Breaking bud dormancy in apple with a plant bioregulator,thidiazuron

The effects of N-phenyl-N′-1,2,3,-thidiazol-5-ylurea (thidiazuron; Dropp; SN49537; TDZ) on metabolic changes in apple buds during dormancy break were determined. The data showed that thidiazuron has the capacity to release lateral buds from dormancy. Decreasing degree of bud break and bud growth with thidiazuron treatment occurred in a basipetal direction, suggesting a gradient of increasingly deep rest from shoot apex to base. The breaking of dormancy by thidiazuron is correlated with increase in DNA, RNA, protein, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC), S-adenosylmethionine (SAM) as well as with greater polyamine formation. Polyamine and ethylene biosynthesis did not seem to be competing for SAM, their common substrate, during bud break and bud development. The release of dormancy in apple bud by thidiazuron was inhibited by cordycepine, 5-fluorouracil, 6-methylpurine and cycloheximide. Inhibition of bud break and bud growth also resulted from treatment with α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO). DFMO was more inhibitory than DFMA.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Changes in the activities of catalase, peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

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.     

Metabolic changes associated with bud break induced by thidiazuron

Bud break in apple (Golden Delicious,Malus domestica Borkh) was induced by thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea). In control and thidiazuron-treated shoots, higher amounts of soluble carbohydrates (sorbitol, fructose, glucose, sucrose) and galacturonic acid were found in the phloem, but higher amounts of starch and cell wall polysaccharides, including cellulose and xylose, were found in the xylem. A decrease in soluble carbohydrates and starch in both phloem and xylem was associated with induction of bud break by thidiazuron. However, little change in cell wall polysaccharides was found. Total carbohydrates were higher in the upper than in the lower portion of shoots. The breaking of dormancy by thidiazuron was also associated with an increase in organic acid content and respiration in buds. KCN inhibited bud respiration during all stages of development. Organic acid content was inversely related to carbohydrate content in developing buds. Axes contained more carbohydrates and organic acids than did scales.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.     

喷施烯效唑对苹果顶芽激素水平和花芽分化的影响

用烯效唑(uniconazol,S3307)1 g/L喷洒“红富士“苹果树降低了顶芽IAA、GA1,3,4,7含量,提高了ZR、ABA含量,从而提高了ZR/IAA、ZR/GA1,3,4,7、ABA/IAA和ABA/GA1,3,4,7比值.烯效唑处理增加了花芽形成百分率,加速了花芽分化的进程,缩短了花芽形成的延续时期,但对花芽生理孕育临界时期长短没有影响.烯效唑处理对花芽的节位数没有影响,但使叶芽节位数增加了1节.     

Changes in endogenous hormones in apple during bud burst induced by defoliation

Under the tropical conditions of East Java, terminal buds of apple burst at any time of the year in response to removal of the subtending leaves. Following two such defoliations, two weeks apart on separate trees, there was a decrease in abscisic acid (ABA), a three-fold increase in gibberellin-like substances (GAs) and only a slight increase in cytokinin-like substances (CKs) in the apex tissue of closed buds. These changes preceded bud opening and the associated increases in fresh and dry weight, and may be causally related to bud burst. In open buds (i.e. young expanding leaves) the concentration of CKs was greater, and the concentrations of ABA and GAs less, than the concentrations in closed buds. As the leaves expanded, ABA increased and GAs and CKs decreased in concentration. The decrease in concentration of GAs and CKs, however, was due to the rise in dry weight of the expanding tissue; the amounts of all three hormones (per apex) increased. During bud burst there was a concurrent decrease in the CKs of subtending stems, suggesting a transfer into the expanding bud tissues. Removal of the old leaves by defoliation may remove the source of ABA and allow the amount of GAs in the apex to rise, bud burst following. Stem CKs may be utilized in the expansion of the new leaves in the bursting buds.     

Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

Foliar-applied Dormex™ or thiourea-enhanced proline and biogenic amine contents and hastened breaking bud dormancy in “Ain Shemer” apple trees

The role of hydrogen cyanamide (Dormex?) or thiourea in the acceleration of dormancy breaking in buds of “Ain Shemer” apple (Malus sylvestris, Mill.) trees, and their effects on metabolic changes in the contents of proline and biogenic amines (BAs; tyramine, tryptamine, histamine, methylbutylamine and serotonin) in buds during bud break were assessed. The efficiency of bud break by these compounds was noticed to varying degrees. Breaking bud dormancy was correlated with the early date of bud break, the short duration of flowering, the high percentages of bud break and fruit set, and the high contents of proline and BAs. This finding was positively reflected in the tree’s yield. Dormex? was found to be more effective than thiourea; therefore, we recommend using Dormex? for early bud break, short period of flowering and high percentages of bud break and fruit set by regulating the contents of proline and BAs in buds.     

