Molecular cloning and functional analysis of a biphenyl phytoalexin-specific O-methyltransferase from apple cell suspension cultures.

MAIN CONCLUSION: This manuscript describes the cloning and functional characterization of a biphenyl phytoalexin biosynthetic gene, 3,5-dihydroxybiphenyl O-methyltransferase from elicitor-treated cell cultures of scab resistant apple cultivar 'Florina'. Apples belong to the subtribe Malinae of the Rosaceae family. Biphenyls and dibenzofurans are the specialized phytoalexins of Malinae, of which aucuparin is the most widely distributed biphenyl. The precursor of aucuparin, 3,5-dihydroxybiphenyl, is a benzoate-derived polyketide, which is formed by the sequential condensation of three molecules of malonyl-CoA and one molecule of benzoyl-CoA in a reaction catalyzed by biphenyl synthase (BIS). This 3,5-dihydroxybiphenyl then undergoes sequential 5-O-methylation, 4-hydroxylation, and finally 3-O-methylation to form aucuparin. A cDNA encoding O-methyltransferase (OMT) was isolated and functionally characterized from the cell cultures of scab-resistant apple cultivar 'Florina' (Malus domestica cultivar 'Florina'; MdOMT) after treatment with elicitor prepared from the apple scab causing fungus Venturia inaequalis. MdOMT catalyzed the regiospecific O-methylation of 3,5-dihydroxybiphenyl at the 5-position to form 3-hydroxy-5-methoxybiphenyl. The enzyme showed absolute substrate preference for 3,5-dihydroxybiphenyl. The elicitor-treated apple cell cultures showed transient increases in the MdOMT (GenBank ID MF740747) and MdBIS3 (GenBank ID JQ390523) transcript levels followed by the accumulation of biphenyls (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexins. MdOMT fused with N- and C-terminal yellow fluorescent protein showed cytoplasmic localization in the epidermis of Nicotiana benthamiana leaves. In scab inoculated greenhouse-grown 'Florina' plants, the expression of MdOMT was transiently induced in the stem followed by the accumulation of biphenyl phytoalexins.

Salicylaldehyde synthase activity from Venturia inaequalis elicitor-treated cell culture of apple.

Salicylic acid (SA) is known to trigger a number of plant defense responses upon pathogen attack. It is well known that apple (Malus domestica) plants respond to pathogen invasion by synthesizing SA, but its biosynthesis is not well understood. In this study, we report salicylaldehyde synthase (SAS) activity from Venturia inaequalis elicitor (VIE)-treated cell suspension cultures of apple (Malus domestica 'Florina'). SAS catalyzes non-oxidative C2-side chain cleavage of 2-coumaric acid to form salicylaldehyde (SALD) in the presence of a reducing agent such as cysteine. The side chain cleavage mechanism was found to be very similar to that of salicylaldehyde synthase activity from tobacco and 4-hydroxybenzaldehyde synthase activity from Vanilla planifolia and Daucus carota. A basal SAS activity was observed in the non-elicited cell cultures, and a 7-fold increase in SAS activity was observed upon elicitation. In parallel to SAS activity, the level of total SA accumulation increased by 5.6-fold after elicitation compared to the untreated control cells. Elicitor treatment further resulted in an 8.7-fold increase in the activity of the phenylalanine ammonia-lyase (PAL) enzyme that preceded the peak of SAS activity and total SA accumulation, suggesting the involvement of the phenylpropanoid pathway in SA metabolism. The preferred substrate for SAS was 2-coumaric acid (Km = 0.35 mM), with cysteine being the preferred reducing agent. In addition, a 1.8-fold enhancement in the SA content and 0.7-fold enhancement in the SALD content was observed when elicited cell cultures were fed with 2-coumaric acid. These observations suggest the involvement of SAS in SALD biosynthesis.

Comparative metabolomics of scab-resistant and susceptible apple cell cultures in response to scab fungus elicitor treatment.

Apple scab disease caused by the fungus Venturia inaequalis is a devastating disease that seriously affects quality and yield of apples. In order to understand the mechanisms involved in scab resistance, we performed gas chromatography-mass spectrometry based metabolomics analysis of the cell culture of scab resistant cultivar 'Florina' and scab susceptible cultivar 'Vista Bella' both prior -to and -following treatment with V. inaequalis elicitor (VIE). A total 21 metabolites were identified to be altered significantly in 'Florina' cell cultures upon VIE-treatment. Among 21 metabolites, formation of three new specialized metabolites aucuparin, noraucuparin and eriobofuran were observed only in resistant cultivar 'Florina' after the elicitor treatment. The score plots of principal component analysis (PCA) exhibited clear discrimination between untreated and VIE-treated samples. The alteration in metabolite levels correlated well with the changes in the transcript levels of selected secondary metabolite biosynthesis genes. Aucuparin, noraucuparin and eriobofuran isolated from the 'Florina' cultures showed significant inhibitory effect on the conidial germination of V. inaequalis. The results expand our understanding of the metabolic basis of scab-resistance in apple and therefore are of interest in apple breeding programs to fortify scab resistance potential of commercially grown apple cultivars.