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New Zealand Journal of Crop and Horticultural Science

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Hydrogen cyanamide improves endodormancyrelease and blooming associated with endogenoushormones in ‘Summit’ sweet cherry trees

Lei Wang, Caixi Zhang, Jiancheng Huang, Lina Zhu, Xiuming Yu, Jiefa Li, YusuiLou, Wenping Xu, Shiping Wang & Chao Ma

To cite this article: Lei Wang, Caixi Zhang, Jiancheng Huang, Lina Zhu, Xiuming Yu, JiefaLi, Yusui Lou, Wenping Xu, Shiping Wang & Chao Ma (2017) Hydrogen cyanamide improvesendodormancy release and blooming associated with endogenous hormones in ‘Summit’sweet cherry trees, New Zealand Journal of Crop and Horticultural Science, 45:1, 14-28, DOI:10.1080/01140671.2016.1229344

To link to this article: http://dx.doi.org/10.1080/01140671.2016.1229344

Published online: 27 Sep 2016.

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RESEARCH ARTICLE

Hydrogen cyanamide improves endodormancy release andblooming associated with endogenous hormones in ‘Summit’sweet cherry treesLei Wanga, Caixi Zhanga, Jiancheng Huangb, Lina Zhua,c, Xiuming Yua, Jiefa Lia,Yusui Loua, Wenping Xua, Shiping Wanga and Chao Maa

aSchool of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; bDepartment ofBiology, Qingdao University, Qingdao 266071, Shandong, China; cShanghai Academy of Agricultural Sciences,Shanghai 200240, China

ABSTRACTA trial was conducted to verify the effect of hydrogen cyanamide(HC) application on endodormancy release and blooming of‘Summit’ sweet cherry trees, as well as on endogenous gibberellicacids (GAs) and abscisic acid (ABA) levels which were identifiedand quantified by gas chromatography/mass spectrometry inbranches with spurs. Results showed that HC efficiently hastenedendodormancy release and budbreak, shortened bloomingduration and improved fruit set. Furthermore, bioactive GAs playdifferent roles in the process of endodormancy release andblooming. GA3 was recognised as the highest isomer of bioactiveGA and was closely related with endodormancy release improvedby HC treatment as well as GA4. GA7 displayed a rapid increasewhen buds went into the burst stage and associated withpromoted budburst and blooming by HC treatment. GA1 showedirregular changes during this process. A higher GAs:ABA ratio wasobserved in HC-treated ‘Summit’ branches from endodormancyrelease until full bloom. However, a reciprocal pattern occurredthereafter due to senescence of the flowers.

ARTICLE HISTORYReceived 20 January 2016Accepted 6 August 2016

KEYWORDSAbscisic acid; blooming;endodormancy release;gibberellins; hydrogencyanamide; sweet cherry

Introduction

Fruit trees from temperate or colder regions have specific genetic winter chillingrequirements to break endodormancy and for economic production (Saure 1985;Erez 1995). Sufficient winter chill along with favourable spring temperature conditionsguarantee full and uniform development of foliage and flowering in deciduous fruittrees such as peach (Chaar & Astorga 2012), apricot (Ruiz et al. 2007), apple(Hauagge & Cummins 1991) and sweet cherry (Alburquerque et al. 2008). Otherwise,the vegetative and reproductive behaviour of the cultivars will be affected negatively(Samish 1954).

Sweet cherries (Prunus avium L.) grown in temperate climates are commercially cul-tivated in more than 40 countries worldwide, mainly between 35°N and 55°S latitudes(Chadha 2003). In China, sweet cherry cultivation has expanded quickly beyond the

© 2016 The Royal Society of New Zealand

CONTACT Caixi Zhang acaizh@sjtu.edu.cn; Chao Ma chaoma2015@sjtu.edu.cn

NEW ZEALAND JOURNAL OF CROP AND HORTICULTURAL SCIENCE, 2017VOL. 45, NO. 1, 14–28http://dx.doi.org/10.1080/01140671.2016.1229344

