Gibberellin-like Activity in the Developing Avocado Fruit

Gibberellin-like activity in avocado (Persea americana) fruit extracts was measured by the barley endosperm bioassay. Fruit tissues were anlyzed separately during fruit growth. The level of the activity found was very high in the endosperm and in the seed coats, but decreased in the latter during fruit growth. No measurable gibberellin-like activity was detected in the mesocarp or in the embryo. It is assumed that the seed coats are a site of production of gibberellin-like substances in the avocado fruit.     

Production of gibberellin-like substance in the embryo of barley during germination

Summary Embryos were excised from barley seeds, their homogenates were incubated with ficin, and their content in gibberellin-like substance was assayed by means of -amylase-producing activity, but no gibberellin-like substance could be detected.Embryos free from endosperm which were cultured for five days produced measurable amounts of gibberellin-like substance. This substance was not produced when a carbon source was absent from the medium, or when air was removed after 2 days of aerobic culture. CCC and Phosfon D (at the concentration of 2×10^-4 M and 0.8×10^-4 M, respectively) inhibited the formation of the gibberellin-like substance in cultured embryos without affecting their growth.Mevalonic acid could be used as a carbon source in the culture of the embryos. The formation of the gibberellin-like substance was in this case inhibited by 0.8×10^-4 M Phosfon D, but was not inhibited by 2×10^-3 M CCC.     

Gibberellin-like Substances in Developing Barley Grain and Their Relation to Dry Weight Increase

Gibberellin-like activity of two varieties of barley (Hordeum vulgare L.) at different stages of grain development was determined by barley endosperm bioassay (acid phosphatase bioassay). Activity of the acidic ethyl-acetate fraction (“free” GAs) in both varieties displayed two maxima, a first maximum at the 9th day and a second one 20 to 21 days after pollination. Activity of the n-butanol fraction (“bound” GAs) first dropped to a minimum level at the 9th day, then increased to reach a maximum 32 days after pollination, and finally decreased again towards maturity. From the 9th day after pollination, a conversion of “free” GAs to “bound” GAs has probably occurred. From the 12th day after pollination, the curves of rate of dry weight increase and of “free” GAs run nearly parallel, but the latter reached its maximum about 2–4 days earlier than the former. The results indicate that gibberellins may participate in the regulation of the accumulation processes into the barley grain.     

Physiological effects of gibberellic Acid. X. The release of gibberellin-like substances by germinating barley embryos

Barley embryos, completely free from endosperm, were excised from germinating grain at various times and allowed to diffuse into an aqueous medium for varying lengths of time. At the end of this time, the embryos and ambient solutions were separately extracted. Gibberellin-like activity in the extracts was determined with the barley endosperm bioassay using seed from the same variety, harvest and treatment schedule as was employed for the embryo diffusion experiments. Gibberellin-like substances were released by embryos throughout the 60 hour germination period, though at no time during this period could sufficient activity be extracted from the embryos themselves to account for the observed release. Solvent partitioning and chromatography identified at least one major acidic component migrating at an Rf similar to that of GA₃.

It is concluded that the endogenous gibberellin-like substance(s) originates within the embryo during germination, and that the release of this substance(s) is temporally consistent with, and quantitatively sufficient to account for the in vivo endosperm mobilization response syndrome. A gibberellin-like substance is undoubtedly the endosperm mobilizing hormone.

     

