Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.)

Gibberellins (GAs) A₁, A₅, and A₂₉ were identified, and also GA₃₂ was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning → paper chromatography → reverse phase C₁₈ high performance liquid chromatography (HPLC) → bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C₁₈ HPLC at Rts where GA_4/7 and GA₈ (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA₃₂ (112 ng of GA₃ equivalents per gram fresh weight), and minor amounts (0.5 ng of GA₃ equivalents) for each of GA₁ and GA₅, respectively, based on the microdrop assay.     

Purification and Biochemical Characteristics of Pectinesterase from Malatya Apricot (Prunus armeniaca L.)

Abstract

Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60°C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70°C. Km and V_max values were 0.77 mM and 1.75 µmol min^?1 mg^?1 for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na₂CO₃, iodine, lauril sulphate, AgNO₃, EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol^?1 for the enzyme. Ascorbic acid, CaCl₂, and KCl showed activatory effect on the apricot PE enzyme.     

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [²H]-ent-kaurenoic acid (KA) and [¹⁴C]-gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [²H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [²H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A₉ (GA₉) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA₉. In contrast, thermo- and noninduced leaves metabolized exogenous [²H]-KA into GA₂₀ equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [¹⁴C]-GA₁₂-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA₁₂-aldehyde.     

Metabolism of Tritiated Gibberellins in d-5 Dward Maize: II. [H]Gibberellin A(1), [H]Gibberellin A(3), and Related Compounds

After 30 minutes of incubation of young leaf sections of d-5 maize (Zea mays L.) in [³H]gibberellin A₁ ([³H]GA₁), the metabolite [³H]GA₈ was present in significant amounts, with a second metabolite, [³H]GA₈-glucose ([³H]GA₈-glu), appearing soon after. A third [³H]GA₁ metabolite, the polar uncharacterized conjugate [³H]GA₁-X, took more than 1 hour to appear. The protein synthesis inhibitor cycloheximide inhibited the production of all [³H]GA₁ metabolites, indicating a possible protein synthesis requirement for [³H]GA₁ metabolism.     

Effect of Abscisic Acid on Uptake and Metabolism of [H]Gibberellin A(1) and [H]Pseudogibberellin A(1) by Barley Half-seeds

Uptake and metabolism of 1,2-[³H]gibberellin A₁ ([³H]GA₁, I) and its 3-hydroxy epimer ([³H]pseudoGA₁, II) by barley (Hordeum vulgare L.) half-seeds were measured after 24 hours of incubation, in the presence or absence of abscisic acid in the media. Uptake of both compounds was enhanced by abscisic acid, and abscisic acid enhanced the extent of metabolism of [³H]GA₁. However, [³H]pseudoGA₁ was not metabolized, even in the presence of abscisic acid. The significance of the stereo-chemistry of the 3-hydroxyl position is discussed.     

中国南疆栽培杏群体遗传结构的荧光AFLP分析

以中国新疆维吾尔自治区南部塔里木盆地边缘库车、喀什和和田3个栽培杏群体的85个品种类型为试材,利用荧光标记AFLP对群体遗传结构进行了研究,结果表明:对64对EcoRⅠ/MseⅠ引物(其中MseI引物为FAM荧光标记物)进行了筛选,其中8对荧光标记引物谱带清晰,多态性高;同一引物在不同群体以及同一群体不同引物扩增多态性均存在显著差异,种级水平多态带百分率P>库车>和田>喀什3个群体水平;种级水平Nei基因多样度指数H和Shannon信息指数I>库车>和田>喀什3个群体水平。种级水平大于种下群体,各群体以库车最高;杏遗传多样性主要存在于群体内,群体间的遗传分化系数GST为0.0882,即群体间的遗传变异占总变异的8.82%,群体间的基因流Nm为5.1689;群体的遗传一致度在0.9772～0.9811之间,遗传距离在0.0191～0.0232之间,说明群体间的相似程度较高,遗传距离较小;UPGMA聚类分析结果表明,库车、喀什、和田3个亚群可能是相对独立的孟德尔群体,但同时存在部分基因交流;各项研究指标都表明库车群体的遗传多样性都最高,它可能是野杏向栽培杏过渡的中间群体;南疆栽培杏群体拥有极为丰富的遗传多样性,为进一步的选择育种提供了更多的种质资源,为该地区杏群体生物多样性保护和利用提供理论依据。     

Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₂₀ ([³H]GA₂₀) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [³H]GA₂₀, the three in highest yield were GA₂₉, an α,β-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA₂₀ → GA₂₉ → compound B → the ketone was indicated. The amount of [³H]GA₂₉ in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [³H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA₂₉, a 2β-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Isolation and Structure of a New Gibberellin from Immature Seeds of Prunus persica

New antibiotics, T1801 A, B, C, and D, were isolated from the culture broth of Pseudomonas sp. SC-1801. Their structures were found by spectroscopic analyses to be tri- and tetra(methylthio) derivatives of hydro quinone (T1801 A and C) or p-benzoquinone (T1801 B and D). They are new quinone and hydroquinone antibiotics and are active against Gram-positive bacteria, some fungi, and yeasts.     

