Endogenous cytokinin distribution patterns at
budburst in Granny Smith and Braeburn
apple shoots in relation to bud growth
Nigel C. Cook
a,*, Dirk U. Bellstedt
b, Gerard Jacobs
a aDepartment of Horticultural Science, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa
b
Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa
Accepted 11 April 2000
Abstract
The possible relationship of branching habit to cytokinin content of apple shoots (Malusdomestica Borkh.) was investigated. One-year old apple shoots are acrotonic (distal branching), more strongly so in Granny Smith than in Braeburn. In the ®rst trial, long, 1-year old Granny Smith and Braeburn apple shoots were sprayed on 29 August 1995 to break rest with dinitro-o-cresol (DNOC) oil (5%). The cytokinin contents of the xylem sap, the combined bark and buds, and the wood were determined in distal and proximal shoot halves over the next 6 weeks. Budburst (terminal and lateral buds) was ®rst visible (green tip) in both cultivars on 20 September 1995. A greater increase in cytokinin content of distal xylem sap, coupled with elevated cytokinin in the distal wood, re¯ect the overall acrotony of both cultivars. The strong acrotony of Granny Smith is re¯ected in the higher cytokinin concentration in distal portion 1 week before the proximal portion of the shoot. The differential distribution of cytokinin re¯ects the pattern of budburst and may be correlated with growth habit. In a subsequent trial, Granny Smith shoots chilled and forced in the absence of roots showed an increase in cytokinin content of the bark and buds, and the wood as growth resumed. This was roughly comparable in magnitude to the increase observed under ®eld conditions. The cytokinin increase in rootless shoots and differential distribution of cytokinin prior to sprouting, support the hypothesis that shoot-derived, rather than root-derived, cytokinins act to trigger spring budburst.#2001 Elsevier Science B.V. All rights reserved.
Keywords: Acrotonic branching; Prolepsis;MalusdomesticaBorkh.
*
Corresponding author. Tel:27-21-808-4763; fax:27-21-808-2121.
E-mail address: nc@land.sun.ac.za (N.C. Cook).
1. Introduction
Acrotony, mesotony and basitony describe distal branching, branching that is more or less evenly distributed over the shoot axis, and branching from the proximal buds, respectively (Rauh, 1939; CrabbeÂ, 1987). Proleptic branching, i.e. from previously dormant buds, in spring on 1-year old apple shoots, is acrotonic (Halle et al., 1978). The expression of acrotony in apple varies among varieties, resulting in a continuum from strongly acrotonic to almost mesotonic (Lespinasse and Delort, 1986). The current understanding is that acrotony is established under the correlative in¯uences that are active during the dormant period and appears to be mediated via a dominance of the apical bud that is established prior to budburst in spring (Cook et al., 1998).
Cytokinins play a central role in budburst in spring (Faust et al., 1997). The exogenous application of cytokinins promotes budburst during late dormancy and spring (Arias and CrabbeÂ, 1975; Steffens and Stutte, 1989). Endogenous cytokinins increase just prior to budburst in spring in the xylem sap of shoots and in the bleeding sap from roots (Skene, 1975; Tromp and Ovaa, 1990; Cutting et al., 1991). This cytokinin peak in spring is believed to originate from the roots, as shown by the increase in cytokinin in the bleeding sap (Skene, 1975), and from the shoot, as shown by the increase in cytokinin in the xylem sap of shoots that had been arti®cially chilled following excision (Hewett and Wareing, 1973). Trees exhibiting delayed foliation show a delay in the increase in the xylem sap cytokinin concentration compared to trees treated with rest breaking agents, which exhibit a more rapid increase in cytokinin (Cutting et al., 1991). While the importance of root-derived cytokinin cannot be excluded, shoot-derived cytokinins may be more directly involved with spring budburst in apple (Tromp and Ovaa, 1990; Cutting et al., 1991; Faust et al., 1997).
With acrotony the terminal buds burst ®rst, followed closely by the lateral buds. In our study, cytokinin content changes of the distal and proximal halves of 1-year old, dinitro-o-cresol (DNOC) oil treated, Granny Smith and Braeburn apple shoots were determined to assess whether cytokinin content changes within the shoots correlate with the pattern of budburst. Cytokinin content of the xylem sap, wood, and pooled bark and bud material was analysed shortly before and after budburst. In a subsequent trial, the cytokinin content of fall-harvested Granny Smith shoots was determined after chilling and subsequent forcing (not DNOC oil treated) in an attempt to con®rm that cytokinins involved in spring budburst are shoot-derived.