The Mechanism of Low Temperature Dormancy in Mature Seeds of Syringa Species

The mechanism of seed dormancy at low temperatures (15-9°C) was investigated in the seeds of Syringa josikaea, S. reflexa and S. vulgaris. Low temperature dormancy in Syringa species was mainly imposed by endosperm embedding the radicle. Different degrees of embryo dormancy may occur in S. reflexa seeds. In most cases the low temperature dormancy was broken completely by removing the endosperm around the radicle. The endosperm did not seem to contain significant quantities of germination inhibitors, and the results indicate that it prevents germination mainly due to its mechanical resistance. The mechanical resistance of endosperm did not change during chilling or during induction of dormancy by high temperature incubation. The strength of the endosperm decreased rapidly in non-dormant seeds before visible germination. Similar changes were not observed in dormant seeds. Generally, the strength of the endosperm was lower in the non- (or less) dormant species S. josikaea and S. vulgaris than in the more dormant S. reflexa seeds. The growth potential of the embryos, measured as their ability to germinate in osmotic solutions (mannitol or polyethylen glycol 4000), was increased by chilling and by GA₃-treatment. The growth potential of untreated S. josikaea and S. vulgaris embryos was generally higher than that of S. reflexa embryos. Acid ethyl acetate fractions of methanol extracts from embryos of all three species contained substances with GA³-like activity in the lettuce hypocotyl test. The activity was found at Rf 0.9–1.0 on paper chromatograms run in distilled water. No significant changes in the activity were detected during chilling or prior to visible germination.     

Evidence for gibberellin-like substances in phloem exudate of higher plants

Summary Samples of sieve-tube sap were obtained as honeydew from aphids feeding on three species of higher plants. The honeydew was extracted, chromatographed and tested in several bioassays for the presence of gibberellin-like substances. The bioassay results indicated that gibberellin-like substances were translocated in the phloem of dandelion (Taraxacum officinale), broad bean (Vicia faba) and willow (Salix viminalis). Results obtained with willow showed that the concentration of gibberellin-like substances in the sieve-tube sap is daylength dependent, high levels being present in plants maintained under long days and low levels in short day plants.     

Cyclic Purine Mononucleotides: Induction of Gibberellin Biosynthesis in Barley Endosperm

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3′,5′-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of α-amylase formation by 3′,5′-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA₃. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase.     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

SEED DORMANCY AND GERMINATION IN MELAMPYRUM LINEARE

Immature seeds of Melampyrum lineare Desr. have very high germination percentages and dormancy is induced in a variable fraction of the seed crop during ripening. Correlated with this is the endogenous gibberellin-like activity which is found in considerable amounts in immature seeds, less in batches of ripe seeds, and is not detectable in batches containing only dormant seeds. For germination dormant seeds require activation followed by cold storage. In the laboratory activation is produced by allowing moist, dormant seeds to respire freely for several weeks at 20 C, or by treatment with exogenous GA₃. Dormancy appears to be most directly related to inability of the embryo to hydrolyze the thickened, mannan-containing endosperm cell walls. Embryos excised from dormant seed can be grown on agar enriched with whole macerated dormant seeds or with the ethanol-extractable materials from these (mostly sucrose and a glycoside). However, dormant seed material does not support growth when extracted to remove benzene- and ethanol-soluble materials.     

Soluble and particulate lysophospholipase in the aleurone and endosperm of germinating barley

Lysophospholipase was measured in extracts of germinating barley by determining the amount of free [¹⁴C]palmitate released from [1-¹⁴C] 1-palmitoyl-lysophosphatidylcholine (LPC). Soluble and particulate lysophospholipase activity was measured at 1-day intervals in extracts from the aleurone and endosperm of barley seeds germinated for 8 days. The soluble and particulate activities of the aleurone increase approximately in parallel with one another and after 8 days of germination have 20–30 times more activity than at day 1. The activity profiles and the distribution of the activity between the soluble and particulate forms of lysophospholipase in the endosperm are markedly different. With the exception of the first 2 days when the aleurone activity is low, the endosperm activity is less than that associated with the aleurone. The soluble activity increases during the first 3 days and is more active than that of the aleurone. Thereafter it diminishes and remains low. The particulate enzyme, however, increases dramatically between days 4 and 5 and remains moderately high. The fourth and fifth day represent that stage of germination when starch-bound LPC is released in concert with the increase in amylase activity. It is proposed that it is this particulate form of the endosperm activity which may be responsible for maintaining the level of free LPC low in the endosperm of the germinating seed.     

