Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.     

Biologically active gibberellins in immature seeds of Pyrus serotin

Biologically active gibberellins were isolated from immature seeds of Japanese pear (Pyrus serotina). The major one was tentatively identified as GA₄ by selected ion monitoring. Evidence for the possible presence of GA₇ in the seeds was inconclusive but GA₃ was not detected by either the rice seedling or barley half-seed bioassay.     

The biosynthesis of the gibberellin precursorent-kaurene in cell-free extracts and the endogenous gibberellins of Japanese morning glory in relation to seed development

The endogenous levels of gibberellins (GAs) determined by a combined HPLC-bioassay procedure and the formation ofent-kaurene, an immediate GA precursor, in cell-free extracts were studied in relation to seed development inPharbitis nil Choisy cv. Violet. Three biologically active GA fractions were obtained, tentatively identified as GA₃, GA₅/ GA₂₀, and a GA fraction, possibly GA₁₉ and/or GA₄₄, which all increased in activity during early seed development and subsequently declined during maturation of the seeds. The total endogenous GA level reached its maximum at 19 days after anthesis, just before the seeds had attained their maximum fresh weight at about 23 days after anthesis. Similarly, theent-kaurene synthesizing capacity showed a rapid increase during the period of rapid growth of the seeds, followed by a decline during maturation. A direct relationship between the endogenous GA levels and theent-kaurene synthesizing capacity of a particular tissue was indicated.     

Kinetics of C-photosynthate translocation in morning glory vines

The movement of ¹⁴C-photosynthate in morning glory (Ipomea nil Roth, cu. Scarlet O'Hara) vines 2 to 5 meters long was followed by labeling a lone mature leaf with ¹⁴CO₂ and monitoring the arrival rate of tracer at expanding sink leaves on branches along the stem. To a first approximation, the kinetic behavior of the translocation profiles resembled that which would be expected from movement at a single velocity (“plug flow”) without tracer loss from the translocation stream. There was no consistent indication of a velocity gradient along the vine length. The profile moved along the vine as a distinct asymmetrical peak which changes shape only slowly. The spatial distribution of tracer along the vine reasonably matched that predicted on the basis of the arrival kinetics at a sink, assuming plug flow with no tracer loss. These observations are in marked contrast to the kinetic behavior of any mechanism describable by diffusion equations.

However, a progressive change in profile shape (a symmetrical widening) was observed, indicating a range of translocation velocities. A minimum of at least two factors must have contributed to the observed velocity gradient: the exchange of ¹⁴C between sieve elements and companion cells (demonstrated by microautoradiography) and the range of velocities in the several hundred sieve tubes which carried the translocation stream. Possible effects of these two factors on profile spreading were investigated by means of numerical models. The models are necessarily incomplete, due principally to uncertainties about the exchange rate between sieve elements and companion cells and the degree of functional connectivity between sieve tubes of different conductivities. However, most of the observed profile spreading may be reasonably attributed to the combined effects of those two factors.

The mass average velocity of translocation (calculated from the mean times of ¹⁴C arrival at successive sink leaves) was about 75% of the maximum velocity (calculated from the times of initial detection at the same sink leaves), which was usually between 0.6 and 1 cm min⁻¹. Owing to tracer exchange between sieve elements and companion cells, the mass average velocity of tracer in the sieve tubes was probably closer to 86% of the maximum velocity, a figure which agreed with a predicted velocity distribution based on calculated sieve tube conductivities and the size distribution of functional sieve tubes.

     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

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.     

Variations in endogenous gibberellins in developing bean seeds I. Occurrence of neutral and acidic substances

Activities of separated and chromatographed substances in the nonacidic, acidic ethyl acetate and acidic butanol fractions from bean seeds, Phaseolus vulgaris L., cv. Bountiful and Kentucky Wonder, were measured in the Progress No. 9 dwarf pea bioassay grown under red light. Activity in the nonacidic fraction was shown to be attributable only to neutral substances and was free of acidic gibberellin-like substances. As the seed matures, neutral substances and one of the acidic butanol-soluble substances (B-I) increase in activity. The acidic ethyl acetate substances and butanol-soluble substance (B-II) initially increase and then almost disappear.     

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.     

