Polypeptide profiles from cotyledons of developing and photoperiodically induced seedlings of the Japanese morning glory (Pharbitis [ipomoea] nil)

An unknown substance(s) produced in the cotyledons of seedlings of the Japanese morning glory (Pharbitis nil) during a defined period of darkness triggers the subsequent initiation of floral buds at apical and axillary meristems. Recent studies have concentrated on characterizing molecular changes as a possible mechanism associated with its synthesis, but these have failed to eliminate interference due to lack of development unity in the sampled population and to consider different kinetic alternatives of those potential changes. The current study demonstrates that numerous age-related changes occur in polypeptides from cotyledons during growth under noninductive conditions, but that these are minimal in older seedlings selected for improved synchrony of the floral response. Polypeptides from older seedlings sampled at various times during and after a dark inductive period were examined by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). No differences associated with floral induction could be detected. The results indicate that changes in gene expression correlated with floral-induction do not occur in the abundant polypeptide fraction within the limitations of the techniques employed.     

Peroxidase isozymes and their indoleacetate oxidase activity in the Japanese morning glory, Pharbitis nil

Distributions of peroxidase isozymes in various organs of theJapanese morning glory (Pharbitis nil CHOISY) are described.Most peroxidase isozymes had indoleacetate oxidase activity. (Received December 29, 1969; )     

Isolation, purification and identification of acetylcholine in Pharbitis nil seedlings

Acetylcholine (ACh), a well known animal neurotransmitter was isolated from tissues of Pharbitis nil using five different methods. Its presence in plant extract was confirmed by infrared spectroscopy and mass spectrometry. For quantitative estimation of ACh in P. nil seedlings pyrolysis-gas chromatography was applied. The presence of ACh was found in all organs of the examined plant: seeds, shoot apex, cotyledons, leaves, shoots and roots. However, the highest level of the investigated substance was noted in the youngest growing parts. In 5-day-old etiolated seedlings they were cotyledons, whereas in 14 day-old green plants - shoot apex and young leaves.     

Fate of radioactive gibberellin a(1) in maturing and germinating seeds of peas and Japanese morning glory

Radioactive gibberellin A₁ (³H-GA₁) was injected into excised fruits of peas and Japanese morning glory. These were then grown in sterile culture to maturity and the label was followed in the seeds during further development and subsequent germination. During development of both pea and morning-glory seeds a large part of the radioactivity became associated with the aqueous fraction, while another part of the ³H-GA₁ was converted into 2 new, acidic, biologically active compounds, designated X₁ and X₂. A relatively small part of the neutral compounds could be converted back to ³H-GA₁, X₁, and X₂ by means of mild acid hydrolysis. During germination of pea and morning-glory seeds, part of the bound compounds was released in the form of ³H-GA₁, X₁ and X₂ while, particularly during rapid seedling growth, a further conversion of ³H-GA₁, mainly to X₁, took place. In pea seedlings, growth during the first 2 to 3 days after imbibition was not affected by Amo-1618, an inhibitor of gibberellin biosynthesis. This, in conjunction with the findings on the interconversions between free and bound ³H-GA₁ suggests that, at least in peas, early seedling growth may at least partly be regulated by gibberellins released from a bound form which was formed during seed development.     

Mechanism of dusky reddish-brown "kaki" color development of Japanese morning glory,Ipomoea nil cv. Danjuro

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 lambdamax around 435 and 535 nm in the visible region.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Isolation of gibberellins A26 and A27 and their glucosides from immature seeds of Pharbitis nil

Summary Two new gibberellins, gibberellins A₂₆ and A₂₇ (GA₂₆ and GA₂₇), and their glucosides have been isolated from immature seeds of Japanese morning-glory (Pharbitis nil), together with GA₈ and its glucoside. GA₂₆, GA₂₇ and their glucosides showed only slight growth-promoting activities on seedlings of rice, dwarf maize and cucumber.     

Polypeptide profiles from cotyledons of developing and photoperiodically induced seedlings of the Japanese morning glory (Pharbitis [ipomoea] nil)

An unknown substance(s) produced in the cotyledons of seedlings of the Japanese morning glory (Pharbitis nil) during a defined period of darkness triggers the subsequent initiation of floral buds at apical and axillary meristems. Recent studies have concentrated on characterizing molecular changes as a possible mechanism associated with its synthesis, but these have failed to eliminate interference due to lack of development unity in the sampled population and to consider different kinetic alternatives of those potential changes. The current study demonstrates that numerous age-related changes occur in polypeptides from cotyledons during growth under noninductive conditions, but that these are minimal in older seedlings selected for improved synchrony of the floral response. Polypeptides from older seedlings sampled at various times during and after a dark inductive period were examined by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). No differences associated with floral induction could be detected. The results indicate that changes in gene expression correlated with floral-induction do not occur in the abundant polypeptide fraction within the limitations of the techniques employed.     