Gibberellins of sugarcane

In our hands a phosphate buffered celite column has given an adequate separation of GA₁ and GA₃. These 2 gibberellins are normally very difficult to separate. Young sugarcane growing under moisture stress contains at least 2 gibberellin-like substances. One is suspected to be GA₅. The other is unknown but has high activity in the barley endosperm assay and is neither GA₁ nor GA₃. Four-month-old cane contains 2 major growth promoters. From their chromatographic, fluorimetric and biological properties these are thought to be GA₁ and GA₃. Rapidly growing 6-month-old cane has a surprisingly low level of gibberellin-like substances.     

Barley endosperm bioassay for gibberellins. I. Parameters of the response system

Parameters of the bioassay based on the gibberellin-induced reducing sugar release of barley endosperm were investigated. Procedures for the rapid handling and processing of up to several hundred treatments without loss in sensitivity of the test are described, and the effects of variations in many aspects of the bioassay were assessed.

In general, the variations in varieties, techniques, additives, conditions, and even gibberellins, all illustrate the stability, sensitivity, and adaptability of the hormone-induced response and emphasize its utility as a gibberellin bioassay.

     

Genetic investigation of contributions of embryo and endosperm genes to malt Kolbach index, alpha-amylase activity and wort nitrogen content in barley

 A genetic model is proposed for the analysis of embryo and endosperm effects as well as GE interaction effects. An investigation of three malting quality traits in grains of seven parents and their F₂s was undertaken in a half-diallel cross of barley (Hordeum distichum L.) over 2 years. The results indicated that the malt Kolbach index (KI), alpha-amylase activity (αAA) and wort soluble nitrogen (Wort-N) are controlled by both embryo genetic effects and endosperm genetic effects. Variance of the endosperm additive effects was obviously larger than that of the embryo additive effects. In the contribution of the embryo genetic effects to variation in malt αAA and Wort-N, the dominance effects were considerably larger than the additive effects. The endosperm dominance effects constituted a major part of the total genetic effect on the KI. Significant endosperm GE interactions were also detected in the malt traits concerned. Endosperm general heritability (h ² _e ) tended to be larger than interaction heritability (h ² _oE or h ² _eE ) for all the traits. Endosperm heterosis was observed to be significantly positive for αAA but negative for Wort-N in the F₂ seed generation. Prediction of main gene effects for seven parents showed that ‘Ganmu 2’ and ‘Supi1’ were suitable parental varieties for malt αAA and Wort-N improvement. Our genetic model for malting quality traits and its application in breeding are discussed. Received: 5 August 1997 / Accepted: 11 September 1997     

Gibberellin-like substances in root exudate of Vitis vinifera

Summary The levels of gibberellin (GA)-like activity in the root exudate of two seedless varieties of Vitis vinifera were examined by the barley endosperm assay, and compared with levels determined for other parts of the plant. That activity was due to GA-like substances was confirmed with dwarf-5 corn.When acidic, ethyl acetate soluble GA-like substances from sap and leaf extracts were chromatogrammed on thin layers of silica gel in chloroform/ethyl acetate/formic acid (50:50:1), activity moved to the same Rf as GA₃ and GA₁ (Rf 0.05–0.25). However, substances inhibitory to the barley endosperm assay were detected in both sap and leaf extracts. In the above solvent system the inhibitor(s) co-chromatogrammed with a GA₁/GA₇ mixture, and with abscisin II. The GA-like activity co-chromatogrammed with GA₃ on paper developed in isopropanol/ammonia/water (10:1:1).Calculations on the rate of gibberellin movement from the roots seemed to be compatible with levels of activity in the leaves, although these levels could also be a reflection of the general gibberellin level in the plant.The relevance of the findings is discussed.     

Distribution of gibberellin-like substances in light-and dark-grown seedlings of Phaseolus multiflorus

Summary Lettuce and barley endosperm bioassays of successive eluates from a phosphate-buffered celite column detected two gibberellin-like compunds, Phaseolus I and Phaseolus II, in extracts of both light-and dark-grown seedlings of Phaseolus multiforus. There were differences in the gibberellin content of light-and dark-grown seedlings, the former containing more Phaseolus I but less Phaseolus II than the latter. An examination of the gibberellin content of leaves and apical buds, stems, cotyledons, and roots of light-and dark-grown seedlings revealed distinct qualitative and quantitative differences in the distribution of Phaseolus I and Phaseolus II.     