Comparison of endogenous gibberellins and of the fate of applied radioactive gibberellin a(1) in a normal and a dwarf strain of Japanese morning glory

The effect of application of GA₃ on hypocotyl growth, the endogenous GAs, and the metabolism of applied ³H-GA₁ were investigated in relation to dwarfism and light-mediated growth inhibition in the normal (tall) strain Violet and the dwarf strain Kidachi of Japanese morning glory (Pharbitis nil). GA₃ applied in a wide concentration range (10⁻⁹ to 10⁻³m) to 4-day-old seedlings caused great extension of the hypocotyls in light-grown plants of both the normal and the dwarf strain. However, the dwarf strain did not attain the same length as the normal one at any given GA₃ concentration, even when saturation was reached. Dark-grown plants of the dwarf strain responded to GA₃, although relatively much less than light-grown ones; dark-grown plants of the normal strain showed no GA₃ response at all.     

Source pool kinetics for C-photosynthate translocation in morning glory and soybean

The kinetic behavior of translocation profiles indicates that their shape is determined largely by the rate at which tracer enters the sieve tubes in the source leaf. Confirmation of this relationship was sought by investigating the kinetics of ¹⁴C in the immediate source pool for translocated sucrose in soybean (Glycine max L., cv. Bragg) and morning glory (Ipomea nil Roth, cv. Scarlet O'Hara) leaves. Quantitative microautoradiography was used to follow the water-soluble ¹⁴C contents of the companion cells in minor veins after pulse-labeling with ¹⁴CO₂. In both morning glory and soybean, the observed kinetics in the companion cells matched reasonably well those expected from the shape of the translocation profiles.

Marked compartmentation of sucrose was evident in soybean leaves in that the specific radioactivity of total leaf sucrose was greatest immediately after labeling and quickly declined, whereas labeling in the companion cells was low at first and did not reach a maximum for about 35 minutes. In morning glory leaves, the kinetics of sucrose specific radioactivity and of companion cell-labeling more closely paralleled one another.

     

Fitness consequences of cotyledon and mature-leaf damage in the ivyleaf morning glory

To understand the evolutionary and ecological consequences of natural enemy damage to plants, it is essential to determine how the fitness effects of damage differ depending on the tissues damaged and the subsequent pattern of damage. In a field experiment with the ivyleaf morning glory, the direct and indirect effects on fitness of herbivore damage to cotyledons and mature leaves was evaluated. Damage to mature leaves had negligible direct effects on fitness and no indirect effects on fitness through other correlated traits. Damage to cotyledons also did not directly affect fitness, but did so indirectly through its effects on plant size. These findings suggest that increased resistance to cotyledon damage or increased compensatory growth following cotyledon damage could be effective strategies for plants of this species to counteract the negative effects of herbivory.     

Ethylene binding changes in apple and morning glory during ripening and senescence

Ethylene binding sites were measured during fruit ripening and morning glory flower senescence. Little change in ethylene binding was noted during these developmental stages, except a slight decline during the later stages of fruit ripening or flower senescence. The concentration of ethylene required to achieve 50% saturation of the binding sites was 0.14 l/liter for both apple pulp and morning glory flowers. Ethylene binding sites were calculated to be 3.2×10^–11 moles/kg and 3.8×10^–9 moles/kg in apple and morning glory, respectively. It does not appear that changes seen in ethylene sensitivity during fruit ripening can be readily ascribed to changes in the number of ethylene binding sites in the tissue.Paper No. 11398 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA.     

Metabolism of gibberellins in early immature bean seeds

The endogenous free gibberellins in two different stages of immature Phaseolus vulgaris seeds were investigated and GA₁₇, GA₂₀, GA₂₉, a     

Ribonuclease in senescing morning glory: purification and demonstration of de novo synthesis

Isolated flower buds and flowers of Ipomoea tricolor went through the same stages of development as those attached to the plant. Ribonuclease activity increased sharply in both cases during the time of flower fading and aging. Affinity chromatography using guanosine diphosphate-Sepharose was employed for fast and efficient purification of ribonuclease. Flowers which were kept on D₂O during the senescence phase incorporated deuterium into ribonuclease as shown by isopycnic density gradient centrifugation in CsCl, suggesting that ribonuclease was de novo synthesized during aging of the flower.