2. Materials and methods
2.1. Plant material Ð 1995 trial
develop in the 1994/1995 growing season such that an ample supply of shoots from both cultivars existed on the same trees. DNOC oil (5%) was sprayed on 29 August 1995 to break rest, and the ®rst shoots were harvested on 30 August 1995. Thereafter, shoots were harvested weekly until 4 October 1995 giving a total of six harvesting dates. One-year old, upright shoots (145058 mm in length) were harvested in the early morning. At each harvesting date, 10 shoots of each cultivar were harvested at random from ®ve adjacent trees. Five replicate shoots were used for the xylem sap analysis, and ®ve replicate shoots were used for both the bark and buds and the wood cytokinin analysis.
2.2. Cytokinin determination
Shoots were cut in half and the xylem sap was vacuum-extracted from both the distal and proximal halves as described by Belding and Young (1989). The xylem sap was extracted, rapidly frozen and stored atÿ808C until analysis. Thet-zeatin riboside (ZR) concentration of the xylem sap was determined by means of radioimmunoassay (Hofman et al., 1986) using a monoclonal ZR speci®c antibody (Eberle et al., 1986). The bark and buds were peeled from the wood separately from the distal and proximal shoot halves of each of the remaining ®ve shoots. Bark and bud (pooled) samples as well as wood samples were cut into 1 cm segments, freeze-dried and milled. All samples were stored atÿ808C until analysis.
ZR and ZROG (t-zeatin riboside-O-glucoside) were extracted from the samples by adding 10 ml of extraction solvent (80% methanol, 20% distilled water, BHT (16 mg/l) and ascorbic acid (10 mg/l)) to 0.5 g freeze-dried material and constantly stirring for 24 h at 48C. Undissolved solids were removed by centrifugation (12 000gn for 10 min at <108C) and the supernatant decanted.
Undissolved solids were re-extracted by the addition of 2 ml of the extraction solvent, centrifugation and removal of the supernatant, which was added to the previous extract. The combined extracts were dried on a Savant centrifugal evaporator (Savant Instruments, Farmingdale, NY). Dried extracts were stored at
ÿ808C until further analysis.
Substances interfering with the radioimmunoassay were removed from the extracts. Brie¯y, the dried samples were redissolved in 5 ml of ammonium acetate buffer (0.01 M, pH 8.2) by shaking for 1 h at 258C. The pH of these extracts was readjusted to 8.2 (using HCl or NaOH). PVP (polyvinylpyrrolidone 0.4 g) was added to remove phenols and the samples were incubated for 10 min at 258C. PVP was removed by ®ltration through a glass ®bre ®lter (Advantec GA55, Toyo Roshi Kaisha, Japan).
Seppak C18 cartridges were preconditioned by running 10 ml of methanol
cartridge followed by a further 10 ml of ammonium acetate buffer and then 10 ml of distilled water. The cytokinins were eluted with 10 ml of methanol. The eluate was dried on a Savant centrifugal evaporator and stored atÿ808C until further analysis. The ZR content of samples was determined by radioimmunoassay (Hofman et al., 1986) using a monoclonal ZR speci®c antibody (Eberle et al., 1986).
The ZROG present in the samples was hydrolysed using sweet-almond b -glucosidase (Sigma Chemicals) according to the method of Palni and Horgan (1983). The ZR in the sample was determined by radioimmunoassay to determine the total cytokinin content in the sample. The ZROG content was then calculated by deduction of the free ZR content from the total cytokinin content in the sample.
The possible presence of interfering contaminants in the RIA for zeatin-type cytokinins was determined using dilution analysis curves and checking for parallelism against a standard curve (Pengelly, 1985). It was found that the samples that had been subjected to the clean-up procedure showed no interference with the RIA.
At regular intervals during the extraction and clean-up process, samples were monitored for recovery of cytokinins by the addition of a small amount of radioactively labelled ZR dialcohol (also used as radiolabelled ZR in the RIA). Cytokinin recovery averaged 50 and 30% for the wood and bark/buds, respectively. All ZR and ZROG values were corrected accordingly.
