Comparison of growth and gibberellin concentrations in shoots from orchard-grown standard and thermosensitive dwarf apple trees

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA₃ did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA₁, GA₃ and GA₈, but standard shoots contained higher concentrations of GA₁₉, GA₂₀ and GA₂₉. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA₁₉, GA₂₀, GA₁ and GA₂₉ levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA₁₉ content of dwarf shoots nearly doubled, whereas the amounts of GA₂₀, GA₁, GA₂₉ and GA₈ declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA₁₉ to GA₂₀ in these genotypes.     

Comparison of seasonal growth and polyamine content in shoots of orchard-grown standard and genetic dwarf apple tress

Seasonal variations in growth rate, and polyamine and arginine content were determined in shoots of standard and genetic dwarf apples ( Malus domestica Borkh.) in orchard conditions. Putrescine, spermidine, spermine and arginine exhibited the same seasonal variations. Polyamine titer and arginine content were correlated. Putrescine, spermidine, spermine and arginine levels increased during March and April, reaching the maximum at the end of April. From the onset of May, they decreased gradually until they reached a steady level at the end of July and beginning August. Then the level increased until September when growth stopped. Shoots of both standard and dwarf trees started to grow in April, elongated slowly during the summer and ceased growth in September. Polyamine and arginine content of the shoots of standard apple trees were higher compared to the shoots of genetic dwarfs, but the concentration of polyamine and arginine were not correlated with the growth rate.     

Drought stress of apple trees alters leaf emissions of volatile compounds

Actively growing potted apple trees ( Malus domestica [L.] Borkh. ev. Delicious) unacclimated to drought stress were subjected to drought to determine changes in emissions of leaf volatile compounds. Drought stress was imposed over a 2-week period by weighing pots every 2 or 3 days and adding water hack to an arbitrary and decreasing traction of the original pot weight. Stem water potential was -2.7. -2.0 and -0.8 MPa for the severely stressed, moderately stressed and control trees, respectively. 13 days alter watering treatments were begun. Water use the last 4 days of the experiment was about one-half for the moderately and severely stressed trees compared to that of the controls Twenty-nine volatile compounds were identified by using gas chromatography and mass spectroscopy. Emission rates of hexanal, (E)-2-hexenal, (E)-2-hexen-1-ol, 1-hexenol, hexyl acetate and (E)-2-hexenyl acetate were 5 to 310 times higher for severely stressed trees compared to those of the controls with the moderately stressed trees intermediate. The large increases in hexanal. (E)-2-hexenal and l-hexanol may be related. In enhanced lipoxygenase activity. Volatile compounds are products of metabolism and measurement of their changes after biotic or abiotic stresses will increase understanding of the relationship of changes in plant metabolism by those stresses.     

Response of genetic dwarf apple plants to GA3

High temperature has been implicated as the major factor responsible for dwarfing of selected apple ( Malus domestica Borkh.) trees of a hybrid population of cv. Goldspur Delicious x cv. Redspur Delicious. Dwarf plants grew only 2.2 cm in 63 days under a ramped temperature regime (night 15°C, day ramped up to 38°C, held for 2 h and ramped down to 15°C—14 h daylength), whereas semi-dwarf plants grew 26.3 cm. At a constant 27°C (14 h daylength), both dwarf and semi-dwarf plants grew 26.3 cm. At a constant 27°C (14 h daylength), both dwarf and semi-dwarf plants grew nearly 50 cm. The gibberellin biosynthesis inhibitor, paclobutrazol, retarded growth of semi-dwarf plants in both ramped and constant environments and dwarf plants in the constant 27°C environment. It did not further reduce the size of dwarf plants growing under the ramped regime. Gibberellin (GA₃) treatment reversed the inhibition of growth caused by paclobutrazol for all plants except it did not restore growth of dwarf plants in the ramped environment. These data suggest that neither pacobutrazoltreated nor untreated dwarf plants growing in the ramped environment (or in the orchard during hot summer months) are able to respond to GA₃. In constrast, GA₃ was utilized by the paclobutrazol-inhibited dwarf plants growing at constant 27°C, enabling shoot elongation to take place. It appears that high temperature may have caused alterations in GA target tissues in dwarf plants so that they no longer had the capacity to respond to GA.     

Benzyladenine-controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin-requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA-treated cells showed a well developed sub-cellular organization and protein synthesis. Upon transfer to fresh cytokinin-free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin-deprived cells was overcome by exogenous BA-treatment. In cells maintained on a cytokinin-free medium for 5 days, BA-treatment enhanced protein synthesis from pre-existing mRNA in a specific way.     

