Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

Abstract

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus 3 domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.

Heartwood‐specific transcriptome and metabolite signatures of tropical sandalwood (Santalum album) reveal the final step of (Z)‐santalol fragrance biosynthesis

“…However, this strategy excludes candidate genes in P450 clades with different or unknown functions. For example, SaCYP736A167 belongs to a sub-family for which biochemical functions were only recently identified, none of which involved a terpenoid substrate (Takos et al, 2011;Sircar et al, 2015). Thus, association by function would not have revealed SaCYP736A167 as the P450 of interest.…”

“…Earlier work in grapevine (Vitis vinifera) and soybean (Glycine max) suggested that CYP736As may be involved in interactions with pathogenic or symbiotic micro-organisms (Cheng et al, 2010;Guttikonda et al, 2010). Recently, two CYP736As with roles in cyanogenic glucoside biosynthesis in Lotus japonicus (Takos et al, 2011) and biphenyl phytoalexin biosynthesis in rowan and apple (Sircar et al, 2015) were functionally characterized. The substrates of these two CYP736As are as different from one another as they are from the sesquiterpene substrates of SaCYP736A167 (Figure 8).…”

“…Overall, SaCYP736A167 does not cluster with previously characterized P450s involved in terpenoid oxidation. The most closely related and functionally defined P450s are L. japonicus LjCYP736A2 (cyanogenic glucoside metabolism; Takos et al, 2011) and S. aucuparia SoCYP736A107 (noraucuparin formation in biphenyl phytoalexin biosynthesis; Sircar et al, 2015). The CYP750B1 sabinene hydroxylase of the gymnosperm Thuja plicata appears to be the most closely related P450, with a known function in terpenoid oxidation (Gesell et al, 2015).…”

The genome sequence of the wild tomatoSolanum pimpinellifoliumprovides insights into salinity tolerance

“…Biphenyl-4-hydroxylases (B4H) have only recently been identified and cloned in rowan (Sorbus aucuparia) and apple (Malus spp.) and were characterized as cytochrome P450 736A proteins that catalyze the 4-hydroxylation of a biphenyl scaffold towards the biosynthesis of biphenyl phytoalexins such as aucuparin in response to pathogen attack (Khalil et al, 2013;Sircar et al, 2015). Research into biphenyl phytoalexins is somewhat scarce, possibly due to the absence of B4H in the model organism Arabidopsis, with most studies restricted to the Malinae subtribe of the subfamily Amygdaloideae (e.g.…”

“…Research into biphenyl phytoalexins is somewhat scarce, possibly due to the absence of B4H in the model organism Arabidopsis, with most studies restricted to the Malinae subtribe of the subfamily Amygdaloideae (e.g. apple and pear) (Kokubun et al, 1995;Hüttner et al, 2010;Sircar et al, 2015;Chizzali et al, 2016). As such, the observed presence and, indeed, expansion of B4H-related genes in S. pimpinellifolium, could be related to the increased pathogen resistance of S. pimpinellifolium and represents an interesting target for further studies.…”