Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance. Received: 12 December 1996 / Accepted: 7 January 1997     

Hormone Profiling by LC-QToF-MS/MS in Dormant <Emphasis Type="Italic">Macadamia integrifolia</Emphasis>: Correlations with Abnormal Vertical Growth

A method for analyzing multiple plant hormone groups in small samples with a complex matrix was developed to initiate a study of the physiology of abnormal vertical growth (AVG) in Macadamia integrifolia (cv. HAES344). Cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA), and auxins were detected in xylem sap and apical and lateral buds using high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS). The extraction method separated compounds with high sensitivity in positive (CKs) and negative (ABA, auxins, GAs) modes of QToF-MS/MS. CK profiles differed in xylem sap and apical and lateral buds irrespective of AVG symptoms. Trans-zeatin riboside (t-ZR) was dominant in sap of normal and AVG trees (∼4 and 6 pmol g⁻¹ FW, respectively). In apical buds isopentenyl adenine (iP) (∼30 pmol g⁻¹ FW) was the most abundant CK, and in lateral buds trans-zeatin (t-Z) (22–24 pmol g⁻¹ FW) and iP (24–30 pmol g⁻¹ FW) were the most abundant. t-Z levels of AVG trees were higher in apical buds (13.88 vs. 6.6 pmol g⁻¹ FW, p < 0.05) and lower in sap (0.16 vs. 0.51 pmol ml⁻¹, p < 0.005) compared to normal trees. ABA in lateral buds was 1.9 times higher (p < 0.001) in AVG. IAA was below quantification, whereas indole-3-butyric acid (IBA) was consistently present. GA₇ was the dominant GA in apical and lateral buds of all trees (100–150 pmol g⁻¹ FW). GA_{3, 4, & 9} were consistently present at low concentrations (<12 pmol g⁻¹ FW) in buds. GAs_{1, 3, & 9} were detected in xylem sap at low concentrations (<0.5 pmol g⁻¹ FW). Differences in sap amino acids (AA) were also assessed. In sap from AVG trees, asparagine and glutamine increased significantly (p < 0.05) in their contribution to total AA. Potential AVG hormone correlations are discussed.     

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [³H]zeatin riboside ([³H]ZR), a water‐soluble blue dye or [³H]H₂O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within 1 h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [³H]ZR and of blue dye in the same bud position was increased 3‐ to 10‐fold relative to intact plants, whereas content of [³H]H₂O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [³H]H₂O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [³H]ZR injected at physiological concentrations. Within 1 h, 80–95% of [³H]ZR was converted to other active CKs (mainly zeatin riboside‐5′phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O‐acetylZR, O‐acetylZRMP and a compound correlated with sites of high CK‐concentrations) and inactive catabolites (adenosine, adenine, 5′AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.     

Abscisic acid, indole-3-acetic acid and cytokinin changes in buds of Pseudotsuga menziesii during bud quiescence release

Bud quiescence release, considered as the ultimate dormancy breaking phase, was achieved in Pseudotsuga menziesii (Mirb.) Franco by a 9-week cold (5°C) treatment, under short daylength (9 h) followed by a transfer to mild temperature (22°C) under long daylength (16 h). Indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin-type (Z) and isopentenyladenine-type (iPA) cytokinin (CK) levels were measured by means of an ELISA technique performed on HPLC-fractionated extracts of terminal and axillary buds. During the cold period, all hormones except IP-type CK levels decreased, whereas the opposite observation was made after transfer to mild temperature and long daylength, when buds started to grow. Some other immunoreactive compounds were also detected and quantified. The ABA-glucosyl ester (ABA-GE) level pattern was similar to that of ABA, but no accumulation occurred at mild temperatures. A putative IAA conjugate, more polar than IAA, was also detected. Its level increased transiently like IAA in terminal buds and, to a lesser extent, in axillary buds during the 10th week of the experiment. In terminal buds, isopentenyladenosine ([9R]-iP) was released by alkaline hydrolysis of a polar immunoreactive compound detected with anti-[9R]iP antibodies. This compound accumulated during the cold period and quickly dropped at 22°C. Relationships between environmental conditions and endogenous hormones are discussed.     