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.     

Non-TIR-NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genes encoding for proteins with nucleotide-binding site and leucine-rich repeat motifs (NBS-LRR) have been suggested to play a general role in plant defence mechanism. In Prunus species, many TIR (Toll / Interleukin-1 Receptor), and only very few non-TIR sequences were identified, which was explained either by the unequal distribution of TIR/non-TIR sequences in the Prunus genome or by the incapability of primers in the amplification of non-TIR RGAs. The objective of this work was to check whether a new semi-nested PCR strategy can be developed for the targeted isolation of non-TIR-NBS-LRR Resistance Gene Analog (RGA) sequences from apricot. Three primers (CUB-P-loop F, CUB-Kin2 F and CUB-HD R) were designed, from which CUB-Kin2 F and CUB-HD R were constructed to anneal selectively to the non-TIR sequences. A colony Polymerase Chain Reaction (PCR) indicated that out of the 96 clones tested 28 showed amplification using the newly developed primers, while no amplification occurred when using the formerly described primers. Half of the 28 positive clones were sequenced and they turned out to represent 11 different non-TIR RGA sequences. A phylogenetic analysis was carried out based on an alignment containing 293 Rosaceae and 21 non-Rosaceaa sequences. A significantly higher ratio (91%) of non-TIR sequences were arranged in multi-genera clades than that of (57%) the TIR groups confirming that non-TIR sequences might be of more ancient origin than TIR sequences.     

Effective pollination period in apricot (Prunus armeniaca L.) varieties

A study of the effective pollination period (E.P.P.) of six varieties of apricot has shown that in the warm climate of the South Mediterranean this parameter is extremely short. The percentage of fruit set of emasculated flowers diminishes rapidly with time. Temperature plays an important role in this process and is responsible for the rapid degradation of the stigma, which hinders pollen germination.     

Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

Changes in Compositional Properties during Fruit Development and On-Tree Ripening of Two Common Apricot (Prunus armeniaca L.) Cultivars

The objective of the present study was to investigate the variations in some major primary (sugars and organic acids) and secondary (phenolics, β-carotene) metabolite contents during fruit development and ripening in two important apricot cultivars (Hacıhaliloğlu and Kabaaşı). The changes in the compositional properties of two apricot cultivars were monitored during fruit development with one-week intervals from 56 to 119 days after blossom. During fruit development, the contents of organic acids and phenolics decreased whereas that of sucrose and sorbitol increased. p-Coumaric acid was the only phenolic compound which increased in concentration during fruit development regardless of the cultivar. The content of the other phenolic compounds decreased in a cultivar-dependent manner. The β-carotene content of the cultivars showed distinct patterns of change such that 3 fold increase in β-carotene content of Kabaaşı cultivar was observed whereas the β-carotene content of the Hacıhaliloğlu cultivar did not show any significant change during fruit development.     

Vernalization and Gibberellin Physiology of Winter Canola (Endogenous Gibberellin (GA) Content and Metabolism of [3H]GA1 and [3H]GA20

Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.     

Polyacetylenes from Immature Seeds of Safflower (Carthamus tinctorius L.)

Six polyacetylenes have been isolated from immature seeds of safflower (Carthamus tinctorius L.) by thin-layer chromatography. They were identified as 1,11-tridecadiene-3,5,7,9-tetrayne, 1,3,11-tridecatriene-5,7,9-triyne, 1,3,5,11-tridecatetraene-7,9-diyne, 1-tridecene-3,5,7,9,11-pentayne, 1,3-tridecadiene-5,7,9,11-tetrayne and 1,3,5-tridecatriene-7,9,11-triyne from the results of their spectroscopic and chemical analyses. Three of these polyacetylenes had not been isolated from Carthamus tinctorius L.

Changes in the polyacetylene content during maturation were followed by the measurement of ultraviolet absorbance. While 1,3,11-tridecatriene-5,7,9-triyne and 1,3,5,11-tridecatetraene-7,9-diyne had already occurred abundantly at the day of flowering, the amounts of the other polyacetylenes reached the maximum values at the fourth to sixth days after flowering. Although the total amounts of the six polyacetylenes were about 0.8 mmole/g lipid at the fourth day after flowering, no polyacetylene was detected in the mature seeds.