Secondary metabolites as quality factor for horticultural products

Abstract

Secondary metabolites play a major role as quality factors in horticultural products by significantly contributing to plant health, optical attributes, sensory attributes and health beneficial effects for the consumer. This thesis concentrated on dihydrochalcones, a rare class of secondary metabolites, which is, however present in particularly high amounts in apple (Malus × domestica), one of the major cash-cows of global fruit production. Dihydrochalcones are assumed to be particularly relevant for both plant health and health beneficial effects for the consumer. In apple, phloridzin (phloretin 2'-O-glucoside) is prevalent representing more than 90% of the soluble phenolic compounds, but small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. An involvement of the latter in defense against plant pathogens was suggested but not unambiguously demonstrated so far. Despite of the large amounts of dihydrochalcones present in apple, their physiological function in planta still remains a puzzle. For systematic investigation, sufficiently comprehensive knowledge of the underlying pathway is yet lacking. Whereas dihydrochalcone biosynthesis has been studied during the past few years, the introduction of a hydroxyl group in position 3 of dihydrochalcones was investigated in this thesis for the first time. Two types of enzymes could catalyse the reaction, polyphenol oxidases (PPOs) and/or cytochrome P450 dependent monooxygenases. 3-Hydroxyphloridzin and the corresponding aglycone 3-hydroxyphloretin occur as intermediates in the oxidation of phloridzin and phloretin by unspecific PPOs, if intact cells are damaged and the enzymes and dihydrochalcones are released from different cell compartments. It seems however unlikely that unspecific enzymes which produce a spectrum of cell-toxic compounds should be involved in the biosynthesis of constitutive 3-hydroxyphloridzin present in Malus sp. Hydroxylation in position 3 of dihydrochalcones shows high similarity to the introduction of a second hydroxyl group in the B-ring of flavonoids and chalcones which are catalysed by the cytochrome P450 dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and chalcone 3-hydroxylase (CH3H). F3´Hs usually show a broad substrate specificity, whereas CH3Hs specifically hydroxylate chalcones. Chalkones clearly show structural relation to dihydrochalcones as both are lacking the heterocyclic C ring of flavonoids. To investigate a potential involvement of CH3H and F3'H in 3-hydroxylation of dihydrochalcones, their substrate specificity was tested in vitro and in planta. Recombinant enzymes obtained by heterologous expression in yeast were able to convert dihydrochalcones to some extent, and higher conversion rates were observed with CH3H than with F3'H. The respective natural substrates were, however, much better accepted. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones also in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from Cosmos sulphureus under the control of the CaMV 35S promotor in cooperation with the Institute of Fruit Breeding at the Julius Kühn Institut Dresden-Pillnitz. The three independent transgenic lines obtained showed an increase of 3-hydroxyphloridzin within the dihydrochalcones thereby unambiguously demonstrating that CH3H can mediate 3-hydroxylation of dihydrochalcones in planta. Increased levels of 3-hydroxyphloridzin correlated with reduced susceptibility to fire blight and apple scab, which are the main bacterial and fungal diseases of apple. To test a possible involvement of the F3'H of apple in the formation of constitutive 3-hydroxydihydrochalcones, cDNA clones of the two types of F3'Hs present in apple was isolated and heterologously expressed in yeast. Despite showing high F3'H activity with various flavonoid substrates, hydroxylation of dihydrochalcones was not observed at standard conditions, indicating that F3'H is not part of the dihydrochalcone pathway. A final clarification will require silencing of the Malus F3'Hs, as previous results have shown that substrate conversion in planta can occur under suitable physiological conditions, even if substrate specificity of the involved enzyme is very low. The findings from the present work will feed into new strategies for the biotechnological production of dihydrochalcones derivatives as well as to innovative plant protection approaches.