Occurrence of proteolytic inhibitors in various tissues of barley

Summary The three groups of proteolytic inhibitors present in resting barley grains, namely, trypsin inhibitors, Aspergillus-proteinase inhibitors, and inhibitors of endogenous proteinases, occur in both the embryo and the two endosperm tissues. There are pronounced quantitative differences, however. The three inhibitor activities in the embryo are, respectively, 6-, 0.1-, and 6-fold of those in the endosperm.During germination at 20° all inhibitor activities disappear from the endosperms in 4–5 days. Young rootlets and coleoptiles contain inhibitors of trypsin and Aspergillus proteinase, but these disappear after 4–5 days' germination. However, the trypsin inhibitor content per seedlings remains roughly constant through the whole period. The Aspergillus-proteinase inhibitors, in contrast, exhibit a pronounced increase of activity per seedling.No inhibitor activities were detected in leaves and roots at later stages of growth.The trypsin inhibitor which we have earlier purified from resting grains occurs exclusively in the two endospermal tissues and is immunologically entirely different from the trypsin inhibitors present in embryos and young seedlings.     

Chemical characterization of the aqueous algistatic fraction of barley straw (Hordeum vulgare) inhibiting Microcystis aeruginosa

The algistatic properties of aqueous barley straw (Hordeum vulgare) extracts have been observed in laboratory studies and in situ. This reported algistatic property has been used by farmers and horticulturists to control algal blooms in various systems and has become standard practice in some areas. However, both inhibition and stimulation of algal growth in freshwater and marine species have been demonstrated. While the number of taxa known to be inhibited by barley straw has increased, comparatively little has been done to isolate and classify the compound(s) responsible for this algistatic effect. A microplate assay system using Microcystis aeruginosa was developed to isolate and identify the inhibitory components of barley straw extract. M. aeruginosa was selected for the bioassay because it is consistently inhibited by barley straw extract in studies conducted by our laboratory and others. The 24-well plate assay utilizes in vivo fluorescence monitoring with a TECAN GENios plate reader to determine chlorophyll-a levels in each culture. Fractionation and partial chemical characterization of inhibitory extracts suggests that the inhibitors are polyphenolics with molecular weights (MW) between 1,000 and 3,000 Da. Percolation of the aqueous extract through a Polyamide CC6 resin or through various MW cutoff filters resulted in the loss of algistatic activity, which confirms this assertion, while hydrolysis resulted in little change in the activity profile. Fractionation by HPLC methods yielded a highly potent multi-compound fraction, showing toxicity at 353 mg L⁻¹ and algistatic activity between 11.1 and 3.53 mg L⁻¹.     

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice,Confers Resistance to the Rice Weevil Sitophilus Oryzae

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R₃ seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.     

Hormonal Control of Germination under Saline Conditions of Three Halophytic Taxa in the Genus Suaeda

The interaction between hormones and salinity on seed germination of three halophytic taxa in the genus Suaeda: S. maritima (L.) Dum. var. flexilis Focke and var. macrocarpa Moq., and S. depressa (Pursh) Wats, was studied. Exogenous applications of kinetin and gibberellic acid (GA3) were applied in order to determine if either of these growth-promoting hormones would promote germination in the two dormant taxa, Suaeda depressa and S. maritima var. flexilis and to see if osmotically induced dormancy by NaCl could be alleviated. Our results indicate that gibberellic acid is capable of breaking dormancy in these species with dormant seeds, but kinetin proved to be ineffective. A seed dormancy that was induced by osmotic stress could also be alleviated by treatments with gibberellic acid. Endogenous concentrations of both cytokinins and gibberellins were measured in seeds exposed to osmotic stress (0.85 M NaCl), and we found a reduction in cytokinin activity in these three taxa. Gibberellin-like activity was reduced in S. depressa when seeds were soaked in 0.85 M NaCl.     

The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work.