2.3. Plant material Ð 1997 trial
In 1997, an additional trial was conducted to determine whether cytokinins increased in shoots independently of the root system. Fifteen Granny Smith shoots were harvested shortly after leaf drop on 3 July 1997, wrapped in moist paper, placed in a plastic bag and stored for 48 days in an upright position at 48C (1150 chilling hours). The bark and buds from ®ve shoots were peeled from the wood immediately after removal from the cold room, and after 6 and 8 days of forcing, respectively. The shoots were forced at 258C with continuous illumination (215mmol mÿ2
sÿ1
PAR, cool±white ¯orescent and incandescent light source). Bark and bud (pooled) samples as well as wood samples were cut into 1 cm segments, freeze-dried and milled. All samples were stored atÿ808C until analysis. The ZR content of the wood and bark/buds samples were determined as described above.
2.4. Statistics
Institute, 1996. SAS1release 6.12. Cary, NC.). LSD (least signi®cant difference, P0.05) and standard error values were calculated where applicable. The harmonic mean was used to calculate LSD values when necessary. Granny Smith wood had one, and xylem sap three missing plots. Braeburn wood had two missing plots.
3. Results
In the initial trial, budburst (green tip) was ®rst visible in both cultivars on 20 September 1995. Following DNOC application, cytokinin generally increased with budburst and dropped thereafter with lea®ng out (Figs. 1±3), as previously reported (Tromp and Ovaa, 1990; Cutting et al., 1991). A sharp increase in xylem sap ZR was associated with the onset of budburst for both cultivars (Fig. 1). Xylem sap ZR tended to increase most rapidly in the distal shoot half.
In both cultivars, the free wood ZR was greater in the distal shoot half than in the proximal shoot half (Fig. 2). In 3 weeks before budburst, this difference was greater in Granny Smith than in Braeburn. In the distal Granny Smith wood, ZR reached a peak a week prior to budburst. Granny Smith proximal wood ZR gradually increased until budburst, peaking a week later than the distal wood. Braeburn free wood ZR ¯uctuated in both distal and proximal shoot portions but tended to increase simultaneously to a peak that coincided with budburst.
Bark/buds free ZR generally increased as budburst approached (Fig. 3). At the beginning of the observation period, Granny Smith distal bark/buds contained more ZR than the proximal bark/buds. This distal ZR peak was a week earlier and larger than the proximal ZR peak. In Braeburn, no ZR peak was observed in the
distal bark/buds and differences between the distal and proximal bark/buds were less evident. In contrast to Granny Smith, the ZR increase in Braeburn was less pronounced, and was more evident in the proximal bark/buds at budburst (20 September).
ZROG content was determined in the DNOC oil treated shoots as a possible indication of stored cytokinin. The bark/buds ZROG showed a trend similar to that of the free cytokinin, but only at a lower level (Fig. 3). The wood ZROG remained roughly constant (Fig. 2). Similarly, bark/buds ZROG and wood ZROG made up a small fraction of the total measured cytokinin. Furthermore, increases observed in the free ZR were not associated with any major drop in ZROG. ZROG as a source of ZR at budburst, played a minor role. ZR has previously been shown to be the predominant cytokinin associated with budburst in apple (Tromp and Ovaa, 1990; Cutting et al., 1991). Only the free ZR content (free cytokinin) will be discussed.
Granny Smith shoots that were cut from the tree shortly after leaf drop and arti®cially chilled in a cold room showed an increase in wood and bark/buds ZR content as growth resumed under forcing conditions (Table 1). Furthermore, the observed ZR increases were roughly comparable in magnitude to the
Fig. 3. Cytokinin content changes of the pooled bark and buds from distal (diamonds) and proximal (squares) halves of Braeburn and Granny Smith 1-year old, DNOC oil apple shoots in 1995. Arrows indicate budburst. Mean separation by LSD at 5% level;n5; d.f.11.
Table 1
Cytokinin content of both the pooled bark and buds, and the wood of Granny Smith apple shoots (full length) harvested after leaf drop and arti®cial chilling (1150 chilling hours) in 1997a
P>F LSD Cytokinin content (ZR ng/g dry mass) 0 days forced
(dormant)
6 days forced (green tip)
8 days forced (leaves unfolding)
Wood 0.0004 13 50 64 83
Bark and buds 0.0001 82 153 520 579
a
The shoots were forced at 258C with continuous illumination (215mmol mÿ2
sÿ1
increases observed in DNOC treated shoots that burst buds in the ®eld (Figs. 2 and 3).
