Molecular and Enzymatic Characterization of Flavonoid 3'-Hydroxylase of Malus × domestica.

Julia Weissensteiner,Lisa Führer,Olly Sanny Hutabarat,Johanna Hausjell,Andreas Spornberger,Christian Molitor,Christian Haselmair-Gosch,Syed Waqas Hassan,Heidi Halbwirth,Silvija Marinovic,Karl Stich,Oliver Spadiut

doi:10.3390/plants10091956

Abstract

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.

Highlights

Malus × domestica accumulates high amounts of dihydrochalcones in leaves, fruits, and barks, with phloridzin (Figure 1) being the prevalent compound [1]
Using transtransgenic M. × domestica plants, we previously showed that an increase in 3-hydroxylated genic M. × domestica plants, we previously showed that an increase in 3-hydroxylated didihydrochalcones correlates with a lower susceptibility for fire blight and apple scab [15]. 3hydrochalcones correlates with a lower susceptibility for fire blight and apple scab [15]
Our studies showed that F30 H from apple have a relatively narrow substrate specificity, as they accept, under in vitro conditions, only the most common substrate classes, flavanones, dihydroflavonols, and flavonols

Summary

Introduction

Malus × domestica (apple) accumulates high amounts of dihydrochalcones in leaves, fruits, and barks, with phloridzin (phloretin 20 -O-glucoside) (Figure 1) being the prevalent compound [1]. Formation of dihydrochalcones occur as a side branch of the flavonoid pathway, with p-coumaroyl-CoA as common intermediate [2]. P-DihydrocoumaroylCoA is further converted by the common chalcone synthase (CHS) to the dihydrochalcone phloretin [3,6], and subsequently glucosylated to provide phloridzin [7,8,9,10]. The special case of apple, with its exceptionally high amount of accumulated dihydrochalcones [11], gave rise to speculation about the physiological function of dihydrochalcones in this 4.0/). The special case of apple, with its exceptionally high amount of accumulated dihydrochalcones [11], gave rise to speculation about the physiological function of dihydrochalcones inrole this in species

Results

Discussion

Conclusion