Jasmonic acid promotes germination and lipase activity in non‐stratified apple embryos

The effects of (−)jasmonic acid (JA) on germination of embryos isolated from dormant seeds of apple ( Malus domestica Borb. cv. Antonówka) cultured in darkness or at 12‐h photoperiod were studied, as well as its effects on the activity of alkaline lipase (AlkL, EC 3.1.1.3) in these embryos. The maximum sensitivity of germination to JA occurred on days 3 and 4 of embryo culture. Both germination and enzyme activity were stimulated by JA, its effect being additive to that of light. Inhibitors of lipoxygenase inhibited embryo germination and AlkL activity, both effects being partially reversed by JA treatment. We suggest that (1) JA is implicated in an endogenous complex controlling apple seed germination, and that (2) it acts independently of the mechanism triggered by light.     

Effect of paclobutrazol on membrane lipids in apple seedlings

The effect of paclobutrazol [( 2RS, 3RS )-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2, 4-triazol-1-yl) pentan-3-ol] on the fatty acid composition of polar lipids and on the sterol content in apple ( Malus domestica Borkh. cv. York Imperial) seedlings was determined. Polar lipids isolated from leaves, stems and roots included mono- and digalactosyldiglycerides and the phospholipids phosphatidylinositol, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids in membrane polar lipids were palmitic (C16:0), linolnic (C18:2) and linolenic (C18:3). The predominant sterol, both free and esterified, was β-sitosterol. There were no significant alterations in the fatty-acid composition of glyco- and phospholipids from paclobutrazol-treated apple seedlings. In contrast, a significant decrease in the content of β-sitosterol and campesterol occurred in treated tissues. The decline in sterol content continued with increasing duration of paclobutrazol treatment, and was most pronounced in the root tissue.     

Influence of an inhibitor of gibberellin biosynthesis, paclobutrazol, on apple seed gibberellin content

Tissue-culture-propagated own-rooted cv. Spartan apple trees (Malus domestica Borkh.) planted in 1979 were treated in 1983 and 1985 via a soil-line trunk drench with the plant growth retardant paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4.4-dimethyl-2-(1,2, 4-triazol-1-yl) pentan-3-ol]. Seeds of immature fruits from untreated and treated trees were sampled in 1989 ca 75 days after full bloom. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified and fractionated via C₁₈ reversed-phase high-performance liquid chromatography (HPLC). Gibberellins A₁, A₃, A₄, A₇, A₈, A₉, A₁₅, A₁₇, A₁₉, A₂₀, A₂₄, A₃₄, A₃₅, A₄₄, A₅₁, A₅₃, A₅₄, A₆₁, A₆₂, A₆₃ and A₆₈ were identified by using C₁₈ HPLC, gas chromatography-selected ion monitoring and Kovats retention indices. Eight of the GAs identified were also quantified by using deuterated internal standards. The paclobutrazol applications caused a 55% reduction of vegetative shoot elongation in 1989, but both treated and untreated trees had developed a biennial bearing pattern by that time (heavy bloom or “on year’in 1989). Levels of early 13-hydroxylation pathway GAs, viz. GA₅₃, GA₁₉, GA₂₀, GA₁ and also GA₃, were not altered by treatment. However, GA₄, GA₇ and GA₉ were increased 13.4, 6.5 and 3.8 times, respectively, in seeds of fruit from treated compared to untreated trees.     

Changes in parameters of the plasmalemma ATPase during cold acclimation of apple (Mains domestica) tree bark tissues

A plasmalemma fraction was isolated from homogenized apple tree (Mains domestica Borkh 'Golden Delicious') hark tissues using aqueous phase partitioning and ultra-centrifugation. Results of marker enzyme assays indicated that a membrane preparation highly enriched in plasma membranes was obtained. ATPase activity in this preparation possessed a high specificity for ATP as substrate, was inhibited by vanadate, diethylstilbcsterol and dicyclohexylcarbocHimide, and was insensitive to inhibitors of mitochondria! and tonoplasl ATPases. Specific activity of the plasma-lemma ATPase increased during cold acclimation prior to the attainment of vegetative maturity. Kinetic parameters (Km, Vln) determined from assays performed at different temperatures (10, 30°C) indicated a differential effect of cold acclimation on enzyme activity. Vm increased during cold acclimation, whereas Km increased when determined at 30°C but declined at 10°C. Acclimation treatments during April and May resulted in alteration of ATPase kinetics in the absence of any increase in bark frost hardiness. Changes in ATPase kinetics may be related more to enhanced low temperature metabolism than to frost hardiness per se.     

