Masateru Shinozaki

The first isolation of lignans, megacerotonic acid and anthocerotonic acid, from non-vascular plants, anthocerotae (hornworts)

Abstract Two new lignans, megacerotonic acid and anthocerotonic acid, were isolated from the Anthocerotae (Hornworts), Megaceros flagellaris and Anthoceros punctatus. Their structures were determined by spectroscopic methods. This is the first known example of lignans isolated from non-vascular plants.

Accumulation of phyllodulcin in sweet-leaf plants ofHydrangea serrata and its neutrality in the defence against a specialist leafmining herbivore

Among wild plants ofHydrangea serrata (Hydrangeaceae) in Japan, there are sweet plants whose leave contain a kind of isocoumarin, phyllodulcin, which happens to be 350 times as sweet as sucrose to the human tongue. In a primary beech forest in Ashu, Kyoto, the spatial distribution of sweet plants and temporal and the spatial distribution of phyllodulcin within and among plants were investigated using a high performance liquid chromatograph. The distribution of sweet plants was confined within a valley and was parapatric with non-sweet plants. A plants characteristic phyllodulcin accumulation did not change, even when transplanted into the different habitats. The phyllodulcin content of the sweet plants varied greatly among plants, and the population mean peaked in July when the plants flowered. Within a plant, phyllodulcin content was elevated by partial defoliation. We examined the possible effect of phyllodulcin on herbivory by a specialist leafmining herbivore,Antispila hydrangifoliella (Lepidoptera: Heliozelidae). We transplanted sweet and nonsweet plants reciprocally between their original habitats, excluded attacks by parasitoids, and compared performance of the leafminer. Leafminer colonization and larval survivorship on transplanted andin situ plants was not significantly different between sites. The fact that accumulation of phyllodulcin did not augment a defensive function, at least against herbivory by the leafminer, and the sporadic distribution of phyllodulcin-accumulating plants, suggest that the genotypes synthesizing phyllodulcin emerged independently at separate localities by mutation, and that the genotypes are almost adaptively neutral in defence against the specialist herbivore.

Interaction of Microsomal Cytochrome P-450 s and N-Phenylcarbamates that Induce Flowering in Asparagus Seedlings

n-Propyl N-(3,4-dichlorophenyl)carbamate, which induces flowering while it inhibits a step or steps in the phenylpropanoid metabolism in Asparagus officinalis L. seedlings, was found to retard the conversion of t-cinnamic acid to p-coumaric acid by high-pressure liquid chromatography of the metabolites in the shikimic acid pathway. The concentrations of the metabolites preceding t-cinnamic acid on the pathway in treated and untreated seedlings were the same, but those of p-coumaric acid and later metabolites were significantly lower in treated plants. The carbamate inhibited phenylpropanoid metabolism when used to treat stem segments that included a shoot apex primordium, where flowers are induced, and when added to a 100,000 × g microsomal fraction prepared from such segments. NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases in the 100,000 × g fraction were not inhibited by the carbamate. The results showed that this compound has its site of action on the endoplasmic reticulum and that it inhibits cytochrome P-450s, including t-cinnamic acid 4-hydroxylase. We examined the flower-inducing activity of known cytochrome P-450 inhibitors, and found that piperonyl butoxide also causes flowering.

Biochemistry of Short-Day and Long-Day Flowering in Pharbitis nil(Physiology and Biochemistry of Flowering, For Further Development of Horticulture in East Asia)

