMBOA, however, does not seem to serve a function within the biosynthetic pathway, as it is already formed as a glycoside. The formation of HBOA-Glc would be a logical starting point for the biosynthesis of lactams. Further hydroxylation and methylation to produce DHBOA-Glc, HMBOA-Glc, and HM 2 BOA-Glc might be performed by the same or similar enzymes as those involved in hydroxamic acid biosynthesis (BX6, BX7, and BX13) or by a yet to be discovered part of the BX enzyme-cluster. The compounds TRIBOA-Glc and TRIMBOA-Glc are intermediates of the biosynthesis and are not typically accumulated and detected in maize tissues ( Cambier et al., 1999; Handrick et al., 2016). Possibly, DHBOA-Glc serves a similar role as an intermediate in HMBOA-Glc synthesis. An analogue of TRIMBOA-Glc as an intermediate for the biosynthesis of HM 2 BOA-Glc is not yet known. The biosynthetic pathway of benzoxazinone lactams presents a gap in our current knowledge. Lactams are the least prevalent subclass of benzoxazinones in maize explaining the lack of research on their biosynthesis, however, lactams are more prominent in other species, such as rye ( Tanwir et al., 2013).

The methyl derivative equivalent of DIBOA, 4- O -Me-DIBOA-Glc, was annotated in wheat seedlings exposed to fungal stress based on LC-MS analysis ( de Bruijn et al., 2016). Thus far, its biosynthesis has not yet been fully elucidated. It was shown that a DIMBOA-Glc 4- O - methyltransferase from wheat was also able to convert DIBOA-Glc to 4- O -Me-DIBOA-Glc in vitro, but the latter compound was not detected in planta in that study ( Oikawa et al., 2002).

3.3. Distribution of benzoxazinones in Poaceae

Several recent reviews have addressed the genetic background of benzoxazinoid production between the different species within the Poaceae family ( Dutartre et al., 2012; Makowska et al., 2015). Three agriculturally important crops that produce benzoxazinoids are maize, wheat, and rye. The profile of benzoxazinones produced between these species varies ( Fig. 4 ). Overall, maize produces the most diverse profile of benzoxazinones, whereas rye possesses the lowest diversity. Based on the biosynthetic pathways involved ( Fig. 3 ), it seems like wheat does not possess an active BX14-like enzyme to perform the conversion of DIM 2 BOA-Glc into HDM 2 BOA-Glc. It is, however, able to produce HDMBOA-Glc which indicates the presence of an active BX10-like enzyme. Rye has not been shown to produce HDMBOA-Glc and other methyl derivatives.

Several other well-known species from the Poaceae family, such as rice ( Oryza sativa ), oat ( Avena sativa ), and barley ( Hordeum vulgare ) do not produce benzoxazinoids. Interestingly, some other members of the genus Hordeum , e.g. Hordeum lecheri , have been found to produce benzoxazinoids ( Grün et al., 2005). There have also been some reports of benzoxazinoid production in sorghum ( Malan et al., 1984; Niemeyer, 1988). Several other less well-known species in the Poaceae (e.g. Aegilops speltoides ) have been reported to produce benzoxazinoids ( Dutartre et al., 2012). As shown by the phylogenetic trees presented by Dutartre and co-workers, the development of the benzoxazinoid biosynthetic cluster does not necessarily follow the general phylogenetic relationships between the different species, as is exemplified by the genus Hordeum ( Dutartre et al., 2012) .

For a more in-depth perspective of benzoxazinoid phylogenomics, we would like to refer readers to the aforementioned reviews ( Dutartre et al., 2012; Makowska et al., 2015) (focussed on Poaceae ). In addition, there are several studies that provide more information about benzoxazinoids in dicots ( Dick et al., 2012; Frey et al., 2009; Schullehner et al., 2008). The main benzoxazinoids produced by dicots are similar to those produced by Poaceae ( Table 1 ). In Section 2 and Table 1 , several notable molecules unique to dicots are shown such as chloro-derivatives of HBOA and DIBOA, and the production of benzoxazinone galactosides rather than glucosides.

Figures

Fig. 3. The biosynthetic pathway of the three subclasses of benzoxazinones in plants (Dutartre et al., 2012; Frey et al., 1997; Handrick et al., 2016; Jonczyk et al., 2008; Meihls et al., 2013; Niemeyer, 2009; Oikawa et al., 2002) and their transformation into benzoxazolinones (Wouters et al., 2016). Structures of biosynthetic intermediates are shown in grey. Different mechanisms have been proposed for the multi-step degradation from the ring-opened configuration of benzoxazinones to the corresponding benzoxazolinone (Wouters et al., 2016). IGP, indole-3-glycerol phosphate.

Fig. 4. Distribution of individual benzoxazinones amongst the three major benzoxazinoid producing species of the family Poaceae (Cambier et al., 1999; de Bruijn et al., 2016; Le-Van and Wratten, 1984; Søltoft et al., 2008; Tanwir et al., 2013; Woodward et al., 1979). The biosynthetic intermediates DHBOA, TRIBOA, and TRIMBOA are not included.