Exogenous dormancy-breaking substances positively ... - Springer Link

Plant Growth Regul (2014) 72:211–220 DOI 10.1007/s10725-013-9852-1

ORIGINAL PAPER

Exogenous dormancy-breaking substances positively change endogenous phytohormones and amino acids during dormancy release in ‘Anna’ apple trees Mohamed A. Seif El-Yazal • Samir A. Seif El-Yazal Mostafa M. Rady

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Received: 1 April 2013 / Accepted: 3 October 2013 / Published online: 11 October 2013 Springer Science+Business Media Dordrecht 2013

Abstract 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 DormexTM, potassium nitrate, mineral oil, calcium nitrate and thiourea was noticed in varying degrees. Although DormexTM 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 DormexTM, 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. Keywords ‘Anna’ apple Dormancy-breaking agents Proline Arginine Phytohormones

M. A. Seif El-Yazal M. M. Rady Botany Department, Faculty of Agriculture, Fayoum University, Fayoum, Egypt S. A. Seif El-Yazal (&) Horticulture Department, Faculty of Agriculture, Fayoum University, Fayoum, Egypt e-mail: [email protected]

Introduction Bud dormancy in woody perennials is a complex process that enables plants to survive long periods of adverse conditions, including the extremes of drought, cold and heat, and is characterized by growth cessation, arrest of cell division, and reduced metabolic and respiratory activity (Arora et al. 2003). In late summer, declining photoperiods and temperatures cause shoot extension growth to cease the initiation of apical buds to protect the apical meristem which defined as paradormancy (PD) (Lang 1987; Heide and Prestrud 2005). A specific signal, endogenous, perceived within the bud, induces and maintains these buds in a state of endodormancy (ED) (Lang 1987; Bohlenius et al. 2006), while the effect of environmental conditions defined as ecodormancy (ECD) (Lang 1987). In temperate perennial species, a period of low temperatures, commonly referred to as winter-chilling, is needed to release buds from ED. In some apple and grape varieties, decreasing photoperiod, as well as warm winters triggers the transition of buds into ED (Bound and Jones 2004; Ku¨hn et al. 2009). In Egypt, bud break values normally reach 50 % and the dormancy-breaking agents (DBA) are applied to the trees to overcome the adverse effect of warm winter temperatures. Hydrogen cyanamide (DormexTM) has been used to break bud and seed dormancy and to improve rooting in several species, responses usually associated with the action of amino acids and plant hormones (Guevara et al. 2008; Seif El-Yazal et al. 2012). Using the DBA (i.e., potassium nitrate, mineral oil, calcium nitrate and thiourea), few studies has measured endogenous hormones and amino acids. On the other hand, many DBA were applied and shown to be the most effective with deciduous fruit trees management (Rufato et al. 2009; Hawerroth et al. 2010; Germchi et al. 2010, 2011; Ben Mohamed et al. 2012a, b; Eshghi et al. 2012).

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The relation of plant hormones and bud dormancy as well as bud break was a vital problem for both theories and production practices. In this respect, the endogenous hormonal change in the bud dormancy inducing and releasing processes was studied by many researchers (Wang et al. 2006; Guevara et al. 2008; Dong et al. 2009; Mornya and Cheng 2011; Okay et al. 2011). They found that the occurrence, termination, regulation, and control of dormancy were regulated by hormones. A few number of studies have been done on the change of endogenous hormones from dormancy releasing to bud opening, but none on the relation between bud break and dynamic change of endogenous hormones, as well as the equilibrium of late-opening apple varieties. Amino acids are the currency of nitrogen exchange between sources and sink tissues in plants, and constitute a major source of the components used for cellular growth and differentiation (Couturier et al. 2010). Recent research has shown that changes in amino acid profiles are associated with the release of buds from dormancy (Judd et al. 2010). Several functions are proposed for the accumulation of proline in tissues exposed to stress: C and N reserves for growth after stress relief (Hossain et al. 2011), free radical scavenging (Chen and Dickman 2005), antioxidation (Hoque et al. 2007) and dormancy break (Ben Mohamed et al. 2010, 2012a). In addition, proline biosynthesis may be associated with the production of NADP? for stimulating the pentose phosphate pathway (Wang et al. 2007; Hossain et al. 2011). Moreover, arginine provides N for the synthesis of other amino acids, some of which are precursors for growth hormones, polyamines, and nitric oxide (Durzan and Steward 1983; Durzan and Pedroso 2002). Several researchers justified the idea that proline and arginine are involved in the regulation of dormancy (Ben Mohamed et al. 2010, 2012a; Kaur et al. 2011; Seif El-Yazal et al. 2012; Seif El-Yazal and Rady 2013) in some plants. Short winter is found to be not meeting the chilling requirements of buds, and cause a delay in opening the buds of ‘Anna’ apple trees until late winter. This condition exposes these buds to damage under the influence of high temperature and/or delays them in entering in dormancy in the following year, and leads to some physiological defects, which may result in bud weakness and death. These negative events threaten the ‘Anna’ apple productivity in Egypt and similar regions. Thus, this work focuses mainly to explain the behavior of the amino acids; proline and arginine, and phytohormone contents in buds and their reflections in the duration to full bud break. In addition, the percentages of bud break and fruit-set were measured as a result of exogenous application with DormexTM, potassium nitrate, mineral oil, calcium nitrate and thiourea for ‘Anna’ apple trees. The impact of these chemicals on hastening the