Abscisic Acid Analysis in Vitis vinifera in the Period of Endogenous Bud Dormancy by High Pressure Liquid Chromatography

In buds and nodes of Vitis vinifera L. cv. Riesling, the content of abscisic acid (ABA) was measured by high pressure liquid chromatography and related to bud dormancy. In the period of endogenous bud dormancy (rate of bud break is low or zero under favourable climatic conditions) the ABA content increased twelvefold. This indicates a causal relationship between endogenous bud dormancy and ABA.     

Shoot bending promotes flower bud formation by miRNA‐mediated regulation in apple (Malus domestica Borkh.)

Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down‐regulated and up‐regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering‐related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees.     

Exogenous dormancy-breaking substances positively change endogenous phytohormones and amino acids during dormancy release in ‘Anna’ apple trees

A 2-season trial was conducted to verify the effects of foliar applications of some dormancy-breaking substances (DBS) on dormancy release in buds of ‘Anna’ apple (Malus sylvestris, Mill) trees, as well as on metabolic changes in the contents of phytohormones, proline and arginine in buds during their release from dormancy. The efficiency of early bud break induced by Dormex?, potassium nitrate, mineral oil, calcium nitrate and thiourea was noticed in varying degrees. Although Dormex? was distinguished, all DBS hastened bud break, shortened flowering duration, improved bud break% and fruit-set%, increased the contents of gibberellic acid, indole-3-acetic acid, proline and arginine, but reduced abscisic acid content in buds as compared to the control. These results were positively reflected in the final tree yield. Accordingly, it is concluded that the use of Dormex?, at a rate of 4 %, could be recommended for reaching bud break as early as possible and improving ‘Anna’ apple tree yield under the short winters in Egypt and similar regions by regulating the contents of proline, arginine and phytohormones in buds.     

The Effect of Chemical Defoliation on the Development of Apple Spurs

An analysis was made of bud development on apple spurs sprayedwith copper sulphate in late spring. The treatments caused thespur buds to break into a second flush of growth and eventuallyto form a new resting bud. The failure of these later buds tobecome fruit buds was correlated with the time in the seasonwhen they began to form, and was determined by the number ofleaves and bud scales preceding the buds. The number of leavesin the second flush was related to the extent to which shoottips and spurs were killed by the sprays; the number of leavesin the first flush and the number of bud-scales between theseflushes of growth was also altered by the chemical. The factorsaffecting the number of nodes in each of these zones of growthare discussed, and it is suggested the sprays reduced the levelof endogenous auxins in the leaves and endogenous gibberellinsin the shoot tips. Differences associated with the vigour ofthe trees and with seasonal factors were also observed. Thedifficulties in estimating these factors limit the commercialvalue of this technique.     

Changes in abscisic acid and indole-3-acetic acid in axillary buds of Elytrigia repens released from apical dominance

Growth of axillary buds on the rhizomes of Elytrigia repens (L) Nevski is strongly dominated by the rhizome apex, by mechanisms which may involve endogenous hormones. We determined the distribution of indole-3-acetic acid (IAA) and abscisic acid (ABA) in rhizomes and measured (by gas-chromatography-mass spectrometry) their content in axillary buds after rhizomes were decapitated. The same measurements were also made in buds induced to sprout by removing their subtending scale leaves. The ABA content tended to be higher in the apical bud and in the axillary buds than in the adjacent internodes, and tended to decline basipetally in the internodes and scale leaves. IAA was similary distributed, except that there was less difference between the buds and other rhizome parts. After rhizomes were decapitated, the ABA content of the first axillary bud declined to 20% of that of control values within 24 h, while the IAA content showed no marked tendency to change. The ABA content also declined within 12 h in the first axillary bud after rhizomes were denuded, while the content of IAA tended to increase after 6 h. These changes occurred before the length of the first axillary bud increased 24–48 h after rhizomes were decapitated or denuded. We conclude that the release of axillary buds from apical dominance in E. repens does not require IAA content to be reduced, but is associated with reduced ABA content.     

Effects of Exogenously Applied Gibberellins and Thidiazuron on Phytohormone Profiles of Long-Shoot Buds and Cone Gender Determination in Lodgepole Pine