4. Discussion
Acrotony in M.9 apple shoots growing in Belgium, a region with an extended ecodormant period, appears to be mediated via dominance of the distal buds re-established during ecodormancy (Cook et al., 1998). The dominance of the distal buds (terminal and/or laterals) associated with acrotony is possibly an effect of polar auxin transport (Bangerth, 1989). Acrotony has been explained in terms of a positional advantage of distal buds on orthotropic shoots, mediated via an auxin effect (Faust et al., 1997). Similarly, gravimorphic responses of shoots inclined from the normal orthotropic orientation have been ascribed to the effects of a redistribution of endogenous auxins (Mullins and Rogers, 1971). It has also been shown that during dormancy a distal disbudded shoot piece maintains an inhibitory in¯uence on lateral buds under forcing conditions (Cook et al., 1998), an effect that can be overcome by girdling (Champagnat, 1983). Notching, known to disrupt polar auxin transport, is a commonly used practice to release lateral buds from the inhibitions associated with acrotony (Greene and Autio, 1994). In short, auxin undoubtedly plays a central role in correlative phenomena involved in the determination of acrotony and thus tree architecture.
The inhibition of lateral bud outgrowth can also be overcome by locally applied cytokinins alone (Steffens and Stutte, 1989), and in combination with gibberellins (Promalin1 [N-(phenylmethyl)-1H-purine 6-amine and gibberellins A4A7] is
used commercially in South Africa to promote lateral branching). Endogenous cytokinin increases in spring just prior to and during budburst in poplar shoots removed from the plant during dormancy (Hewett and Wareing, 1973); in dormant almond shoots stored at low temperatures for prolonged periods (Van Staden and Dimalla, 1981); in apple trees exhibiting delayed foliation (Cutting et al., 1991); and in non-chilled apple trees (Young, 1989). Provided that buds are receptive, i.e. not endodormant, and conditions are conducive to growth, buds can respond to this cytokinin peak, overcome correlative inhibitions and grow. This cytokinin peak appears, thus, to act as a trigger for resumed growth (Faust et al., 1997).
are stored in the shoot prior to becoming dormant, as has previously been suggested by Van Staden and Dimalla (1981).
The higher rate of cytokinin increase in the distal xylem sap (Fig. 1), and the higher distal wood ZR content (Fig. 2), re¯ect the overall acrotonic growth habit of both cultivars. The distribution of cytokinins in pea and bean stems has been reported to be under the control of polar auxin transport (Li et al., 1995). Auxins appear to prevent the movement of cytokinins into the lateral buds under the in¯uence of apical dominance. It is possible that auxins exported by the shoot apex before and/or after endodormancy may be associated with the differential distribution of cytokinins after chilling.
Branching in Granny Smith is strongly acrotonic. Shoot growth is restricted to the dominant distal shoots with the development of few spurs and a latency of proximal lateral buds, i.e. ``blind wood'' (Oosthuyse et al., 1992). This was re¯ected by a large, initial difference between the distal and proximal wood ZR content (distal contained more ZR, Fig. 2), and an enhanced ZR peak in the proximal wood and bark/buds (Figs. 2 and 3). In the more ``spur type'' Braeburn, distal shoots and proximal spurs are more numerous than in Granny Smith. In Braeburn, which exhibits less dominance, a simultaneous increase of wood ZR of both shoot halves (Fig. 2), and generally less bark/buds ZR with a greater increase at budburst in the proximal half was observed (Fig. 3). The differential distribution of cytokinin in the shoot may be involved in the pattern of budburst and thus growth habit.
Delayed heading is commonly used to reduce the effects of acrotony. Heading 1-year old Granny Smith shoots from budburst to shortly after full-bloom increases the number of shoots formed with a subsequent reduction in the total length of new growth (Oosthuyse et al., 1992). The resultant shoots originate from buds that would otherwise remain latent. Heading allows buds to burst in a portion of the shoot with lower available cytokinin levels, the result of the gradient observed in these data, at a time generally associated with less cytokinin avail-ability, i.e. after the cytokinin peak. Possibly, buds are released under endogenous conditions less favourable for the development of dominance phenomena.
In conclusion, while auxins play a central role in acrotony and thus tree architecture, evidence presented here suggests that shoot-derived cytokinins may also be involved in the acrotonic branching habit of apple. The cytokinin increase in rootless shoots and differential distribution of cytokinin prior to sprouting, support the hypothesis that shoot-derived, rather than root-derived, cytokinins act to trigger spring budburst.
Acknowledgements
fuÈr P¯anzenphysiologie, Ruhr UniversitaÈt, Bochum, Germany for supplying the monoclonal ZR speci®c antibody, and both the FRD and the Deciduous Fruit Industry for ®nancial support.
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