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traditional temperate regions surrounding Bohai Bay to higher latitude areas in thesouthwest due to increasing consumer demand and the high profit for growers (Hanet al. 2008; Zhang et al. 2013; Wang et al. 2014). The ability to cultivate sweet cherriesaround Shanghai, a southern warm winter region with a low number of chill hours, hasbeen evaluated in recent years (Li et al. 2010; Zhang et al. 2013). One of the majorlimitations of cultivating sweet cherries successfully in this region is satisfying thechilling requirements, which are generally from 600 to 1400 h by a 7.2 °C model(Baldochi & Wong 2008). Symptoms of delayed foliation, deformed flowers or abortionof flowers and subsequent poor fruit set i.e. a reduction in potential yield, have beenwidely observed (Wang et al. 2004; Li et al. 2010).

Warm winters impair the breaking of endodormancy, possibly due to inadequate chil-ling accumulation (Yamamoto et al. 2010). Various methods have been used to sup-plement chilling. Hydrogen cyanamide (HC), an effective dormancy-breaking agent,has been used widely in grape (Vitis vinifera L.) (Dokoozlian et al. 1995), kiwifruit (Acti-nidia deliciosa L.) (Mcpherson et al. 2001), apple (Malus domestica L.) (Krisanapook et al.1995), peach (Prunus persica [L.] Batsch.) (Siller-Cepeda et al. 1992), pear (Pyrus spp.)(Singh & Mann 2002) and sweet cherry (Godini et al. 2005). Previous studies revealedthat applying HC to fruit trees 2–5 weeks before field budburst resulted in early anduniform budbreak, increased flower and fruit numbers, faster flowering and fruiting,and improved fruit quality (Shuck & Petri 1995; Pramanik et al. 2009). Recent studiesin grapevines have shown that the application of HC increased H2O2 levels and inhibitedcatalase activity, along with up-regulation of a series of transcriptions, including grapedormancy-breaking related protein kinase (GDBRPK), a sucrose non-fermentingprotein kinase (SNF-like protein), pyruvate decarboxylase (PDC), alcohol dehydrogenase(ADH), thioredoxin h (Trxh), glutathione S-transferase (GST), ascorbate peroxidase(APX), glutathione reductase (GR) and sucrose synthase (SuSy) (Or et al. 2000; Neillet al. 2002; Pérez & Lira 2005; Keilin et al. 2007; Halaly et al. 2008; Pérez et al. 2008).However, the biological mechanism that underlies its dormancy-breaking effect has notbeen fully described. In pea seedlings andMalus sylvestrisMill. ‘Anna’ trees, the responsesto HC are usually associated with the action of plant hormones (Guevara et al. 2008; SeifEl-Yazal et al. 2014).

Plant hormones play pivotal roles in plant growth, development, and response to bioticand abiotic cues (Davies 1995, 2004). Endogenous hormone changes in the process of buddormancy induction and release have been studied (Fernie & Willmitzer 2001; Yamazakiet al. 2002; Suttle 2004a, 2004b; Qin et al. 2009). Previous studies showed that gibberellicacids (GAs) often release endodormancy, whereas abscisic acid (ABA) is associated withendodormancy induction and maintenance. However, there is little information about therelationships between HC and endogenous phytohormones during endodormancy releaseand blooming (flowering) in sweet cherries. Therefore, the objectives of this study are toexplain how changes in GAs and ABA levels in the branches of sweet cherry are associatedwith the regulation of endodormancy and blooming following HC application in‘Summit’ sweet cherry trees, which are classified as a moderate-high chill variety withan 800 chilling hour requirement. These trees exhibit developmental defects in flowers,fruit sets and yields in the Shanghai region (Li 2011; Zhang 2011). In addition, theeffect of HC on the release of bud dormancy under mild winters in Shanghai wasinvestigated.

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Materials and methods

Plant materials and treatments

The research was carried out on 6-year-old trees of ‘Summit’ grafted on Daqingye (Prunuspseudocerasus L.) rootstock by using P. avium L. ‘Lapins’ and P. avium L. ‘Tieton’ as pol-linisers in a sweet cherry commercial orchard located in Shanghai (31°14′N, 121°29′E) in2012. Trees were trained to a spindle system, spaced 5 × 6 m and regularly irrigated andfertilised.

