The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

Effects of abscisic acid on flowering in Pharbitis nil

Under continuous light, flowering of Pharbitis seedlings wasnot induced by abscisic acid (ABA) treatment. Under short daytreatment, flowering was slightly enhanced by ABA at 0.2 and0.4 mg/liter. Stem elongation was considerably inhibited by25 and 50 mg/liter of ABA irrespective of day length. (Received October 21, 1972; )     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis

Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.     

Flowering of Pharbitis nil is controlled by an endogenous semidian rhythm

Abstract Flowering of Pharbitis nil after an inductive dark period is greatly influenced by far-red (FR) irradiation during the preceding light period. The response to FR is rhythmic in otherwise constant conditions, and the period of the oscillation is approximately 12 h (i.e. semidian). The rhythm also appears to operate under daily light-dark cycles. The expression of this novel rhythm depends on the time from the beginning of FR pretreatment to the onset of the inductive dark period. The cotyledons are the site of response to both the pretreatment and inductive darkness, and both these conditions must be perceived by the same cotyledon.     

Gibberellins in Relation to Growth and Flowering in Pharbitis nil Chois

Flowering can be modified by gibberellins (GAs) in Pharbitis nil Chois. in a complex fashion depending on GA type, dosage, and the timing of treatment relative to a single inductive dark period. Promotion of flowering occurs when GAs are applied 11 to 17 hours before a single inductive dark period. When applied 24 hours later the same GA dosage is inhibitory. Thus, depending on their activity and the timing of application there is an optimum dose for promotion of flowering by any GA, with an excessive dose resulting in inhibition. Those GAs highly promotory for flowering at low doses are also most effective for stem elongation (2,2-dimethyl GA₄ GA₃₂ > GA₃ > GA₅ > GA₇ > GA₄). However, the effect of GAs on stem elongation contrasts markedly with that on flowering. A 10- to 50-fold greater dose is required for maximum promotion of stem elongation, and the response is not influenced by time of application relative to the inductive dark period. These differing responses of flowering and stem elongation raise questions about the use of relatively stable, highly bioactive GAs such as GA₃ to probe the flowering response. It is proposed that the `ideal' GAs for promoting flowering may be highly bioactive but with only a short lifetime in the plant and, hence, will have little or no effect on stem elongation.     

Flowering Response of Pharbitis nil to Agents Affecting Cytoplasmic pH

Permeant weak acids and auxins have been shown to reduce the cytosplasmic pH in several systems. Lactic, citric, formic, butyric, salicylic, parahydroxybenzoic, propionic acid, and sodium propionate inhibited the flowering response of Pharbitis nil seedlings when applied immediately before an inductive dark period. The acidic auxins IAA, indolebutyric, and α-naphtaleneacetic acid, as well as the nonacidic auxin α-naphtaleneaceteamid, also inhibited the flowering response. Inhibition was generally more pronounced with a 12-hour than with a 16-hour dark period. Salicylic acid and sodium propionate shifted the response curve of the dark period by about 2 hours. Salicyclic acid, sodium propionate, and indolebutyric acid were inhibitory when applied during the first few hours of the dark period. The permeant weak bases NH₄Cl, procaine, and trisodium citrate enhanced the flowering response. NH₄Cl reduced the length of the critical dark period. The inhibition of flowering by acids and auxins as well as the promotion of flowering by bases was obtained even when only the cotyledons had been treated. The inhibition of floral induction by auxins may not be dependent on their effect on the cytoplasmic pH.     

Chloroplastic activity in response to gibberellic acid treatment of dwarf and normal cultivars of pea (Pisum sativum L.)

