Eckard Wellmann

Photocontrol of flavonoid biosynthesis

The flavonoids are derived from the flavan or isoflavan skeleton (Fig. 1) and comprise a large group of secondary metabolites from higher plants (Harborne et al. 1975). In the literature the term flavonoid is often used to mean all flavonoids except anthocyanins and for the sake of convenience we have followed this usage. Anthocyanins are 3- or 3,5-glycosides of anthocyanidins (Fig. 2) and include the principal red (R), violet and blue (B) plant pigments (λ 520-545 nm).

Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension culture of parsley (Petroselinum hortense)

Abstract 1. 1. In cell suspension cultures of parsley ( Petroselinum hortense ) the degree of increase of activity of phenylalanine ammonia-lyase caused by white light depends on the growth stage of the cultures. Maximum enzyme stimulation is observed at approximately the tenth day of growth. 2. 2. Changes in the activities of eight enzymes directly related to the biosynthesis of apiin ( 7-O-[β- D -apiofuranosyl -(1 → 2)-β- D -glucosyl ]-5,7,4′- trihydroxyflavone and graveobiosid B (3′-methoxyyapiin) were measured over a period of 24 h after illumination of cell suspension cultures of parsley. 3. 3. The enzymes investigated were phenylalanine ammonia-lyase, trans -cinnamic acid 4-hydroxylase, p -coumarete:CoA ligase, chalcone-flavanone isomerase, UDP-glucose:apigenin 7- O -glucosyltransferase, UDP-apiose:apigenin 7- O -glucoside-[1 → 2]-apiosyltransferase, UDP-apiose synthetasem and S -adenosylmethionine:luteolin3′- O -methyltransferase. 4. 4. Two groups of enzymes could be distinguished on the basis of their responses to light. The first group comprises the three enzymes acting on substrates of the phenylpropanoid type (phenylalanine, cinnamic acid and p -coumaric acid); the second group includes all those enzymes involved exclusively in the formation of flavonoid compounds and their glycosides. 5. 5. A hypothesis is discussed which would correlate three other enzymes assumed to take part in the same biosynthetic pathway with one of these groups of enzymes.

UV-B Radiation and Adaptive Mechanisms in Plants

Further examples of UV-B dependent formation of UV-B screening pigments have been investigated. This active protective mechanism also has been found to be present in the leaf epidermis of various lightgrown vegetables (greenhouse conditions excluding UV-B radiation). Typical action spectra for this kind of positive UV-B effect are interpreted in terms of effectiveness under natural growth conditions’ and also with respect to the role of interaction with longer wavelengths. The importance of photorepair of UV-B damage is demonstrated for two systems, bean leaves and mustard cotyledons. Photorepair phenomena are further characterized and their importance for adaptation to an increased UV-B environment are pointed out. UV-B effects on growth and the hypersensitivity reaction (isoflavonoids, phytoalexins) in bean are discussed both from the viewpoint of damaging and beneficial consequences for the plant.

ANALYSIS OF LIGHT‐CONTROLLED ANTHOCYANIN FORMATION IN COLEOPTILES OF Zea mays L.: THE ROLE OF UV‐B, BLUE, RED AND FAR‐RED LIGHT

Abstract— Light‐induced anthocyanin formation in Zea mays L. coleoptiles was investigated in seven different varieties of this species. Under the test conditions, four varieties showed practically no response to any waveband used (UV, continuous red and continuous far‐red), two responded strongly to both UV and far‐red, and one showed a strong response only to far‐red. The radiation‐sensitive varieties showed, however, only a very weak response to continuous red light. In those varieties sensitive to far‐red light, a pretreatment with continuous red light led to a greatly enhanced response to UV or in one case the manifestation of a response to UV that was previously lacking. Further investigations in one radiation‐sensitive variety (INRA) showed that the UV response was to UV‐B radiation below 350 nm. The UV response, as well as the far‐red and blue responses in this variety, showed fluence‐rate dependency. Red light was almost ineffective and showed only a very weak fluence‐rate dependency.

Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light

SummaryUltraviolet light was demonstrated to stimulate flavone glycoside synthesis in Petroselinum cell suspension cultures. The data presented suggest the involvement of phytochrome in this response: Flavone glycoside formation resulting from 1 h of ultraviolet irradiation was increased by subsequent continuous far-red light irradiation. However, the ultraviolet effect was reduced by a subsequent irradiation with 10 min of far-red. This far-red effect was fully reversed by a sub-sequent irradiation with 10 min of red. Red and far-red irradiations were ineffective without ultraviolet preirradiation. It is concluded that in this system ultraviolet irradiation is required in order to change the cells in such a way as to allow a physiological effectiveness of the phytochrome system.

Chlorogenic acid biosynthesis: Characterization of a light-induced microsomal 5-O-(4-coumaroyl)-d-quinate/shikimate 3′-hydroxylase from carrot (Daucus carota L.) cell suspension cultures

Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsibl for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation.

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A Pfr/Ptot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of Pfr in order to be fully effective.

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Abstract Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.

Light-induced flavone biosynthesis and activity of phenylalanine ammonia-lyase and UDP-apiose synthetase in cell suspension cultures of Petroselinum hortense

SummaryThe formation of flavone glycosides in cell suspension cultures from parsley leaf petiols after illumination with high intensities of white light was demonstrated. Changes in the activities of two of the enzymes involved in flavone glycoside biosynthesis (phenylalanine ammonia-lyase and UDP-apiose synthetase) were measured over a period of 14 days. The two enzymes reached maximum specific activities after significantly different periods of time.