On 8 February, nine sweet cherry trees were selected completely randomly and treatedwith a spray application of HC, which was approximately 30 days before the natural onsetof budbreak in the Shanghai region. Each group of three trees was designated as one repli-cate. Each treatment was repeated in triplicate. Spray applications of HC were conductedonce as follows: each tree was divided into two spray areas with polyethylene barriers. Oneside was sprayed to the point of run-off (3 L tree−1) with 2% (v/v) hydrogen cyanamide(Ningxia Darong Chemicals & Metallurgy Co) using a 16 L knapsack sprayer. Anotherside received the equivalent volume of distilled water as a control. The applicationswere performed early in the morning on non-windy days to avoid significant drift, andno precipitation was recorded in the following 48 h.

To identify the effect of HC on dormancy release and blooming, the preliminary studieswere conducted in both 2010 and 2011. HC was applied once or twice at different rates. Asingle application of HC at a concentration of 2% (v/v) was found to be the most effectivetreatment for later bud growth in ‘Summit’ sweet cherry trees (data not shown).

Phenophase observation, sampling and dormancy status testing

Five fruiting branches were randomly selected from the outer canopy approximately1.5–1.8 m above the ground of nine trees in each treatment (HC and control). The budphenophase of ‘Summit’ trees was recorded every 3 days until post bloom (senescenceof the flowers). The floral bud phenophase of the sweet cherries was assessed and cate-gorised into eight stages: first swelling, side green, green tip, tight cluster, first white,first bloom, full bloom and post bloom, according to the stages described by WashingtonState University (http://cahnrs-cms.wsu.edu/StoneFruit/research/Pages/GenesSpringFloralBuds.aspx) with minor modifications. The period of phenological growth of the wholebranches was defined as the time when 50% buds or flowers on the branches reachedthe classified stages, except for the ‘first bloom’ period, during which approximately 5%of flowers opened. In addition, the dates at which approximately 75% and 100% offlowers opened were recorded in each treatment. Endodormancy release means thebuds of temperate trees can burst in a suitable environment after a certain period of chil-ling. Budbreak was defined as the time when the green tips break through the brown scaleand are just visible. Blooming duration indicated the days from ‘first bloom’ to ‘postbloom’. The final fruit set was calculated 4 weeks after the full bloom stage as thenumber of persistent fruits per 100 flowers under open pollination by bees.

To assess biochemical changes, two 2-year-old fruiting branches uniformly distributedover the periphery canopy at 1.5–1.8 m in each tree (HC and control) were chosen andtagged. Approximately 5 cm of each cleaned branch was sampled at 0, 14, 21, 28, 32,36, 40, 44, 48, 52, 56 and 60 days after HC treatment. The buds, flowers and leaves of

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each sampled branch were removed to avoid the changes of bud status after budburst andthe confounding effects of their density variation on the endogenous hormone concen-tration. These branches were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.

To test the dormancy status of ‘Summit’ trees on each sampling date, nine 2-year-oldbranches with spurs in similar growth vigour were collected and put into vials containingdistilled water after being cut, and were cultivated under controlled growth chamber con-ditions with a light intensity of 40 µmol m−2 s−1. The temperature ranged from 25 °Cduring the light period (16 h) to 18 °C in the dark, and the relative humidity was 80%–90%. The water was renewed every 2 days. After 4 weeks of cultivation, the budburstrate of floral buds was investigated, and a budburst rate greater than 50% indicated thatendodormancy had been released.

In addition, to evaluate the behaviour of sweet cherry under Shanghai climatic con-ditions, macroclimate data including monthly average temperatures and total rainfallduring shedding, dormancy and blooming were obtained from the local weather stations.

Branch water content

Branch water content was determined by drying a part of each sample in a convectionoven at 80 °C for 96 h until constant weight was attained.