Effect of thidiazuron on abscisic acid content in apple bud relative to dormancy

The effect of N -phenyl- N '-1,2,3,-thidiazol-5-ylurea (thidiazuron, Dropp, SN 49537) on abscisic acid (ABA) level in apple ( Malus domestica Borkh. cv. York Imperial) buds associated with bud break and bud development was determined. The data showed that increased ABA content in apple buds was associated with thidiazuron-induced bud break and bud development. ABA stimulated growth of apple buds that had emerged from dormancy by thidiazuron treatment. The ABA in apple buds was confirmed by GC-EIMS and GC-CIMS.     

Polyamines and ethylene in the removal of embryonal dormancy in apple seeds

Putrescine (Put), spermidine (Spd) and spermine (Spm) were found in seeds of apple ( Malus domestica Borkh. cv. Antonovka), in amounts that increased in the order given. The levels slowly decreased during 30 days of stratification. Exogenous polyamines (PAs) affected germination of isolated embryos in a way dependent on the type of polyamine, its concentration, and the state of the embryo dormancy. The effect of Put and Spd on germination was stimulatory, while that of Spm was inhibitory. Stimulation of germination was also observed when embryos were treated with arginine, ornithine and methionine. Canavanine inhibited germination, and this effect was reversed by arginine or Put. Ethephon, aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) present during seed stratification had no effect on the levels of endogenous PAs. Put and Spd did not change ethylene production, neither during seed stratification nor during embryo germination, whereas Spm reduced ethylene evolution. The data suggest that Spm plays a role in the maintenance of dormancy by preventing ethylene production, while Put and Spd participate in dormancy removal, independently of ethylene.     

Characterisation of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG; >90 mol%) which was purified by selective precipitation with Fehling’s solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.     

Controlling plant growth via the gibberellin biosynthesis system – I. Growth parameter alterations in apple seedlings

'York Imperial' apple seedlings ( Malus domestica Borkh.) were continuously supplied via the roots with paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol)], a triazole GA biosynthesis inhibitor, at 0.68 μ M in a nutrient solution. In comparison to controls, seedlings treated with paclobutrazol for 66 days showed a 91% reduction in shoot length, a 66% reduction in leaf area but only a 17% reduction in leaf number. This effect could be reversed by GA₃ applied to the foliage at 71.4 μ M 0, 19 or 35 days after paclobutrazol was initially supplied and leaf area values for paclobutrazol-treated seedlings given both treatments did not differ significantly from controls. Plots of growth data indicate linearity of shoot longitudinal growth of GA₃-treated seedlings. Leaf area increase was non-linear after GA₃ treatment up to approximately 30 days, when the rate dropped. On a per shoot basis, leaf weight closely followed leaf area but on a per unit area basis, paclobutrazol-treated leaves were heavier than controls; GA₃ applications temporarily reversed this trend.     

The embryonic axis controls lipid catabolism in cotyledons of apple seeds during germination

Activities of alkaline lipase (AlkL, EC 3.1.1.3), isocitrate lyase (ICL, EC 4.1.3.1), glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and pyruvate kinase (PK, EC 2.7.1.40) were determined in embryos of apple ( Malus domestica Borb. cv. Antonówka) during culture in darkness or at 12 h photoperiod; in both cases either in the presence of gibberellin A₃ (GA₃) or AMO 1618 (inhibitor of GA synthesis). AlkL and ICL were stimulated by light and GA₃; light stimulation was reversed by AMO. G6PDH and PK were not affected by culture conditions. Almost all the activity of all enzymes was found in the cotyledons; only PK was distributed between axis and cotyledons. GA-like activity was found almost exclusively in the embryo axis. Cultured isolated cotyledons lost their sensitivity to light and AMO, but AlkL and ICL were still stimulated by GA₃. Translocation of GA from axis to cotyledons during the culture of embryos is postulated.     

Effects of cyanide and cold treatment on sugar catabolism in apple seeds during dormancy removal

The ratio of glycolysis to the pentose phosphate pathway (PPP, C₆/C₁ ratio), and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and pyruvate kinase (PK, EC 2.7,1.40) were determined in apple seeds ( Malus domestica Borb, cv. Antonówka) submitted to cold and warm stratifications. Our results indicated that the elimination of embryonal dormancy in apple seeds was connected with a change from domination of PPP to domination of glycolysis in sugar catabolism during cold stratification. Cyanide pretreatment affected the C₆/C₁ ratio and the activities of the enzymes under study in such a manner that the maxima of PPP and glycolysis appeared earlier during stratification. We suggest a regulatory role of cyanide in removal of dormancy.     