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Plant Growth Regul (2014) 72:211–220

release of buds from dormancy under short winters in Egypt and similar regions was elucidated.

Materials and methods Trees selection and treatments Uniform, 12-year-old ‘Anna’ apple trees (Malus sylvestris Mill.) grafted on Malling-Merton 106 (MM 106) rootstock were selected at random, for a preliminary study in 2008/2009 and for the main research studies in the 2009/2010 and 2010/2011 seasons. All trees (n = 36) were grown in the Orchard (newly reclaimed saline calcareous soil) of the Horticultural Station at Aboksah in Abshawai, Fayoum, Egypt (29170 N; 30530 E). For the main 2-season study, selected trees of each treatment (n = 6) were labelled in November 2009 and 2010, received the foliar treatments during December of the same 2 years and sampled from 5 January–19 February 2010 and 2011. Trees chosen for the study in the first season is not the same trees that were selected for the second season. Each tree was designed as one replicate, and each treatment included six trees (total n = 36). Foliar-spray applications (6 l tree-1; six trees per treatment) were conducted as follows: treatment 1; control trees did not receive any of the five DBA compounds, only tap water; trees of treatment 2 were foliar sprayed to runoff with 4 % (v/v) hydrogen cyanamide (DormexTM; Dr. A. F. Straße, Trostberg, Germany); trees of treatment 3 were foliar sprayed with 8 % (w/v) potassium nitrate [KNO3; containing 13 % (w/w) N and 44 % (w/w) K]; trees of treatment 4 were foliar sprayed with 6 % (v/v) mineral oil (MO; Guangzhou Hanglian Chemical Industry Co. Ltd., Guangzhou, P. R. China); trees of treatment 5 were foliar sprayed with 6 % (w/v) calcium nitrate [Ca(NO3)2; containing 15.5 % (w/w) nitrogen]; trees of treatment 6 were foliar sprayed with 2 % (w/v) thiourea (molecular weight 76.12, Assay 99–100 %, Sulphated ash 0.1 %, produced by Jinan Hongfangde Pharmatech Co., Ltd, China). All spray treatments were applied twice, on 11 December in each season and 2 weeks later, using 20 LKnapsack Sprayer. Triton B [0.1 % (v/v)] was added as a wetting agent to each spray solution. A preliminary study was conducted in 2008/2009 in which DormexTM, KNO3, MO, Ca(NO3)2 or thiourea were applied once or twice at different rates for each. The rates of 4 % (v/v), 8 % (w/v), 6 % (v/v), 6 %(w/v) or 2 % (w/v) respectively, applied twice were found to be the most significant concentrations for later bud growth in ‘Anna’ apple trees (data not shown). These treatment levels were used later for the main study.