Gibberellic acids and abscisic acid extraction and purification

The branch samples were ground by a cryo-grinder (Freezer Mill 6870, SPEX SamplePrep)in liquid nitrogen. Hormone extraction and purification were based on the method devel-oped by Zhang (2007) with minor modifications. The powder (approximately 2 g freshweight [FW]) was extracted at 4 °C in the dark overnight in 10 mL 80% cold aqueousmethanol containing 0.2 g L−1 butylated hydroxyltoluene (BHT), which served as an anti-oxidant. At this point, 100 ng deuterated GAs ([17-2H2]GA1, [17-

2H2]GA3, [17-2H2]GA4

and [17-2H2]GA7) and 100 ng [2H6]ABA were added to the extract, serving as internalstandards for estimating the recovery after purification. The standards were purchasedfrom OlChemIm. The solution was then centrifuged and the residue re-extracted twicewith 5 mL of cold extraction solvent to ensure the complete extraction of the chemicaland bioactive compounds. After centrifuging, the combined extracts were concentratedby a rotary evaporator under reduced pressure at 42 °C to remove organic solvent. Theaqueous solution was adjusted to pH 6–7 and partitioned against hexane three times.The pH of the combined aqueous phases was acidified to 2.5 with 1 MHCl and partitionedagainst ethyl acetate. The combined ethyl acetate fraction was partitioned against 0.5 Mphosphate buffer (pH 8.3). Then insoluble PVPP was added to the combined bufferedphases and filtered. The filtered aqueous solution was adjusted to pH 2.5 by adding6 M HCl and partitioned against ethyl acetate again. After drying with Na2SO4 and filter-ing, the combined ethyl acetate solution was evaporated in vacuo. The residue was dis-solved in a small amount of 80% aqueous methanol and passed through a C18 Sep-Pakcartridge (Waters). The cartridge was pre-wetted with methanol, water and 80% water,and followed by drying under a stream of nitrogen. The residue was redissolved in asmall amount of 45% methanol-water containing 0.1% acetic acid and was further

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loaded on to a Bondesil DEA column (diethylaminopropyl, 40 µm, Agilent Technologies).After loading the sample, the column was eluted with distilled water, followed by metha-nol. The eluent was reduced to dryness under a nitrogen stream and stored at −20 °Cbefore further purification by high-performance liquid chromatography (HPLC).

High-performance liquid chromatography analysis

The crude GAs and ABA were redissolved in methanol and subjected to a reversed-phaseSenshu-Pak ODS-4253-D HPLC column (10 mm i.d. × 250 mm, Senshu Scientific). Theywere eluted with 0.1% acetic acid in 30% aqueous methanol (solvent A) and 100% aqueousmethanol (solvent B) at 40 °C as follows: 0–3 min, elution with solvent A; 3–30 min, lineargradient of 0% to 100% solvent B; 30–50 min, elution with 100% solvent B. The flow rateof the solvent was 3 mLmin−1 and eluate fractions were collected every minute. The reten-tion times of ABA, GA1, GA3, GA4 and GA7 were 22, 17, 15–16, 29 and 28 min, respect-ively, and these times were identified by running authentic standards under the sameconditions.

Gas chromatography/mass spectrometry-selected ion monitoring

The bioactive GA-like fractions were dissolved in 50 µL methanol, then methylated with2 µL of (trimethysilyl) diazomethane (2 M solution in hexane, Alfa Aesar China [Tianjin]Co) at room temperature for 30 min followed by trimethylsilylation with 50 µL of SylonBFT (BSTFA + TMCS, 99:1, Supelco) at 70 °C for 1 h in a sealed glass vial, and theywere then used for gas chromatography/mass spectrometry (GC-MS) (7890A/5975C,Agilent Technologies) analysis. 1 µL of each silylated sample was injected into a DB-1capillary column (0.25 mm i.d. × 30 m; 0.25 µm film thickness; J&W Scientific). Theoven temperature for GAs was held at 80 °C for 3 min and then programmed to increasefrom 15 °C per minute to 300 °C, followed by 5 min at 300 °C. Helium was used as a carriergas at a linear flow of 1 mL min−1. The GC was directly interfaced to a mass-selectivedetector with an interface and source temperature of 280 °C, an electron energy of 70 eVand a dwell time of 100 ms.