Endogenous polyamines in apple and their relationship to fruit set and fruit growth

The distribution of free and bound polyamines was investigated from blooming until harvest on flower, fruitlets and fruits of Malus domestica Borkh cv. Golden Delicious, Relationships between polyamines and fruit set and growth were also investigated. The level of free polyamines was high only during the first weeks after full bloom and then decreased gradually. The amount of bound trichloroacetic acid-insoluble polyamines was much higher than free polyamines. Bound spermine in particular showed a high value for almost 40 days after full bloom, while spermidine and putrescine were no longer detectable even a few days after full bloom.
In relation to fruit set, it was possible to observe that abscission peaks took place when free polyamine levels were low or decreasing. Insofar as fruit growth is concerned, the most substantial variations in polyamine levels occurred very early in the season when fruit dry weight and protein amount were also changing rapidly and fruit diameter was almost impossible to measure.     

Diffusible IAA and dominance phenomena in fruits of apple and tomato

The relationships between indole-3-acetic acid (IAA) diffusing out of the fruit and competition among fruits, and between fruits and shoot tips were investigated using apple ( Malus domestica Borkh. cv. Jonagold) and tomato ( Lycopersicon esculentum Mill.) plants. Dominant fruits always had more diffusible IAA than subordinated, inhibited fruits. Alterations in dominance – by fruit- or shoot tip removal – led to significant changes in diffusible IAA by the remaining fruits. This change could be detected one day after dominance modification.
It is suggested that diffusible IAA is involved in the correlative signal regulating dominance relationship between fruits, and between fruits and shoots in apple and tomato.     

Toxicity of ethanol during proliferation and adventitious root formation in apple microcuttings in vitro

We have examined the toxicity of ethanol in tissue culture of the apple rootstock ‘Jork 9’. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the genetic diversity and structure of the Spanish apple genetic resources suggests the existence of an Iberian genepool

The nature and structure of genetic diversity in the Spanish apple germplasm preserved at the national level was widely unknown, since studies performed to date on this topic have been exclusively carried out at the regional scale. Here, 1453 accessions from Spanish collections of Malus × domestica were evaluated with a common set of 13 SSR (Simple Sequence Repeats) markers in order to estimate genetic diversity, to identify the underlying genetic structure and to unravel the relationships among them and among a wide set of international cultivars for reference. In total, 737 unique genotypes were identified, 581 diploids and 156 triploids. Using a model‐based Bayesian clustering procedure, two reconstructed populations were obtained for diploid genotypes; one retaining only Spanish cultivars (42% of genotypes), and a second containing all foreign cultivars the latter exhibiting evidence supporting the existence of a secondary sub‐structure. Similarly, analysis performed on the 156 triploid genotypes also revealed two reconstructed populations; one exclusively associated with local Spanish genotypes (44%). The Jaccard coefficient allowed clustering by UPGMA (Unweighted Pair Group Method) diploid and triploid genotypes, and remarkable differences in allelic composition among the different partitioning levels were found. AMOVA analyses showed moderate but significant differentiation among the main groups (0.08 ≤ F_ST ≤ 0.12). Our results highlight an important fraction of the Spanish apple germplasm that constitutes a differentiated genepool with respect to the international and commercial apple cultivars. Moreover, the extent of the Spanish genetic diversity was spatially distributed along the northern Iberian Peninsula, suggesting an extensive migration of genotypes along the country. This study is the first valuable action for genetic conservation of apple at the national scale, and constitutes a decisive step towards the definition of a Spanish core collection that will be useful for further studies in dissecting the genetic control of important horticultural traits through genome‐wide association analysis in apple.     

Changes of fatty acids and sterols in apple buds during bud break induced by a plant bioregulator, thidiazuron

The effects of N-phenyl-N'-l,2,3,-thidiazol-5-ylurea (thidiazuron; Dropp; SN 49537) on fatty acids of membrane lipids and sterol content in apple ( Malus domestica Borkh cv. Golden Delicious) buds associated with bud break and bud development were determined. The predominant fatty acids in the membrane lipids of apple buds were palmitic acid (C16:0), linoleic acid (C18:2) and linolenic acid (C18:3). β -Sitosterol and sitosteryl ester were the predominant sterols. An accumulation of unsaturated polar membrane fatty acids started after thidiazuron treatment. A decrease in the percentage of the sitosterol was accompanied by an increase in campesterol and stigmasterol at the beginning of rapid growth. An increase in the ratio of campesterol and stigmasterol to sitosterol and a decrease in the ratio of free sterols to membrane lipids upon breaking of dormancy also occurred in apple buds induced by thidiazuron.