Morphological characteristics and yield measurements Bud count was made for each tree (n = 6) in each treatment. The dates on which floral and vegetative buds started to open were recorded. In addition, the dates at which flowering reached 25, 50, 75 and 100 % of the total flowers were estimated in each treatment. The dormant buds were also counted and were expressed, with opened buds, as a percentage of the total number of buds. The final fruit set was calculated 6 weeks after full bloom stage as a number of persisted fruits per hundred spur and lateral buds (Westwood 1978). At harvest stage, apple fruits were harvested, counted and weighed for each examined tree. Physical properties; fruit size (cm3), fruit weight (g), fruit length (mm) and fruit diameter (mm) were also determined. Extraction and determination of endogenous hormone in apple buds Bud samples were collected, 8-day intervals, beginning from 4 January up to 19 February for determining the metabolic changes in the hormonal content in buds. Buds were randomly sampled and immediately transported to the laboratory. Floral bud samples were taken from each tree of each treatment and were analyzed for endogenous levels of gibberellic acid (GA3), indole-3-acetic acid (IAA) and abscisic acid (ABA). The extraction and purification were made following the method of Kettner and Doerffling (1995). Samples (1.0 g) were collected, from each treatment, and ground, at 4 C, in 80 % methanol containing 0.1 g l-1 an antioxidant, butylated hydroxyl toluene (BHT). They were extracted at 4 C in dark for 72 h with subsequent change of solvent. The extracted samples were centrifuged and the supernatant was reduced to aqueous phase using rotary thin film evaporator (RFE). The pH of aqueous phase was adjusted to 2.5–3.0 and partitioned three times with 1/3 volume of ethyl acetate. The ethyl acetate phase was dried down completely using RFE. The dried sample was re-dissolved in 1 ml of methanol (100 %) and was analyzed on HPLC (Shimadzu, C-R4A Chromatopac; SCL-6B system controller) using UV detector and C18 column (39 9 300 mm). For identification of hormones, 100 ll samples filtered through 0.45 Millipore filters were injected into column. Pure IAA and GA3 (Sigma, USA) were used as standards for identification and quantification of these hormones. The identification was made on the basis of retention time and peak area of the standards. Methanol, acetic acid and water (30:1:70, respectively) were used as a mobile phase. The flow rate was adjusted at 0.5 ml min-1 with an average time for 15 min sample-1. The wavelength used for the detection of IAA was 280 nm, while for GA3 was 254 nm. For ABA, samples were injected onto a C18 column and eluted with a

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linear gradient of methanol (30–70 %), containing 0.01 % acetic acid, at a flow rate of 0.8 ml min-1. The retention time of ABA was determined by using authentic standards, monitoring the elution of standard at 254 nm. Extraction and determination of free proline Free proline was determined according to the method described by Bates et al. (1973), with a slight modification of Ennajeh et al. (2006). Briefly, sample (200 mg) of frozen bud was extracted with 5 ml of 40 % (v/v) methanol heated to 80 C for 30 min in hermetically sealed tubes. The supernatant (1 ml) was mixed in a reaction test tube with 2 ml glacial acetic acid, 1 ml ninhydrin solution (25 mg ml-1) and 2 ml of a mixture consisting of 24, 60 and 16 % (v/v/v) of distilled water, glacial acetic acid and orthophosphoric acid, respectively. The tubes were closed and heated for 30 min in a water bath set to 100 C. The sample was cooled on ice, then 3 ml of toluene was added and the mixture was shaken vigorously. The colored toluene phase (upper phase) was saved and dehydrated with anhydrous Na2SO4. The extracts were kept in the dark for a minimum of 2 h before their absorbance was measured at 528 nm. Proline content of the fresh bud was calculated based on a standard calibration curve with concentrations ranging from 0 to 0.025 mg ml-1. Extraction and determination of arginine Bud samples were collected at 15 days intervals, from 4 January–19 February 2010 and 2011, to determine seasonal changes in arginine. Buds were sampled at random and immediately transported to the laboratory to determine their contents of arginine. Arginine was measured after each bud sample (2 g) had been freeze-dried and ground to a fine powder. Samples (0.5 g) were then extracted, at room temperature, by shaking for 24 h with 50 ml of a single-phase 12:5:3 (v/v/v) mixture of methanol:chloroform:water (Bieleski and Turner 1966). Norleucine (0.5 ml of a 4 mM solution in 0.01 M HCl) was added prior to extraction as an internal standard. After extraction, the colourless aqueous methanolic-phase (containing the amino acids) was separated from the chloroform-phase (containing pigments and lipids; Redgwell 1980). The solid residue was boiled gently for 10 min in 40 ml water to extract the residual amino acids (Oland 1959). After cooling to room temperature and centrifugation at 10,0009g, the aqueous extract was combined with the methanolic extract and made up to 100 ml. A 20 ml aliquot was loaded onto a cation-exchange column (Dowex-SOW 8 %; 200–400 mesh) with a bed volume of 3 ml. The column was washed with 45 ml 0.01 M HCl, followed by 5 ml water, then eluted with 30 ml 2 M NH4OH to release