A portion of the ABA equivalent fraction from ODS-HPLC was subjected to GC/SIManalysis after esterifying with 2 µL of 2 M trimethylsilyl-diazomethane solution in n-hexane. The temperature programme was set as follows: 3 min held at 60 °C, followedby an increase at a rate of 20 °C per minute to 290 °C and 5 min of constant temperature.

Quantification of biologically active endogenous GAs and ABA

To identity the eluted GAs using their retention times, diagnostic ions of endogenous GAsand deuterated GAs were monitored. The levels of endogenous GAs were determined bymeasuring the abundance of the following ion pairs: m/e 506/508 for GA1; m/e 504/506 forGA3; both m/e 418/420 and 284/286 for GA4; and m/e 416/418 and 222/224 for GA7. Inthe GC/SIM analysis of ABA, the characteristic ions m/z 190/194 were monitored.

The endogenous levels of the detected hormones were calculated by multiplying thepeak area ratios of the endogenous hormones by their standard quantities and dividingby the tissue fresh weight (mg). Each treatment was repeated in triplicate.

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Statistical analysis

Pairs of values were compared by Student’s t-test using SAS 9.1.3 software (SAS Institute).Differences were considered significant at the P = 0.05 level.

Results

Effect of HC on dormancy release, floral budbreak and fruit set

Shanghai has a humid subtropical monsoon climate. The monthly average temperatureand precipitation during shedding, dormancy and blooming are presented in Table 1.

When release from endodormancy occurs, the buds of fruit trees can burst in a suitableenvironment. However, if the environmental temperature is low, buds cannot burst, a situ-ation referred to as ecodormancy. In the Shanghai region, the budburst rate of ‘Summit’sweet cherry was approximately 30% on 8 February (Table 2), indicating that ‘Summit’was still in the endodormancy stage at 0 days afterHC treatment. Subsequently, the budburstrate increased to 72% and 96% under HC treatment, and 42% and 68% under the controltreatment on 22 February and 7March, respectively. Therefore, the release date of bud endo-dormancy underHC treatmentwasmid-February, whereas it was earlyMarch in the controlgroup, which was approximately half a month later than the HC treatment.

In addition, the spray application of HC to endodormant ‘Summit’ sweet cherry treeswas found to advance the phenological development compared with the controls, in whichparts of the trees were sprayed with distilled water (Table 3, Figure 1). Application of HCinduced budbreak initiation 11 days earlier than the control. Application of this agent alsosignificantly induced early blooming compared with the control. Blooming occurred 5days earlier in the treated trees compared with the controls. HC was found to be effectivein causing full bloom to occur earlier. The blooming duration was also shortened to 8 dayscompared with 10 days in the controls. Furthermore, HC significantly increased the fruitset by 359.6% compared with the controls.

Effect of HC on branch water content

Water is one of the basic determining factors for bud development because it is quantitat-ively the major component of plant tissues (Zeuthen 2001). Dormancy of woody plants isconsidered to be closely related to changes in water movement (Welling & Palva 2006).Branch water content changes are shown in Figure 2. Similar trend of water contentchanges were found in HC-treated branches and in the controls, except that the increasedwater content induced by application of HC was significantly higher than the reducedwater content in the controls at the beginning. However, there is a rapid increased afterbudbreak. Additionally, branch water content was consistently higher in HC treatmentthan in the controls.

Table 1. Monthly average temperature (°C) and precipitation (mm) during shedding, dormancy andblooming in Shanghai, China.

Average value

2011 2012

October November December January February March April

Temperature (°C) 19.1 12.8 6.8 4.3 5.2 9.2 15.3Precipitation (mm) 63.3 54.1 38.4 37.3 62.2 84.4 104.9

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Changes in endogenous biologically active gibberellic acids and abscisic acid