Compensatory growth responses of seedlings of Pharbitis purpurea (Convulvulaceae) to tissue removal at different seeding depths of seeds

Tissue damage to seedlings can limit their later growth, and the further effects may be greater with increasing seedling age. Seedlings, however, can minimize the effect of damage through compensatory growth. Seedlings of Pharbitis purpurea grow in frequently disturbed habitats and generally tolerate damage to leaf tissues. We evaluated the compensatory responses of the cotyledon to different levels of defoliation and their effect on seedling growth and development. We also examined the relationship between seeding depth and compensatory growth. We tested seven defoliation treatments with one or both cotyledons and/or the apical meristem of seedlings removed from seeds buried at a seeding depth of either 2 or 5?cm. We then measured 12 growth traits of the seedlings to assess development and growth compensation. The area, thickness, biomass, and longevity of the remaining cotyledon were also measured to quantify increased growth as result of treatment effects at both seeding depths. The results showed that defoliation reduced seedling height, belowground length, and total biomass significantly in subsequent growth in all treatments. However, defoliation treatments had direct positive impacts on growth at 2?cm depth compared with 5?cm depth. In contrast, the compensation of cotyledon area (C _area), biomass (C _mass), and thickness (C _thickness) was greater at 5?cm depth than at 2?cm depth. The results thus indicate that P. purpurea seedlings adopted a compensatory growth strategy to resist leaf loss and minimize any adverse effects using the remaining cotyledon. Increasing seeding depth can aggravate the compensatory growth of remain cotyledon after partial defoliation.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Distribution of Three Auxin Protector Substances in Seeds and Shoots of the Japanese Morning Glory (Pharbitis nil)

The existence of substances which inhibit the enzymatic destruction of auxin in shoots of the Japanese morning glory (Pharbitis nil Choisy) has been confirmed, as has the fact that these substances are distributed in a gradient diminishing from apex to base in a manner indicating a regulatory role in internode elongation and tissue maturation. In addition to the 2 auxin protector substances reported previously (protectors I and II) which appear to account for most of the inhibition of the enzymatic destruction of auxin in young, elongating stem tissue, a third substance, designated as protector A, has been found to be highly active in seeds, and shoot tips of mature plants: In germinating seeds, no protector I or II activity was observed; in stem tips, no protector II and only slight protector I activity was observed. In contrast, old tissue contained no detectable amounts of protector A, but did contain protectors I and II. Between these extremes along the shoot axis, mixtures of the 3 substances were found. The evidence can be interpreted to mean that protector A is degraded into protectors I and II and perhaps translocated in this form. Gel filtration studies indicate that protector A has a molecular weight exceeding 200,000 gm/mole.     

Incorporation of C-Kaurene into the Gibberellin of a Higher Plant (Pharbitis nil Chois)

Enzymic formation of ¹⁴C-kaurene from 2-¹⁴C-mevalonate was carried out with a cell-free system of Cucurbita pepo L. It was shown that either heating of the enzyme system or the addition of the growth retardants (2-chloroethyl)-trimethylammonium chloride and 2′-isopropyl-4′ (trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate prevented the synthesis of ¹⁴C-kaurene. Experiments in which ¹⁴C-kaurene was applied to seedlings of Pharbitis nil revealed that the kaurene is converted to at least two compounds present in the acidic ethyl acetate fraction, containing free gibberellins, as well as in the second acidic ethyl acetate fraction, containing the released bound gibberellins. One of the compounds cochromatographed with gibberellic acid; the other compound is possibly a break-down product of gibberellic acid with no biological activity.     

Stem Internode Elongation in the Japanese Morning Glory (Pharbitis nil Choisy) in Relation to an Inhibitor System of Auxin Destruction

Further investigations on a wild-type strain of the Japanese morning glory (Pharbitis nil Choisy) to ascertain the relationship between stem internode maturation, decreasing rate of internode elongation, and increasing auxin destruction, have established the following: There exists in young, elongating, internode tissue, substances which prevent the destruction of indoleacetic acid by enzymes normally found in stem tissue. Almost all of the protecting activity can be attributed to two substances, one of them possessing an apparent molecular weight in the 5000 to 10,000 g/mol range, the other, in the 1500–5000 g/mol range. Both are water soluble, and heat labile, at least in vitro. It is further suggested that associated with Japanese morning glory stem maturation, is the loss of these auxin-protecting substances, and as a consequence of this loss, the loss of further endogenous auxin-induced elongation.