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the amino acids. The flow rate was maintained at approx 1.0 ml min-1 using a small vacuum pump. Columns were regenerated by washing sequentially with 5 ml water, 10 ml 1.0 M HCl, 5 ml water, 40 ml 0.2 M NaOH, 5 ml water, 10 ml 1.0 M HCl, and finally 5 ml water (Lazarus 1973). The ammoniacal eluates were lyophilized and stored at -10 C until analysis, at which time they were resolubilized in 0.2 M lithium citrate loading buffer (pH 2.2; LKB Biochrom, Cambridge, UK). Analyses of amino acids was performed using an Alpha Plus amino acid analyzer (LKB Biochrom) fitted with a stainless steel column (200 mm 9 4 mm) filled with ion exchange resin (Ultropac 8; particle size 8 lm; LKB Biochrom). The content of arginine (in mg 100 g-1 DW) was measured using ninhydrin positive compounds. The reagent coil temperature was 135 C. Data acquisition and peak integrations were evaluated using Baseline chromatography software (Waters Dynamic Solutions, Ventura, CA, USA) on an IBM 286 AT computer (Henle et al. 1991). Statistical analysis Values of the determined characters were subjected to statistical analysis, following the standard procedure described by Gomez and Gomez (1984). The ‘F’ test was applied to assess the significance of the treatment, at 5 % level of probability.

Results Date of floral bud break The foliar application with hydrogen cyanamide (DormexTM), potassium nitrate (KNO3), mineral oil (MO), calcium nitrate [Ca(NO3)2] or thiourea to apple trees was found to hasten the floral bud break as compared to the control in which trees were sprayed with tap water (Table 1). The period to the first floral bud break was shortened in both seasons by 27 and 33, 25 and 25, 23 and 23, 15 and 15, and 14 and 15 days with DormexTM, KNO3, MO, Ca(NO3)2 and thiourea, respectively when compared with the control trees. In addition, these stimulants hastened 50 % bud break as compared to the control. This earliness reached, in both 2010 and 2011 seasons, 31 and 32, 28 and 27, 27 and 30, 16 and 16, and 16 and 18 days, respectively compared to the control trees. DormexTM was found to be most effective in shortening the period up to full flowering. In both seasons the flowering period was shortened to 20 and 21 days as compared to 24 and 23, 24 and 23, 23 and 23, and 24 and 25 days for KNO3, MO, Ca(NO3)2 and thiourea, respectively. On the other hand, all

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these stimulants obviously exceeded the control with which 31 and 30 days were recorded. Floral bud break and fruit-set Table 2 shows that, all tested substances significantly increased the percentages of floral bud break and fruit-set, while decreased the percentages of dormant buds when compared to the control. However, DormexTM was the most effective in this concern. It significantly surpassed the control; tap water, in both seasons, by 14.47 and 14.28 % for bud break, and 46.02 and 62.36 % for fruit-set. Thiourea was found to be less effective, although it exceeded the control, in both seasons, by 8.65 and 9.42, and 6.52 and 12.90 % for bud break and fruit-set, respectively. Fruit yield and quality Tables 2 and 3 show that, all DBA increased the number of fruits tree-1, total fruit yield tree-1, fruit weight, fruit size and fruit dimensions when compared to the control. DormexTM was found to be most effective in this concern. It exceeded the control in both 2010 and 2011 seasons by 24.23 and 33.48 % for number of fruits tree-1, 42.37 and 41.97 % for fruit yield tree-1, 11.99 and 19.37 % for fruit weight, 30.32 and 38.71 % for fruit size, 4.87 and 8.08 % for fruit diameter, and 13.15 and 16.17 % for fruit length. Thiourea was found to be less effective when compared to all other tested substances, although it exceeded the control in both seasons by 4.14 and 3.34, 1.11 and 4.33, 1.11 and 1.92, 11.69 and 18.27, 2.35 and 1.15, and 3.78 and 8.82 % for aforementioned characters, respectively. Bud hormonal content Foliar-applied DormexTM, KNO3, mineral oil, Ca(NO3)2 or thiourea increased the contents of indole-3-acetic acid (IAA) and gibberellic acid (GA3) in floral buds of ‘‘Anna’’ apple trees as compared to the control in which trees were sprayed with tap water (Table 4). Bud contents of these two hormones obtained with DormexTM were surpassed their contents recorded with all other treatments in most of the sampled dates. Maximum contents of IAA and GA3 were obtained from buds treated with DormexTM, collected in 4 February (buds were released from dormancy), while minimum contents were obtained from buds treated with Ca(NO3)2 and thiourea, sampled in 4 January (buds were still dormant). For ABA, the opposite situation was found. In addition, maximum ratios of IAA/ABA and GA3/ABA were noted in the 4 February sample (buds were released from dormancy), while minimum ones were observed in the 4 January samples (buds were still dormant).