Levels of GAs isomers in branches of ‘Summit’ sweet cherry tree showed different patternsduring the dormancy release and blooming, as shown in Figure 3. Four bioactive gibber-ellic acids (GA1, GA3, GA4 and GA7) were detected in branches during this process. Gen-erally, GA3 (Figure 3B) was the most abundant gibberellin, followed by GA7 (Figure 3D),then GA4 (Figure 3C), and the concentration of GA1 (Figure 3A) was the lowest. Fromendodormancy release to blooming, levels of GA1, GA3 and GA4 tended to decline inboth HC-treated and the control branches, and GA7 increased rapidly after buds burst.During endodormancy release and the initial phase of blooming, the level of GA3 inHC-treated branches was always higher than that in the controls. However, this differencediminished gradually with budburst. GA3 levels were even lower than in the controls afterfull bloom. For GA1 and GA4, there were significant differences between HC-treated andcontrol branches sometimes, but there was no regular change during the processes ofendodormancy release and blooming. Before budburst, no significant difference wasobserved in GA7 levels between HC treatment and controls. However, GA7 levelsrapidly increased in HC-treated branches and were significantly higher compared withcontrols from the burst stage to full bloom. After full bloom, the levels of GA1, GA3,GA4 and GA7 in HC-treated branches were lower than those in the controls in mostcases. As for total amount of bioactive GAs (Figure 3E), it displayed a similar curve toGA3, and HC treatment had higher total GAs levels than the controls before full bloom.

Changes in ABA levels in branches during the dormancy release and blooming areshown in Figure 4. The ABA levels declined rapidly, and no significant difference wasfound between HC treatment and the controls at the early stages of endodormancyrelease (41 days before blooming). However, the ABA concentrations in HC treatmentswere lower than those in the controls from 31 days before the start of blooming untilfull bloom. Then, they were higher than the controls as the flowers became senescent.

In addition, the ratio of GAs:ABA (Figure 5) in natural conditions was relatively low,with little fluctuation during endodormancy release and blooming. However, the GA:ABAratio in HC-forcing conditions increased continuously before the ‘green tip’ stage, and itwas higher than that in the control before ‘first bloom’.

Table 2. Rates of budburst in HC-treated and control branches of ‘Summit’ sweet cherry trees, sampledin 2012.

Treatment

Budburst rate (%)

8 February 22 February 7 March 14 March

Control 34% 42% 68% 90%HC 30% 72% 96% -

Table 3. Effect of spray-applied HC on floral budbreak, blooming and fruit set of ‘Summit’ sweet cherrytrees in 2012.

Treatment Date of floral budbreak

Date of blooming

Blooming duration (days) Fruit set (%)5% 50% 75% End

Control 14 March 8 April 9 April 10 April 18 April 10 3.24 ± 0.53HC 3 March 3 April 5 April 6 April 11 April 8 14.89 ± 0.75*

All values are means ± SEM (n = 5).*Significant differences at P = 0.05 by a standard t-test.

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Discussion

In temperate woody perennial plants, endodormancy release by exposure to low tempera-tures involves complicated mechanisms that are regulated by many factors. Dormancy

Figure 1. Spray application of HC on blooming of ‘Summit’ sweet cherry trees.

Figure 2. Effect of spray-applied HC on branch water content of ‘Summit’ sweet cherry trees in 2012.The stages of floral budbreak are indicated by arrows (solid, HC-treated; dotted, control); full bloomstages are indicated by lines (solid, HC-treated; dotted, control). All values are means ± SEM (n = 3).* and ** indicate significant differences at P = 0.05 and P = 0.01 by a standard t-test, respectively.FW, fresh weight.

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Figure 3. Effect of spray-applied HC on levels of: A, endogenous gibberellic acid 1 (GA1); B, GA3; C, GA4;D, GA7; E, total bioactive GAs, in branches of ‘Summit’ sweet cherry trees. The stages of floral budbreakare indicated by arrows (solid, HC-treated; dotted, control); full bloom stages are indicated by lines(solid, HC-treated; dotted, control). All values are means ± SEM (n = 3). * and ** indicate significantdifferences at P = 0.05 and P = 0.01 by a standard t-test, respectively. FW, fresh weight.

Figure 4. Effect of spray-applied HC on endogenous abscisic acid (ABA) in branches of ‘Summit’ sweetcherry trees. The stages of floral budbreak are indicated by arrows (solid, HC-treated; dotted, control);full bloom stages are indicated by lines (solid, HC-treated; dotted, control). All values are means ± SEM(n = 3). * and ** indicate significant differences at P = 0.05 and P = 0.01 by a standard t-test, respectively.FW, fresh weight.