Levels of ferricyanide reduction, cyclic and non-cyclic photophosphorylation were measured in chloroplasts of two cultivars of pea and a comparison of their P/2e⁺ ratios were made. No differences were observed in cyclic photophosphorylation or ferricyanide reduction but non-cyclic photophosphorylation was lower in chloroplasts from the dwarf than the normal cultivar. Thus the P/2e⁺ ratio of the dwarf was lower than the normal. Dwarf seedlings treated with gibberellic acid (GA₃) had similar rates of cyclic photophosphorylation as the untreated dwarf but non-cyclic photophosphorylation was lower as was ferricyanide reduction. This resulted in P/2e⁺ ratios that were higher in chloroplasts from the GA₃ treated dwarf seedlings than the untreated, and were the same as the untreated normal. Addition of GA₃ directly to the chloroplasts did not alter the activity in any way. Hence gibberellins do not directly affect changes in chloroplastic activity but may conceivably be involved in a feed-back control system.     

Effects of Light and Temperature on Flower-opening of Pharbitis nil

Flower buds of Pharbitis nil cut from plants growing in thefield opened rapidly when kept in darkness for 8 hr followedby continuous light at 20–25°C, but those kept indarkness for 4 hr opened promptly oniy when the temperatureduring the following light period was kept at 23°C or lower.Buds exposed to continuous light at 25°C did not open, butthose exposed to continuous light at 23°C opened slowly.At a lower temperature, the buds opened rapidly even in continuouslight. When the buds were placed in darkness at 25°C at13:30, 17:30 and 21:30 (artificial light from 17:30 to 21:30),they opened about 10 hr after the onset of darkness regardlessof the time of the onset of darkness, but when the buds werekept at 20°C in light from 13:30, 17:30 and 21:30, theyopened at 3:30–5:30 regardless of the time of transferto the lower temperature. The biological clock which controlsthe time of flower-opening is suggested to be easily reset bya light-off signal, but not by a shift from a normal to lowertemperature (20°C). At the lower temperature, the time offlower-opening probably is determined by the time of the latestpreceding light-off (or light-on) signal. ¹Dedicated to Professor Dr. Erwin Biinning on the occasion ofhis 75th birthday. (Received October 23, 1980; Accepted December 15, 1980)     

The Initiation of an Endogenous Rhythm Affecting Flower Bud Formation in Pharbitis nil

When seedlings of Pharbitis nil are presented with an inductive dark period at varying times, they show a circadian fluctuation in the number of flower buds initiated. This study determines if this fluctuation is due to the plant's perception, at the time of the inductive dark period, of either a rhythmic, external, environmental stimulus or of an endogenous rhythm. Using experimental designs in which the time of planting, the time of seedling emergence from the soil, and the time at which the presentation of an inductive dark period are varied, this fluctuation in flower bud formation is shown to be due to an endogenous rhythm initiated or synchronized by some event associated with the emergence of the seedlings from the soil. The results are inconsistent with the hypothesis that the plants are responding to rhythmic external stimuli.     

Establishment of photoperiodic sensitivity by benzyladenine and a brief red irradiation in dark grown seedlings of Pharbitis nil Chois.

Flowering of etiolated seedlings of Pharbitis nil resulted followinga single, brief red irradiation prior to an inductive dark period.Following this irradiation benzyladenine sprayed on the seedlingsenhanced flowering dramatically and this effect was maximalfor concentrations between 44 and 120µM. In the presenceof benzyladenine a brief (4 to 10 sec) low energy red irradiation(2.6 Wm^–2) resulted in flowering and repeated far-redphotoreversal of this red promotion provided clear evidenceof the sole involvement of phytochrome. However, after suchbrief irradiations the critical dark period for flowering waslonger than is normally found in seedlings grown in light whichindicated that additional photoresponses might be importantin natural conditions. An examination of seedling photosynthesisand assimilate transport indicated that the benzyladenine effecton flowering may relate to its promotion of assimilate and floralstimulus transport to the shoot apex. ¹ Present address: Faculty of Agriculture, Mie University, TsuCity, Mie Prefecture, Japan. (Received August 21, 1978; )     