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Table 1 Effects of foliar-applied DormexTM (4 %), potassium nitrate (KNO3; 8 %), mineral oil (MO; 6 %), calcium nitrate [Ca(NO3)2; 6 %] and thiourea (2 %) on dates of the floral bud opening and flowering period of ‘Anna’ apple trees in 2010 and 2011 Treatments

Date of flower bud opening Beginning

25 %

2010

2010

2011

2010

2011

2010

2011

2010

2011

2010

2011

2011

50 %

75 %

End

Flowering period (days)

Control

23 Feb

26 Feb

3 March

4 March

13 March

15 March

15 March

19 March

26 March

28 March

31

30

DormexTM

27 Jan

24 Jan

28 Jan

3 Feb

10 Feb

11 Feb

12 Feb

15 Feb

16 Feb

14 Feb

20

21

KNO3

29 Jan

1 Feb

3 Feb

5 Feb

13 Feb

16 Feb

19 Feb

23 Feb

22 Feb

24Feb

24

23

MO

31 Jan

3 Feb

4 Feb

8 Feb

14 Feb

13 Feb

18 Feb

20 Feb

24 Feb

26Feb

24

23

Ca(NO3)2

8 Feb

9 Feb

12 Feb

11 Feb

25 Feb

27 Feb

28 Feb

1 March

3 March

4 March

23

23

Thiourea

9 Feb

11 Feb

19 Feb

16 Feb

23 Feb

27 Feb

3 March

6 March

5 March

8 March

24

25

Table 2 Effects of foliar-applied DormexTM (4 %), potassium nitrate (KNO3; 8 %), mineral oil (MO; 6 %), calcium nitrate [Ca(NO3)2; 6 %] and thiourea (2 %) on bud break %, dormant buds %, fruit-set % and number of fruit tree-1 of ‘Anna’ apple trees in 2010 and 2011 Treatments

Control Dormex

TM

Bud break (%)

Dormnt buds (%)

Fruit set (%)

No. of fruit tree-1

2010

2011

2010

2011

2010

2011

2010

2011

75.56c

76.95c

24.44a

23.05a

11.95b

12.09b

174.95c

198.88c 265.48a

86.50a

87.94a

13.50c

12.06c

17.45a

19.63a

217. 35a

KNO3

82.73b

84.55b

17.27b

15.45b

16.02a

18.50a

192.20ab

230.15a

MO Ca(NO3)2

83.65b 82.90b

86.15a 85.55a

16.35b 17.10b

13.85c 14.45b

15.88a 14.48a

17.21a 15.11a

188.20b 188.20b

214.11b 211.88b

Thiourea

82.10b

84.20b

17.90b

15.80b

12.73b

13.65b

182.20b

205.54c

Mean pairs followed by different letters are significantly different (p = 0.05) by Duncan’s test; n = 6

Table 3 Effects of foliar-applied DormexTM (4 %), potassium nitrate (KNO3; 8 %), mineral oil (MO; 6 %), calcium nitrate [Ca(NO3)2; 6 %] and thiourea (2 %) on total yield tree-1, fruit weight, fruit size and fruit dimensions of ‘Anna’ apple trees in 2010 and 2011 Treatments

Total yield tree-1 (kg)

2010

2011

Fruit weight (g)

2010

2011

Fruit size (cm3)

2010

2011

Fruit dimensions Diameter (cm)

Length (cm)

2010

2010

2011

2011

Control

18.81c

20.99c

101.80c

104.15c

109.15c

111.22c

5.95a

6.06a

6.08b

6.12b

DormexTM

26.78a

29.80a

114.01a

124.33a

142.25a

154.28a

6.24a

6.55a

6.88a

7.11a

KNO3

22.62b

26.21b

107.28b

109.10b

125.11b

130.10b

6.19a

6.23a

6.61a

7.01a

MO

21.65b

25.15b

104.41b

107.15b

121.55b

128.14b

6.11a

6.19a

6.43b

6.88a

Ca(NO3)2

19.50b

22.14c

103.33c

107.00b

123.16b

129.10b

6.10a

6.17a

6.34b

6.71a

Thiourea

19.02b

21.90c

102.94c

106.16b

121.91b

131.55b

6.09a

6.13a

6.31b

6.66a


Free proline content in buds Proline was increased with foliar application of DormexTM, KNO3, mineral oil, Ca(NO3)2 or thiourea when compared to the control (Table 5). DormexTM was found to be most effective in this regard. It exceeded the control by 31.17, 57.40, 45.03 and 65.45 % for the sampled dates of 4 January, 20 January, 4 February and