Pharbitis nil has been widely used as a typical short-day plant in studies of flowering. It can be induced to flower by a single dark treatment even in seedling with just unfolded cotyledons. This plant is also capable of inducing to flower under continuous light by giving various treatments such as poor nutrition (PN), high-intensity light (HL), low-temperature (LT) and physical or chemical inhibition of root elongation etc. Furthermore, the responses to these treatments were different among strains. Therefore, it is a suitable experimental material to study the mechanism of floral induction. We are investigating to elucidate the mechanism of floral induction under various conditions by using Pharbitis nil, strain Violet, Kidachi and Tendan. In this symposium, we will report two subjects on flowering in Pharbitis nil, (1) the mechanism of floral induction of long-day (LD) flowering by PN, HL and LT, (2) our recent works on short-day flowering. Pharbitis nil, strain Violet, is induced to flower under continuous light by PN, HL and LT, but strains Kidachi and Tendan were not by PN and HL, though they initiated floral buds under LT. HL has interesting effects on flowering, that is, (1) induction, (2) promotion of LT flowering, (3) inhibition of short-day flowering. The cotyledons are necessary for flowering under these conditions. Therefore, we widely analyzed the extracts from the cotyledons by using HPLC equipped with various detectors. As the results, we found several substances which closely correlated with flowering. Floral induction under PN and HL is closely correlated with the accumulation of some phenylpropanoids such as chlorogenic acid (CGA), p-coumaroylquinic acid (CQA) and pinoresinol-β-D-glucoside (PRG), sugars such as glucose, fructose and sucrose, and polyamine such as putrescine (PUT). Under LT, feruloylquinic acid (FQA) and ascorbic acid instead of CGA and PRG increased preceding floral initiation. Promotive and inhibitory effects of HL on flowering can be explained by the action of ascorbic acid. High level of sugars is necessary condition and the accumulation of CGA, PRG, FQA and/or PUT seems to be sufficient condition, that is, floral initiation under long-day condition always occurred when necessary-sufficient condition was fulfilled. In order to elucidate the physiological roles of these phenyl-propanoids, we remarked PRG, which is known as an inhibitor of phospho-diesterase (PDE). Inhibitors of PDE such as papaverine, theophyline and caffeine promoted flowering under PN. Moreover, FQ and some benzoic acid derivatives which promoted LD flowering inhibited the activity of PDE, suggesting the involvement of c-AMP in LD flowering. It will be described about the endogenous level of c-AMP in relation to PN flowering. In pattern analysis by using HPLC, it was difficult to find out florigen-like substance in extracts from photoperiodically induced leaves. Recently, we found that the inhibitors of biosynthesis and degradation of catecholamines (CA) inhibited floral initiation when they were applied before and during the dark period. Thus, we introduced a coulometric array system (ESA-Coulochem), which can measure CA at f mol level, for the quantitative measurement of CA. Unexpectedly we failed to detect CA, but we found many substances which seem to be florigen. It will be referred to their results.

cDNA cloning and expression analysis of a non-photosynthetic ferredoxin gene in morning glory (Pharbitis nil)

A full-length cDNA encoding a non-photosynthetic ferredoxin was isolated from apical buds of morning glory (Pharbitis nil), a short-day plant, by differential screening under flower-inducing and non-inducing conditions. Northern analysis and in situ hybridization showed that the transcript was abundant in shoot apices and root tips. The transcript level in the apical buds decreased with the flower-inducing light treatment.

Effects of the Inhibitors of Biosynthesis and Degradation of Catecholamines on Photoperiodic Induction of Flowering in Pharbitis nil

Summary The effects of the inhibitors of biosynthesis and degradation of catecholamines (CA) on photoperiodic induction of flowering were examined in Pharbitis nil , a short-day plant. The inhibitors of CA biosynthesis such as iodotyrosine, methyldopa, methyltyrosine, carbidopa, NSD-1015, and 6-hydroxydopamine inhibited floral initiation when applied before and during a dark period but not after dark treatment, suggesting that these compounds inhibited the production of a flowering hormone in cotyledons. Inhibitors of CA degradation such as SK&F525A, amitriptyline, pargyline, gallic acid, n-butyl gallate, OR-486 and Ro-41-0960 also inhibited flowering, and these inhibitors were effective even when applied after dark treatment, although they did not inhibit floral initiation when applied at 48 h after the end of dark treatment. This result suggested that the inhibitors of CA acted in bud and inhibited the expression of flowering hormone in bud, or the transformation of it into an active substance. The inhibitors used in the present experiment inhibited flowering without the inhibition of vegetative growth. Exogeneous noradrenaline did not induce floral initation under continuous light but partially reversed the inhibitory effect of 6-hydroxydopamine.

Genes for dwarfing and photoperiod flowering response inPharbitis nil choisy

Dwarfing and sensitivity to the duration of a single inductive dark period for flowering ofPharbitis nil in F2 progeny of a cross between the tall strain Tendan, and the dwarf, Kidachi appear to be controlled by the alleles at two independent loci. Progeny of a similar cross between the tall strain Violet and the dwarf Kidachi at F2 and F3 also showed single locus segregation for tall: dwarf plants. In this cross, differences in photoperiodic response could be identified in F3 families but they were not simply inherited. There was some evidence of difficulties with classification of the F2 plants, but also, the flowering of the F1 between the two less sensitive strains Tendan and Violet indicated complex inheritance of their photoperiodic response. Complementary dominant alleles at three independent loci may be necessary for flowering in even shorter dark periods with the sensitive strain Kidachi.The dwarf strain Kidachi has a reduced gibberellin (GA) content (Barendse and Lang 1972), it flowers in a short dark period without terminal flowering, and it responds positively to GA application both for flowering and growth. However, since control of dwarfing and photoperiodic sensitivity can be separated genetically, there is no strick link between the gibberellin responsiveness of Kidachi for its growth and flowering. Despite the complexity of flowering genetics in Violet×Kidachi, a short-dark-period-sensitive, terminal flowering and tall F7 line was obtained in a pedigree previously held heterozygous for the dwarf: tall character but not selected for flowering time. Thus, flowering in a short dark period can also be obtained in the presence of the non-dwarfing allele from strain Violet, again demonstrating genetic independence.