Cytokinins and bud morphology in Pinus radiata

Cytokinins (CKs) play essential roles in the regulation of plant growth and development. In the previous paper (Zhang et al. 2001), we reported the detection and identification of a wide spectrum of CKs, including several novel forms, in the buds of Pinus radiata D. Don. In this paper we examine the relationship between the CKs and buds from juvenile and adult trees of P. radiata. During development the morphology of buds alters significantly, from buds bearing primary needles during their juvenile phase to buds sealed in scales at the adult phase. The morphology of adult buds is a very stable character, as fascicle meristems released from apical dominance, or cultured in vitro, produced only secondary needles. However, exogenous CK causes the adult buds to revert to juvenile bud development in vitro . Analyses of the endogenous CKs revealed that juvenile buds had a relatively higher level of isopentenyladenine and isopentenyladenosine, extremely low levels of phosphorylated CKs and a relatively low level of novel CK glycosides. The adult buds contained lower levels of free base and riboside CKs but very high levels of phosphorylated CKs and novel CK glycosides. Possible roles for CKs in the regulation of bud development are discussed.     

Periodicity of response to abscisic acid in lateral buds of willow (Salix viminalis L.)

Aseptically cultured lateral buds of Salix viminalis L. collected from field-grown trees exhibited a clear periodicity in their ability to respond to exogenous abscisic acid (ABA). Buds were kept unopened by ABA only when the plants were dormant or entering dormancy. Short days alone did not induce bud dormancy in potted plants but ABA treatment following exposure to an 8-h photoperiod prevented bud opening although ABA treatment of buds from long-day plants did not. Naturally dormant buds taken from shoots of field-grown trees and cultured in the presence of ABA opened following a chilling treatment. In no cases were the induction and breaking of dormancy and response to ABA correlated with endogenous ABA levels in the buds.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography - LD long day - MeABA methyl ABA - PAR photosynthetically active radiation - SD short day     

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.     

Branch development in Lupinus angustifolius L.#II. Relationship with endogenous ABA, IAA and cytokinins in axillary and main stem buds

The concentrations of indole-3-acetic acid (IAA), cytokinins (CK) and abscisic acid (ABA) were measured in buds of different regions (main stem and lateral branches) of Lupinus angustifolius L. (cv. Merrit) and at different stages in the development of branches. In lupin, branching patterns are the result of discrete regions of axillary branches (upper, middle and basal) which elongate at much different rates. Early in development only the main shoot elongates, followed usually by basal branch growth and then rapid upper branch growth. Branches in the middle of the main stem grow only weakly or fail to develop. Levels of IAA were generally high in the apical buds of slowly growing branches and low in buds from strongly growing branches, whereas CK levels showed the opposite relationship. CK:IAA ratio showed a closer relationship with the rate of growth of a particular branch better than the levels of either CK or IAA alone. During early stages of growth ABA concentration did not follow the rate of branch growth. However, later in development, where growth did not closely match the ratio of CK:IAA, ABA level showed a strong negative relationship with growth. A significant decrease in ABA was associated with continued strong growth of the main stem apex following a decline in CK:IAA ratio. Overall, the best relationship between the level of growth factors in apical buds and branching pattern in lupin was the ratio of CK:IAA, implying that high CK:IAA at a given bud would promote growth. ABA level appeared to play a secondary role, as a growth inhibitor.     

Apical dominance in Pssu-ipt-transformed tobacco.

In Pssu-ipt-transformed tobacco, apical dominance was released by defoliation of the upper nodes, while the apex remained intact. After defoliation, the concentration of cytokinins (CKs) increased whereas IAA remained constant, evoking an increase in the CK/IAA ratio in the buds. Moreover, defoliation resulted in a tremendous increase in the concentrations of aromatic amines (AAs): tyramine (TYR), phenethylamine (PEA) and an as yet unidentified compound. Although the total aliphatic monoamine and polyamine (PA) concentration remained constant, putrescine (PUT) and spermidine (SPD) concentrations in the axillary buds decreased, whereas the concentration of spermine (SPM) increased. Similar changes in PAs and AAs could be observed in the buds of untransformed SR1 plants after decapitation, whereas defoliation without removal of the apex had no effect. This is the first report on the possible involvement of PAs and AAs in apical dominance.     