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status testing and observation of phenological stages in this study showed that 2% (v/v) of theaqueous solution ofHCwas able to advance endodormancy release, budbreak and bloomingof ‘Summit’ sweet cherry trees (Figure 1; Tables 2 and 3). Moreover, HC also shortened theblooming duration of ‘Summit’ under the natural field conditions of Shanghai. However, theblooming advancement induced by HCwas lower than the endodormancy release and bud-break advancement. This may indicate that HC sapped its effect in inducing endodormancyrelease, whereas climatic factors began to influence the blooming processes from budbreakonward, generally decreasing the initial advancement (Godini et al. 2005).

In previous works, HC was found to play a role in inducing enzyme activity, promotingthe retranslocation of stored reserves and increasing the uptake of nitrogen (Yang et al.1990; Seif El-Yazal & Rady 2012). Catalase activities were increased by applying HC atan appropriate dose (Bartolini et al. 1997), which results in the detoxification of H2O2

in plant tissues by a chain of reactions that link to the pentose phosphate pathway,leading to increased turnover of the pathway and increased budbreak (Bichler 1999).Although no visible phenological differences were observed between HC treatment andthe controls early in the experiment, significant differences were found in branch watercontent. Furthermore, significantly higher water content was found under HC-forcingconditions in most of the phenological phase (Figure 2). Similar results were obtainedby Trejo-Martinez et al. (2009), indicating that the changes were favoured by HC sprays.

Although there is no generally accepted mechanism to explain the endodormancyresults in perennial plants, hormones have been regarded as one of the key factors associ-ated with dormancy based on concentration measurements and expression analysis ofhormone biosynthesis and hormone-responsive genes (Rinne et al. 2011). It has beensuggested that GAs may function in the timing of dormancy establishment and chil-ling-induced release (Hazebroek et al. 1993; Zanewich et al. 1995; Yamazaki et al. 2002;Suttle 2004a, 2004b). In contrast, ABA was found to be correlated with the inhibitionof bud growth and dormancy induction and maintenance (Yamazaki et al. 2002; Suttle

Figure 5. Effect of spray-applied HC on the ratio of endogenous total bioactive GAs and abscisic acid(ABA) levels in branches of ‘Summit’ sweet cherry trees. The stages of floral budbreak are indicated byarrows (solid, HC-treated; dotted, control); full bloom stages are indicated by lines (solid, HC-treated;dotted, control).

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2004b) and a high ABA level was found in dormant buds. However, ABA declines at theend of the dormancy stage (Fernie & Willmitzer 2001; Yamazaki et al. 2002). In thecurrent study, both GAs and ABA in the branches of ‘Summit’ declined during the endo-dormancy release stage, but they increased slightly before budbreak and increased con-tinuously during blooming under natural climate conditions (Figures 3 and 4), whichindicated that a reduced ABA level is not always associated with growth resumption.The results of the present study are consistent with the findings of Yamazaki et al.(2002), which indicate that endogenous ABA levels increase concomitantly with budelongation after dormancy release.

The major isoform of endogenous bioactive GA varies among plant species. GA1 is con-sidered to be the main GA involved in stem elongation, with low flowering activity,whereas GA3 has higher florigenic activity (Evans et al. 1993). In addition, GA3 can par-tially or totally substitute for the action of low temperature in beech nuts (Fagus sylvaticaL.) (Bonnet-Masimbert & Muller 1976) and it hastens floral bud development and short-ens the time to anthesis at the late flower bud developmental stages during winter dor-mancy in peach (Reinoso et al. 2002). However, GA4 is thought to be the main GAinvolved in shoot elongation in hybrid aspen (Israelesson et al. 2004) and it is able toreverse the obstructed transport paths in the symplasm and cell walls. It also induces cano-nical budburst and development in a concentration-dependent way, whereas GA3 fails toinduce the same response (Rinne et al. 2011). GA7 has often been associated with bloom-ing, especially when applied exogenously in conifers (Ross 1992; Cecich et al. 1994). Ouranalysis of GA isomers showed that there were remarkable differences in the patterns ofendogenous bioactive GA concentrations (GA1, GA3, GA4 and GA7) during the endodor-mancy release and blooming stages (Figure 3). In ‘Summit’ sweet cherry, GA3 was foundto be the most abundant isomer of bioactive GA, followed by GA7 and GA4. The concen-tration of GA1 was lowest during the endodormancy release and blooming stages. Thissuggests that GAs may have selective effects on endodormancy release and blooming,and GA3 may play a major role in endodormancy release and blooming in ‘Summit’.In addition, a sharp and transient increase in both GA7 and total GAs in branches of‘Summit’ were observed during blooming (Figure 3D–E). This suggests the involvementof gibberellin in induction of earlier blooming in sweet cherry.