19 February, respectively. In contrast, thiourea was found to be less effective.

Arginine content in buds Floral buds of ‘‘Anna’’ apple trees sprayed with DormexTM, KNO3, mineral oil, Ca(NO3)2 or thiourea had

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Table 4 Effects of foliar-applied DormexTM (4 %), potassium nitrate (KNO3; 8 %), mineral oil (MO; 6 %), calcium nitrate [Ca(NO3)2; 6 %] and thiourea (2 %) on hormonal content and ratio in buds of ‘Anna’ apple trees in 2010 and 2011

Table 5 Effects of foliar-applied DormexTM (4 %), potassium nitrate (KNO3; 8 %), mineral oil (MO; 6 %), calcium nitrate [Ca(NO3)2; 6 %] and thiourea (2 %) on proline and arginine contents in buds of ‘Anna’ apple trees in 2010 and 2011

Treatments

Treatments

Sample date 4 Jan

20 Jan

4 Feb

19Feb

Indole acetic acid (IAA) (lg g-1 DW)

Sample date 4 Jan

20 Jan

4 Feb

19 Feb

2.75c

Proline (mg/g-1 FW)

Control

0.80c

2.07c

2.26c

1.90c

Control

4.33c

2.16c

6.35c

DormexTM

0.99a

2.36a

2.70a

2.13b

Dormex

5.68a

3.40a

9.21a

4.55a

KNO3

0.85b

2.33a

2.64a

2.19a

KNO3

4.74b

3.10b

8.11b

3.77b

MO

0.88a

2.34a

2.66a

2.15b

Mineral oil

4.84b

3.15b

8.24b

3.84b

Ca(NO3)2

0.83b

2.31b

2.62b

2.13b

Ca(NO3)2

4.71b

3.01b

8.09b

3.68b

Thiourea

0.81c

2.29b

2.59b

2.12b

Thiourea

4.68b

3.00b

7.99b

3.61b

Gibberellic acid (GA3) (lg g-1 DW)

Arginine (mg/100 g DW)

Control

0.53c

0.79b

1.73b

1.29b

Control

119c

96c

221c

210c

DormexTM KNO3

0.64a 0.60 a

0.89a 0.83b

1.80a 1.75b

1.35a 1.31b

Dormex KNO3

300a 274b

210a 131b

356a 320b

219b 215b

MO

0.62a

0.86a

1.78a

1.33a

Mineral oil

290a

134b

335a

233a

Ca(NO3)2

0.57b

0.82b

1.78a

1.30b

Mineral oil

290a

134b

335a

233a

Thiourea

0.56b

0.81b

1.76b

1.30b

Ca(NO3)2

271b

129b

318b

213b

Thiourea

270b

126b

314b

210b

Abscisic acid (ABA) (lg g-1 DW) Control

2.79a

3.93a

1.78a

1.66a

DormexTM

2.36c

3.44c

1.61b

1.51c

KNO3

2.54b

3.61b

1.63b

1.54c

MO

2.47c

3.53b

1.62b

1.52c

Ca(NO3)2

2.57b

3.65b

1.67b

1.56b

Thiourea

2.61b

3.67b

1.69b

1.57b

Control

0.28c

0.52c

1.26c

1.14c

DormexTM

0.41a

0.68a

1.67a

1.41a

KNO3 MO

0.33b 0.35b

0.64b 0.66a

1.61a 1.64a

1.42a 1.41a

Ca(NO3)2

0.32b

0.63b

1.56b

1.36b

Thiourea

0.31b

0.62b

1.53b

1.35b

Control

0.18c

0.20b

0.97b

0.77c

DormexTM

0.27a

0.25a

1.11a

0.89a

KNO3

0.23b

0.22b

1.07a

0.85a

MO

0.25a

0.24a

1.09a

0.87a

Ca(NO3)2

0.22b

0.22b

1.06a

0.83b

Thiourea

0.21b

0.22b

1.04a

0.82b

IAA/ABA ratio

GA3/ABA ratio


increased content of arginine as compared to the watersprayed controls (Table 5). DormexTM treatment increased the arginine content to a greater extent than all other treatments in most sampling dates.