Effect of chilling on the opening and abscisic acid content of dormant lateral buds of willow

A reduction in abscisic acid (ABA) content was not a pre-requisite for the breaking of dormancy of vegetative lateral buds of both field-grown trees and shoots of willow (Salix viminalis L.) maintained in controlled conditions. Similar variations in bud ABA levels were observed whether the shoots were stored in a warm (22 ± 1 °C) or cold (6 ± 0.5 °C) environment. Following transfer to a growth room the ABA content of chilled buds declined more rapidly than did that of non-chilled buds.     

Polyamine levels in buds and twigs of Tilia cordata from dormancy onset to bud break

The fluctuations of free and bound polyamines (PAs) were studied in vegetative buds and underlying twigs of linden (Tilia cordata L.) from August to May, to assess the connection between PA levels and seasonal cycles of growth and dormancy. Outer and inner bud scales and shoot tips (short shoot tips with leaf initials in contiguous short internodes) were analyzed separately, as were phloem with cortex and xylem with pith tissue from twigs. Seasonal variations in PA levels were present in buds and twigs during the research period. The most abundant PA in buds and twigs in free and bound forms was spermidine followed by putrescine. PA amounts were low in buds and twigs in autumn. In twig tissues, free PAs were predominant whereas in bud scales, bound PAs accumulated over free PAs in autumn, first in inner scales and later in outer scales as well. PA levels did not increase dramatically during the onset of dormancy in autumn but lower temperatures and probable cold hardening correlated positively with bound PAs in bud scales. In shoot tips with leaf initials, and contiguous short internodes, free putrescine and spermidine levels rose simultaneously with bud burst and new growth, while bound PAs diminished quite radically from temporary bud scales and from growing shoot tips.     

The effect of decapitation on the levels of IAA and ABA in the lateral buds of Betula pendula roth

The level of IAA and ABA in lateral buds of birch shoots 24 h and 5 days after the decapitation of the apical bud was determined. Twenty four hours after decapitation, when visible signs of outgrowth of lateral buds were not observed yet, an increase in the level of IAA and a decrease of ABA, as compared with the buds of non-decapitated shoots, was found. Five days later, when lateral buds were in the period of intensive outgrowth, a decrease in the levels of IAA and ABA was observed. It has been suggested that removing the source of auxin, by the decapitation of the apical bud makes possible the lateral buds to undertake the synthesis of their own auxin. It could lead to the decrease in the content of ABA. These all events could create suitable conditions for the outgrowth of lateral shoots.     

Dormancy and seasonal changes of plant growth regulators in hazel buds

Levels of indole-3-acetic acid (IAA), abscisic acid (ABA) and total phenolic compounds have been determined in hazel ( Corylus avellana L. cv. Negreta) buds. IAA and ABA were quantified by flame ionization detector gas-chromatography, and the phenolic content determined by a colorimetric technique. The highest level of free ABA occurs in autumn, approximately at the onset of winter dormancy, and it is lowest just before bud burst; suggesting that ABA plays an important role in the induction and maintenance of winter dormancy. The opposite result was obtained for IAA; the level of this regulator was low in autumn and showed a sharp increase just before budbreak. With respect to phenols, an apparent correlation can be observed between them and ABA in at least some of the fractions studied (acid and residual), so that they may enhance the inhibitory effect of ABA. The IAA/inhibitor balance showed a clear correlation with dormancy. In autumn (dormant buds), this balance is in favour of the inhibitors while the balance shifts towards IAA at the end of winter.     

剥鳞和激素处理对大樱桃花芽休眠解除及内源激素变化的影响

采用高效液相色谱法(HPLC)分析了剥鳞与激素处理对大樱桃花芽休眠解除及内源生长素(IAA)、赤霉素(GAD、玉米素(ZT)和脱落酸(ABA)变化的影响。结果表明,花芽中的ABA主要分布于鳞片内,鳞片中的GA3和ZT含量远低于去鳞芽,也低于完整芽。剥鳞能明显增加休眠花芽中内源GA2和ZT的含量,降低ABA的含量,对IAA含量的影响不大。剥鳞降低了ABA／GA3、ABA／ZT的比值,使花芽向促进生长、抑制休眠的方向转化。同时,休眠前、后期剥鳞均能明显提高萌芽率,中期剥鳞效果不明显。剥鳞后施用外源激素随休眠时期不同而有不同的破眠效果,早期剥鳞GA3的效果最好,6-BA次之,IAA最差;中期破眠效果不如早期,GA。和6-BA没有明显差别;后期以6-BA效果最好,其次是GA3和IAA;3次处理中ABA均明显抑制花芽萌发。     

Changes of Abscisic Acid and Gibberellin Contents during the Release of Dormancy in Winter Buds of Populus tomentosa Carr.

