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The mechanism of dusky reddish-brown “kaki” color development of morning glory, Ipomoea nil cv. Danjuro, was studied. Three major known anthocyanins were isolated as glucosylated pelargonidin derivatives. Measurement of the vacuolar pH with proton-selective microelectrodes revealed the vacuolar pH of the colored cell of open flowers to be 6.8, while that of buds was 5.8. Mixing of the three anthocyanins according to the composition ratio in petals at pH 6.8 allowed the identical color to that of petals to be reproduced. The typical “kaki” color development was mostly caused by 5-OH free acylated anthocyanins, which have two λmax around 435 and 535 nm in the visible region.

Two new acylated and three known anthocyanins were isolated from the slate flowers of Pharbitis nil cultivars. The new anthocyanins were determined to be peonidin 3-O-[6-O-(trans-3-O-(β- d - glycosyl)caffeyl )-β- d - glucoside ] as a major pigment, and cyanidin 3-O-[6-O-(trans-3-O-(β- d - glycosyl ) caffeyl )-β- d - glucoside ]-5-O-[β- d - glucoside ] as a minor component. The three known pigments were peonidin 3-glycosylcaffeylglucoside-5-glucoside as a major pigment, and the 3- and 3,5-diglucosides of peonidin as minor pigments. A survey of five slate flower cultivars of this plant showed that peonidin 3-glucosylcaffeylglucoside and peonidin 3-glucosylcaffeylglucoside-5-glucoside were the dominant flower pigments.

In an investigation of the white, cream and pale yellow flowers of 11 acyanic cultivars and 10 acyanic strains of Pharbitis nil, eight flavonoids and six caffeic acid derivatives were detected. From the aqueous methanolic flower extracts five flavonoids were identified: 2′,4,4′,6′-tetrahydroxychalcone, aureusidin 4′-glucoside, R- and S-naringenin 7-glucoside, quercetin 4′- and 7-glucosides, together with chlorogenic acid and caffeic acid. These acyanic cultivars and strains were classified into five groups on the basis of their flavonoid constituents and flower colour. The regulation of anthocyanin biosynthesis in the flowers of P. nil was discussed in relation to the present flavonoid findings.

Six pelargonidin glycosides, four new acylated and two known anthocyanins, were isolated from the maroon flowers of Pharbitis nil cultivars. The four new anthocyanins were determined to be pelargonidin 3- O -(6- O -( trans -3- O -β- d -glucosyl) caffeyl)-β- d -glucoside) as the main pigment and pelargonidin 3- O -(6- O -( trans -3- O -(β-D-glucosyl) caffeyl)-β- d -glucoside)-5- O -(β- d -glucoside), pelargonidin 3- O -(6- O -( trans -caffeyl)-β- d -glucoside), and peonidin 3- O -(6- O -( trans -(β- d -glucosyl) caffeyl)-β- d -glucoside)-5- O -(β- d -glucoside) as the minor pigments. The two known pigments were pelargonidin 3-glucoside and 3,5-diglucoside. A survey of 16 maroon cultivars showed that pelargonidin 3-glucoside and its caffeyl congeners were the dominant flower pigments.

Five peonidin glycosides were isolated from purple to violet flowers of Pharbitis nil and their structures determined by spectral methods. Three pigments were identified as the 6-trans-caffeate and 6,6″-trans-dicaffeate of peonidin 3-sophoroside-5-glucoside and heavenly blue anthocyanin, respectively; the other two pigments were peonidin 3-sophoroside-5-glucoside and its 4′-glucosylcaffeate which have been reported previously. These five anthocyanins were variously present in 18 bluish flowered cultivars. Heavenly blue anthocyanin was the most stable of the five pigments, when measured in neutral solution.

A new acylated anthocyanin was isolated from the violet-blue flowers of Pharbitis nil as a major anthocyanin. The structure of this pigment was determined to be peonidin 3[6″-(4-glucosyl- trans -caffeyl)sophoroside]-5-glucoside. Peonidin 3-sophoroside-5-glucoside was also detected as a minor pigment.

Five acylated peonidin glycosides were isolated from the pale gray-purple flowers of a duskish mutant in the Japanese morning glory (Ipomoea nil or Pharbitis nil) as major pigments, along with a known anthocyanin, Heavenly Blue Anthocyanin (HBA). Three of these were based on peonidin 3-sophoroside and two on peonidin 3-sophoroside-5-glucoside as their deacylanthocyanins; both deacylanthocyanins were acylated with caffeic acid and/or glucosylcaffeic acids. By spectroscopic and chemical methods, the structures of the former three pigments were determined to be 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside], 3-O-[2-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-glucopyranoside], and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside] of peonidin. The structures of the latter two pigments were also confirmed as 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside, and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside of peonidin. The mutation affecting glycosylation and acylation in anthocyanin biosynthesis of Japanese morning glory was discussed.

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.

A new acylated anthocyanin was isolated from the violet-blue flowers of Pharbitis nil as a minor anthocyanin. The structure of this pigment was determined to be cyanidin 3-O-(2-O-(beta-D-glucopyranosyl)-6-O-(trans-4-O-(beta-D- glucopyranosyl)caffeyl)-beta-D-glucopyranosyl)-5-O-(beta-D-glucopyranosi de) based on spectroscopic methods.

A method for the qualitative and semi-quantitative analysis of gibberellins (GAs) was examined, and a systematic method consisting of six steps was established. By this method endogenous GAs in some organs of Pharbitis nil were quantitatively analysed.

Growth-promoting effects of gibberellins and their glucosides isolated from immature seeds of Japanese morning glory ( Pharbitis nil ) were compared in six bioassay systems. GA 3 glucoside exhibited much less activity than GA 3 in the dwarf rice (under aseptic conditions), dwarf maize ( d 1 , d 2 and d 5 ), cucumber and dwarf pea assays. GA 8 , GA 26 , GA 27 and GA 29 showed low activities in all the bioassay systems, while their glucosides were even less active. Thus gibberellin glucosides do not appear to be active in growth regulation.

Pharbitis cotyledons contain methanol-soluble substances which inhibit the oxidation of 3-indolylacetic acid (IAA) by peroxidase. Such substances are much more abundant in etiolated than in light-grown plants, and transfer of etiolated plants from dark to white light results in their prompt decrease. Surprisingly, transfer of a light-grown plant from white light to darkness also results in a marked decrease in the titer of such substances. Thus, it appears that there is a metabolic regulation such that the synthesis of IAA oxidase inhibitors proceeds best in darkness in dark-grown plants and best in light in light-grown plants. Approximately twenty phenolic compounds can be detected in methanolic extracts of cotyledons after two-dimensional paper chromatography. Only five are active as inhibitors of IAA oxidation, and of these, three are increased dramatically in quantity by far-red light. Red light itself produces only small effects, but when given prior to far-red, it greatly diminishes the effectiveness of the far-red. The possible relevance of such changes in photomorphogenesis are considered.

Squalene- 14 C was converted to β-sitosterol by Pharbitis nil seedlings. The β-sitosterol was isolated by chromatography and its radiochemical purity established by dilution with carrier material and crystallization to constant specific activity.