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Discussion During the process of bud release from dormancy, many changes in some chemical components in floral buds, particularly the contents of endogenous hormones (IAA, GA3 and ABA) and amino acids (proline and arginine), found to occur for playing a vital role in regulating dormancy and bud break. Several studies focused on the relationship between the endogenous hormones and/or amino acids and dormancy in buds (Dong et al. 2009; Seif El-Yazal et al. 2012). Endogenous hormones help plants to respond to environmental signals (Horvath et al. 2003). Endogenous gibberellins (GAs) play a role in many developmental processes and have been proved to participate in the regulation of dormancy (Wang et al. 2006). The control of apple bud dormancy induction, maintenance and release therefore is mediated, at least in part, by changes in hormone signaling as it is also known for tree bud dormancy (Horvath et al. 2003; Rohde et al. 2007). Gibberellic acid (GA3) and ABA signaling as well as GA3/ABA ratios are important as known for tree bud sprouting (Horvath et al. 2003; Rohde et al. 2007). GAs are necessary to floral bud break in cherry. They found in the highest increase directly before bud break (Duan et al. 2004). This result is found to be in an agreement with our results (Table 4). The


present results show that growth-promoting hormones (GA3 and IAA) found to be gradually increased, but growth-inhibiting hormones (ABA) decreased during bud break. During the whole testing period, IAA and GA3 contents in buds of ‘‘Anna’’ apple at their release from dormancy were higher than their contents observed before bud break. In contrast, ABA content in floral buds was higher before bud break than its content at dormancy releasing (Table 4). This suggested that higher IAA and GA3 contents and lower ABA content were needed for release of ‘‘Anna’’ apple buds from dormancy. In addition, the ratios of IAA/ABA and GA3/ABA in apple buds were decreased in dormant buds, while increased in opening ones (Dong et al. 2009). Our results showed that when the dormancy released, IAA/ABA ratio had the same changing tendency to IAA content (Table 4). The hormone IAA may have an effect on transcription of nuclear DNA that can contribute to cell enlargement, promote fruit development, and are involved in apical dominance. Like IAA, GA3/ ABA ratio in buds had the same changing tendency to GA3 content during release from dormancy. GAs could increase the activity of hydrolytic enzymes such as a- and b-amylases, proteinase, peptidase, Lipase, ribonuclease and isocitrate lyase … etc. These enzymes and an unknown factor cooperate with GA3 to relieve the inhibition of bud break and assure the transcription process, and consequently promote the synthesis of mRNA and protein (Alexopoulos et al. 2008). In contrast, ABA could inhibit the production of certain RNA indispensable to the synthesis of a-amylase, and the main role of ABA in the process of germination is restraining GA3 and inducing the transforming of reserve substance (Gubler et al. 2005). High levels of endogenous IAA and GAs which noticed in ‘‘Anna’’ apple trees treated with some DBA are agreed with the explanation of some authors (Kuroi 1985; Yang et al. 1990). They concluded that, cyanide ion may play a role in inducing the enzyme activity, promoting the re-translocation of stored reserves and the uptake of nitrogen with water for bud break. In addition, DormexTM is directly involved in nitrogen metabolism and the production of protein. Guevara et al. (2008) concluded that IAA was the only hormone whose endogenous concentration responded to lower hydrogen cyanamide (HC) concentration. IAA levels measured in control plants were found to be decreased, while those with HC treatments tended to increase. This may indicate that IAA, possibly together with H2O2, was among the initial reactions of the plant to the HC treatment. This increase in IAA could be a response to stress, such as the one mentioned above, as pointed out by Havlova´ et al. (2008) using tobacco. This IAA upsurge might trigger other hormonal changes. Moreover, Foott (1987) found that DormexTM is easily absorbed in the buds and initiated the processes leading to bud break. Fuchigami

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and Nee (1987) suggested that, HC breaks dormancy by decreasing the endogenous ABA in buds, and by its involvement in the conjugation of the thiol group which is assumed to be involved in breaking dormancy. Variations in the levels of proline and arginine have been observed in apple buds during dormancy and subsequent to its release. These variations correlate well with those observed in the present study in relation to dormancy and bud break in ‘‘Anna’’ apple buds. In general, low levels of proline and arginine seem to accumulate at the beginning of dormancy and opposite situation was noticed during dormancy release (Table 5). Higher relative levels of free proline and arginine in apple buds were observed subsequent to dormancy release. Free proline accumulation is a common stress response in plants. Proline can contribute to the osmotic balance between the cytosol and the vacuole and can act as an osmoprotectant of the subcellular structures, since it can be used to quench reactive oxygen species formed under stress conditions (Kavi et al. 2005). Thus, the role of proline and arginine could be more important subsequent to dormancy release, i.e. during active growth. This result coincides with the earlier observations that, within plant higher amino acids concentrations are associated with bud break (Millard et al. 1998). Our results suggest that the seasonal changes in proline and arginine from the first sample till bud break indicate that they may play an important role in chilling tolerance during winter months. In this connection, Durzan (1989) reported that, high levels of arginine, proline and guanidino compounds were also probably present in decoctions prepared in the severe winter. In addition, Seif ElYazal and Rady (2012) indicated that nitrogen compounds, including amino acid, were noticed at low levels in buds during dormant stage, but reached maximum levels just prior to bud break. Regarding our results, foliar application with DormexTM and other substances increased the contents of proline and arginine in buds of ‘‘Anna’’ apple trees when compared with the control; water application (Table 5). This finding found to be agreed with suggestions of Wunsch and Amberger (1968). They reported that DormexTM is rapidly metabolized in the plant and helps in the synthesis of amino acids. It may be metabolized into urea, arginine and probably further into guanidinium compounds. It caused a rapid increase of nitrogen level and growth promoting substances (Kuroi 1974), and may be involved in oxidative processes, which are a prerequisite for bud break. Foott (1987) found that HC penetrates the bud scales, gets absorbed in the buds and initiates the processes leading to bud break. It is rapidly metabolized in the plant and helps in the synthesis of amino acids. Moreover, Walton et al. (1991) assumed that the plant was scavenging nitrogen from HC. Elevated levels of proline in HC-treated