Regarding the effects of HC on endogenous phytohormone concentration, Luna et al.(1993) indicated that the favourable effects of HC (Dormex) on the date of floral budopening may be due to the stimulatory effects on natural gibberellins. HC may releasebuds from dormancy by decreasing the ABA concentration (Nashaat 1996). Our resultsshowed that there were higher concentrations of gibberellins and lower concentrationsof ABA in branches that were sprayed with HC than in control branches (Figure 3).This finding agrees with the report of Mohamed et al. (2014), who found that HC(Dormex) is rapidly metabolised in the plant and increases the synthesis of GAs, whichpromotes the activity and biosynthesis of α-amylase, whose products are either a necessarysubstrate or a signal for accelerated growth that contributes to budbreak and the metab-olism of ABA (Goggin et al. 2011; Rentzsch et al. 2012).

Plant dormancy is controlled by numerous integrated plant structures and functions(Simpson 1990; Crabbe 1994) and is induced and released by changes in the balancebetween inhibiting and stimulating endogenous substances. It is reported that GAs oftenrelease dormancy and stimulate bud growth (Fernie & Willmitzer 2001). In contrast, ABA

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is considered to be antagonistic with GAs in their roles during the processes of dormancyinitiation in plants (Anderson et al. 2001; Jacobsen et al. 2002; Seo et al. 2006). The ratio ofthe endogenous hormonesGAandABA is considered to be a relevant factor regulating endo-dormancy. In contrast to the changes inGAandABA levels, the ratio of GA:ABA in branchesincreased during endodormancy release and in the budbreak stage, but decreased after fullbloom due to senescence of the flowers. This suggests that the relatively higher GA concen-trations and lower ABA concentrations were needed for endodormancy release in ‘Summit’sweet cherry. Qin et al. (2009) reported that the ratio of GA3:ABA in apple buds decreased indormant buds, then increased in opening buds. In the current study, hydrogen cyanamideeffectively reduced ABA levels but slowed the decline of bioactive GA levels, which resultedin a sustained increase in the GA:ABA ratio during the process of endodormancy release.

Conclusion

In this study, spray application of HC on ‘Summit’ sweet cherry trees hastened bud releasefrom endodormancy, shortened blooming duration and improved bud growth and fruitset. The current study showed that GAs might have selective effects on endodormancyrelease and blooming. GA3 was the most abundant GA isomer and it actively respondedto the application of HC as well as GA4, which might be closely related to endodormancyrelease, which is improved by HC application. GA7 displayed a rapid increase when budswent into the budburst stage until full bloom, and it also responded positively to HC andwas associated with budburst and blooming improvement induced by HC application. TheGA1 level had no regular changes during the process of endodormancy release and bloom-ing. Furthermore, a higher GA:ABA ratio was observed in HC-treated ‘Summit’ branchesfrom the phase of endodormancy release until full bloom. However, a reciprocal patternoccurred afterwards due to senescence of the flowers. Therefore, if it is economically feas-ible, hydrogen cyanamide could be an option for the production of sweet cherries withhigher chilling requirements, including ‘Summit’ in low winter chill regions.

Acknowledgements

Authors wish to thank the anonymous reviewers for their helpful comments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The authors would like to thank the Foundation of ‘948’ Project of Ministry of Agriculture of China(grant number 2013-Z23) for financial support.

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