In long-shoot buds of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone-bud initiation and gender differentiation occur in a site-specific manner: female cone buds are normally restricted to the distal portion, whereas male cone buds are located in the proximal portion. Application of a paste containing two plant growth regulators gibberellins A₄ + A₇ (GA_4/7) combined with thidiazuron (TDZ) to long-shoot buds prior to cone-bud gender determination altered endogenous phytohormone profiles and induced female cone-bud formation in the proximal portion of the long-shoot bud, where male cone buds normally occur. Induced cone clusters observed in the following spring were either entirely female or a mixture of both female and male cones. Endogenous phytohormones in the long-shoot bud tissues were quantified by the stable isotope dilution method using high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction monitoring mode. Applied GA_4/7 + TDZ led to increased concentrations of endogenous zeatin-type cytokinins, that is, trans-zeatin riboside and dihydrozeatin riboside, whereas concentrations of abscisic acid (ABA) and its catabolite, ABA glucose ester, were decreased, all relative to control, in untreated long-shoot bud tissue. Concentrations of extractable GA₄ and GA₇ declined in long-shoot bud tissues over 4 weeks following treatment with exogenous GA_4/7. This study demonstrates that high levels of endogenous zeatin-type cytokinins, together with reduced levels of ABA, both induced by applied GA_4/7 + TDZ, are positively associated with an increased female cone-bud formation in long-shoot buds.     

Spatial and temporal changes in multiple hormone groups during lateral bud release shortly following apex decapitation of chickpea (Cicer arietinum) seedlings

Although the co-ordination of promotive root-sourced cytokinin (CK) and inhibitory shoot apex-sourced auxin (IAA) is central to all current models on lateral bud dormancy release, control by those hormones alone has appeared inadequate in many studies. Thus it was hypothesized that the IAA : CK model is the central control but that it must be considered within the relevant timeframe leading to lateral bud release and against a backdrop of interactions with other hormone groups. Therefore, IAA and a wide survey of cytokinins (CKs), were examined along with abscisic acid (ABA) and polyamines (PAs) in released buds, tissue surrounding buds and xylem sap at 1 and 4 h after apex removal, when lateral buds of chickpea are known to break dormancy. Three potential lateral bud growth inhibitors, IAA, ABA and cis -zeatin 9-riboside (ZR), declined sharply in the released buds and xylem following decapitation. This is in contrast to potential dormancy breaking CKs like trans -ZR and trans -zeantin 9-riboside 5'phosphate (ZRMP), which represented the strongest correlative changes by increasing 3.5-fold in xylem sap and 22-fold in buds. PAs had not changed significantly in buds or other tissues after 4 h, so they were not directly involved in the breaking of bud dormancy. Results from the xylem and surrounding tissues indicated that bud CK increases resulted from a combination synthesis in the bud and selective loading of CK nucleotides into the xylem from the root.     

Reduction of abscisic acid content and induction of sprouting in potato,Solanum tuberosum L., by thidiazuron

The effect of [(N-phenyl-N-1,2,3-thidiazol-5-ylurea)] (thidazuron) on sprouting of potato (Solanum tuberosum L.) tubers and the role of ABA in bud break and subsequent bud growth were studied. Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) from the peel of potato tubers. The ELISA results were also validated by gas chromatography-electron capture detector and confirmed by gas chromatography-mass spectrometry and by a lettuce hypocotyl bioassay. The degree of rest in the tubers was associated with ABA content in the peel. Basal portion (where tuber was attached to mother plant) contained the highest amount of ABA. Thidiazuron reduced ABA content and induced potato tuber sprouting. Exogenously applied ABA stimulated growth of buds that had emerged from dormancy.On leave from the Department of Horticulture, South China Agricultural University, Guangzhou, People's Republic of China.     

Distribution of free and bound forms of cis-trans and trans-trans abscisic acid in broad-bean plants in relation to apical dominance

Levels of endogenous abscisic acid (ABA; free and bound forms) have been determined by gas chromatography in stems and buds of broad-bean plants ( Vicia faba L. cv. Aguadulce) in relation to apical dominance. A downward gradient of free cis-trans ABA occurred along the stem, from the apical bud to the roots. Except for the actively growing apical bud the levels of free cis-trans ABA were higher in the buds than in the corresponding nodes. An inverse correlation can be set up between levels of free cis-trans ABA and growth of buds, except for the cotyledonary ones. High levels of bound ABA ( cis-trans form) are correlated with the growth of the apical bud and that of the axillary bud ax1. The hormonal regulation of the growth of the cotyledonary buds, which contained high levels of trans-trans ABA in bound forms, is apparently different from that of the other buds.     

Changes in membrane polar lipids associated with bud break in apple induced by nitroguanidines

The predominant lipids in membranes obtained from apple buds were galacto- and phospholipids. The major galactolipid components in apple bud were monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the major phospholipids in the apple buds. -Linolenic acid (C 18:3) was the major fatty acid in MGDG, DGDG, and PC. Phosphatidylglycerol (PG) is the only lipid to contain significant amounts of palmitic acid (C 16:0) in the dormant buds. An increase in the galacto- and phospholipids and the ratio of the unsaturated fatty acids to the corresponding saturated fatty acids of the buds occurred as a result of induction by 1-(3,5-dichlorophenyl)-3-nitroguanidine or 1-(-ethylbenzyl)-3-nitroguanidine during bud break. The identities of fatty acids in apple buds were confirmed by gas chromatography-mass spectrometry.