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plants could be associated with a greater stimulation of bud breaks (Linsley-Noakes 1989). Proline synthesis promotes pentose-phosphate pathway being responsible for dormancy release, while its later degradation upon stress recovery can be considered a major source of carbon and energy for newly growing tissues. It seems that transient proline accumulation was implicated in the process of dormancy release which coincided with the development of stress triggered by HC, and then, this proline was used as a source of nitrogen, carbon and energy during bud break and shoot development. Our results show that, spraying apple trees with DormexTM, KNO3, mineral oil, Ca(NO3)2 or thiourea increased the measured bud opining characters (Tables 1, 2). This result may be attributed to the fact that these treatments resulted in more availability of macronutrients (N, K and Ca) to plants. Application of these chemicals for plants may partially replace the chilling requirement, and consequently solve this problem. Number of flowers and inflorescences increased when plants were treated with KNO3 (Khayat et al. 2010). The mechanism is still obscure; however, as it has been mentioned earlier, the essential element K has a great regulatory role within plant cells and organs, such as activation of more than 50 enzymes, osmoregulation, photosynthesis, and loading and unloading of sugars in phloem (Mengel 2007). Moreover, both Ca(NO3)2 and KNO3 increase the amount of solutes within the tree’s cells, triggering cell expansion through changing the osmotic potential, and thus triggering the tree to bloom by causing the required hormone imbalance. In addition, mineral oil gives a similar effect on bloom timing and works in a different way. Favorable effect of the tested substances on date of floral bud opening (Table 1) may be due to their stimulating effect on natural GAs (Table 4). Subha-drabandhu (1995) agreed with our results that some different spray treatments may break dormancy by decreasing ABA content in buds. In our study, DormexTM, KNO3, mineral oil, Ca (NO3)2 or thiourea positively affected the date of flower bud opening. This may be due to the increase in GA3 and IAA and the decrease in ABA contents (Table 4). In this respect, Hartmann et al. (2011) reported that, the induction and maintenance of tuber bud dormancy seem to involve ABA, while bud dormancy release seems to involve GAs and cytokinins. The improving effect of DormexTM and other tested chemicals on yield and its components was mainly attributed to its positive action on enhancing growth and flowering parameters (Table 1, 2, 3). Skene (1969) reported that when a bud opens and attains the shape of a shoot, its tip acts as a strong sink for metabolites and thus being interception center for photosynthates and nutrients results in earlier start of the bloom. Also, George and Nissen (1990) found that nitrogenous compounds such as cyanamide

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application to grapes doubled yields by increasing bud burst on cordons and number of spurs, number of shoot/ spur and higher numbers of bunches per shoot on cordons and spurs.

Conclusion Foliar application with DormexTM, KNO3, mineral oil, Ca(NO3)2 or thiourea for ‘Anna’ apple trees, in this study, found to be hastened the bud break, shortened the period of flowering and improved the bud growth and fruit-set. DormexTM was found to be the most effective, significantly improved the endogenous proline, arginine, IAA and GA3 and reduced the endogenous ABA. This led to an increase in the percentages of bud break and fruit-set, and a reduction in the period of flowering, and finally the increase in the tree yield. Therefore, we recommend using DormexTM for the increase in ‘Anna’ apple tree productivity. It may provide a well strategy for improving the ‘Anna’ apple tree productivity against high temperatures in late winter in Egypt and similar regions.

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