Changes of abscisie acid (ABA) and gibberellin (GA3) contents during the release of dormancy in winter bud of Populus tomentosa Carr. were determinedwith GC. After leaf fall in autumn, content of ABA in the bud was 888.0 μg/kg. fr.wt. Obvious decrease in ABA content was observed during the bud released from dormancy. The bud kept in room temperature opened about two months earlier thanthat under natural condition; and the rate of decrease of ABA content in these budwas also more rapid. The ABA contents of buds with similar outer appearance werecompared, either the bud from outside under natural condition or under room temperature, they were similar, although time of their occurrence was quite different, withalmost a difference of about two months. From this fact it has been assumed that thereis a close relationship between the release of dormancy and the decrease in ABA content. Another fact was noticed before Dec. 6, no GA3 could be detected in the dormantbud. From Jan. 9 and thereafter, GA3 content increased gradually and reached itsmaximum (20 μg/kg. fr.wt) by Mar. 19, the bud was inflated. GA3 decreased again as the bud was opened, it seems to be that the process of releasing dormancy in bud mightbe promoted by GA3.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

Effect of partial rootzone drying on the concentration of zeatin-type cytokinins in tomato (Solanum lycopersicum L.) xylem sap and leaves

Decreased cytokinin (CK) export from roots in drying soil might provide a root-to-shoot signal impacting on shoot physiology. Although several studies show that soil drying decreases the CK concentration of xylem sap collected from the roots, it is not known whether this alters xylem CK concentration ([CK(xyl)]) in the leaves and bulk leaf CK concentration. Tomato (Solanum lycopersicum L.) plants were grown with roots split between two soil columns. During experiments, water was applied to both columns (well-watered; WW) or one (partial rootzone drying; PRD) column. Irrigation of WW plants aimed to replace transpirational losses every day, while PRD plants received half this amount. Xylem sap was collected by pressurizing detached leaves using a Scholander pressure chamber, and zeatin-type CKs were immunoassayed using specific antibodies raised against zeatin riboside after separating their different forms (free zeatin, its riboside, and nucleotide) by thin-layer chromatography. PRD decreased the whole plant transpiration rate by 22% and leaf water potential by 0.08 MPa, and increased xylem abscisic acid (ABA) concentration 2.5-fold. Although PRD caused no detectable change in [CK(xyl)], it decreased the CK concentration of fully expanded leaves by 46%. That [CK(xyl)] was maintained and not increased while transpiration decreased suggests that loading of CK into the xylem was also decreased as the soil dried. That leaf CK concentration did not decline proportionally with CK delivery suggests that other mechanisms such as CK metabolism influence leaf CK status of PRD plants. The causes and consequences of decreased shoot CK status are discussed.     

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.     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.     

Changes of endogenous hormones in lateral buds of chrysanthemum during their outgrowth

The character of branching for two chrysanthemum (Chrysanthemum × morifolium) cvs. Jinghai and Jingyun was observed, and the changes of endogenous hormones in apical and lateral buds were investigated to determine the relationship between the pattern of hormone distribution, apical dominance, and lateral bud outgrowth. The growth rate of Jinghai lateral buds was higher than that of Jingyun. In vegetative growth stage, IAA level in apical buds of Jingyun was significantly higher than in Jinghai. After flower induction, IAA level in apical buds of two cultivars decreased remarkably, but the IAA level decreased in Jingyun faster than in Jinghai. These results showed that the higher was the IAA level in apical buds the stronger was inhibition of lateral bud outgrowth. An increase in IAA and iP/iPA and a decrease in ABA concentrations were closely associated with lateral bud growth alterations in chrysanthemum.