Neolignan and monoterpene glycoside from the seeds of Pharbitis nil

Phytochemical investigation of an EtOH extract of Pharbitis nil seeds (Convolvulaceae) resulted in the isolation and identification of a new neolignan, 7R,8S-threo-dihydroxydehydrodiconiferyl alcohol (1), and a new monoterpene glycoside, (3Z,7S)-7-hydroxy-3,7-dimethyl-3,8-octadienyl-β-d-glucopyranoside (2), together with a known compound, ethyl α-l-arabinofuranoside (3). The chemical structures of these compounds were unambiguously determined using physical data, HR-ESI–MS and spectroscopic evidence, including 1D and 2D NMR experiments. The anti-inflammatory activities of the isolates were evaluated by estimating the inhibition of nitric oxide (NO) production. Compounds 1 and 2 reduced NO levels in lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. In addition, compound 2 showed weak cytotoxicity against the HCT-15 cell line with an IC₅₀ value of 28.6 μM.     

粳稻矮秆品种苗期和成株期对赤霉素反应的研究

本试验对28个粳稻矮秆品种对GA3的反应进行了研究,结果表明粳稻矮秆品种对GA3反应较为复杂,苗期与成株期反应,大多数品种表现为趋势基本一致,但也存在着其它种类型表现;在粳稻中,对GA3反应敏感的矮秆品种并不一定携带含有sd-1等位基因,但含有sd-1基因的品种表现为敏感,不含有sd-1基因的表现为低敏感或不敏感,据此可以初步确定矮4和紫叶矮含有与sd-1非等位的矮秆基因,是一种新的矮源;GA3对株高构成因素的影响表现因品种不同而异,但以基部一、二节间为主.     

Cloning and structural analysis of the snap-back DNA of Pharbitis nil

We isolated and cloned DNA fragments that exist as inverted-repeat structures in the genome of Pharbitis nil. The method used exploited the fact that if inverted repeat DNA is present in the DNA fragment, intramolecular double-stranded structures can be partly formed within single-stranded DNA molecules after denaturation and rapid renaturation of the fragment. The rapidly renaturing DNA fragments (termed snap-back DNA) were isolated by hybroxylapatite column chromatography and treatment with mungbean nuclease and were cloned into the pUC9 vector. Four snap-back DNA members out of thousands of independent clones obtained were characterized with respect to the reiteration frequency and the nucleotide sequences. When used as probes in Southern hybridization experiments, some of the members identified restriction fragment length polymorphism among the cultivars, suggesting that these sequences might be fluid in the genome. One of the four clones has regions of nucleotide sequence homology to those of inverted-repeat regions in the transposon Taml of Antirrhinum majus.     

Changes in ATP in relation to floral induction and initiation in Pharbitis nil

No changes in metabolism of adenosine phosphates as a function of short day induction were detected in cotyledons of Pharbitis nil Chois strain Violet. A gradual increase in ATP level was detected throughout the dark period in plumules. A rapid decline of ATP pool size was observed in induced plumules shortly after floral induction. The decline occurred close to the 14th hour of the dark period, 1 to 1.5 h after the dark period length required for a 90% flowering response, which is thought to be the minimum time required for transport of the floral stimulus (and assimilates) from the induced cotyledons to the plumule. Transport of the major adenylates from the cotyledons was verified using [¹⁴C]-adenine. Estimates of the amount, and rate, of adenylate transport suggest that the cotyledons could be an important source of adenylates to re-establish the ATP pool size in evoked plumules.     

The metabolism of inhibitor of flowering and prostaglandin biosynthesis, acetylsalicylic acid, in Pharbitis nil cotyledons

Acetylsalicylic acid, which applied to cotyledons of the short day plant Pharbitis nil prior to an inductive 16-h dark period inhibits flowering by 90 %, is converted to salicylic acid and to a lesser extent to gentisic acid in the cotyledons during this 16-h dark period. Our results confirmed that salicylic acid and gentisic acid are responsible for the inhibition of flowering. They also inhibit prostaglandin biosynthesis. This revised version was published online in July 2006 with corrections to the Cover Date.