Role of hydrogen cyanamide (HC) in grape bud dormancy release: proteomic approach

Abstract

In the present study, we identified changes in protein expression patterns of grapevine buds when treated with hydrogen cyanamide (HC). HC induced a shift of more than 2-folds in the expression of 1250 proteins out of approximately 7000 detected proteins. The majority of the differentially expressed proteins (DEPs) were localized in the chloroplast (419) and cytoplasm (347). Most of the detected DEPs were linked with energy metabolism, redox activity, hormone, and stress signaling. Particularly, the DEPs associated with defense and sugar metabolism showed significantly higher expression in HC-treated buds. Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed significant enrichment for circadian rhythm, ribosome, and metabolic pathways. Moreover, the antioxidant activity of peroxidase (POD) increased at initial stages but declined at later stages (18 days post-treatment). This study identified several dormancy-related proteins that regulated signaling, as well as metabolic pathways upon HC application. The outcome of this study provides insights into the role of HC in dormancy release for grapevine production, hence useful to alleviate yield losses in mild winter regions.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02194-5) contains supplementary material, which is available to authorized users.

Introduction

Dormancy is a transitory suspension of viewable growth in plants that enhances persistence and developmental synchronization during adverse environmental conditions. The dormancy regulation has key importance for the survival and productivity of perennial plants including commercially valuable fruit plants. It is generally governed by environmental factors, i.e. temperature and photoperiod (Lang et al. 1987; Horvath et al. 2003; Rohde and Bhalerao 2007).

Grapevine is considered as an economic fruit crop worldwide due to its vast commercial uses. Grapes are berry fruits that have high nutritional value. In recent times, the consumption of value-added grape products in China has increased significantly. Despite being used for wine production, the grape industry of China is mainly based on the production of table grapes that constitutes about 80% of its total production (Wu et al. 2018).

Mild temperatures and warm winters have an erratic impact on the dormancy behavior of plants. The insufficient chilling temperature during the winter season is possibly a leading cause of irregular blooming and bud burst (Fadón and Rodrigo 2018). Inadequate chilling condition is the primary reason behind the failure of achieving regular fruit production round the year under protected cultivation system in southern China (Khalil-Ur-Rehman et al. 2019). To overcome this issue, grapevine growers generally rely on the artificial application of synthetic chemicals to equilibrate chilling requirements (Khalil-Ur-Rehman et al. 2017b). Hydrogen cyanamide (HC) is considered as one of the most suitable rest-breaking chemicals in the table grape industry that offers a uniform effect on bud breaking (Ben Mohamed et al. 2010). HC hinders catalase activity that induces oxidative stress through the production of hydrogen peroxide (H₂O₂), which is a stimulus of growth initiation (Or et al. 2002; Halaly et al. 2008; Prassinos et al. 2011).

Different factors linked with mechanisms of plant development that have been explored via omics-based studies (Karami and Saidi 2010). Several hormonal and environmental elements, regulating the initiation of bud growth, including abscisic acid (ABA) level, low temperature, and photoperiod, have been well characterized (Kermode 2005; Bi et al. 2011). Dormancy maintenance and release are associated with several physiological and biochemical processes that are regulated by protein synthesis, hormonal signaling, and energy metabolism under various environmental conditions (Zhuang et al. 2013; Chang et al. 2018).

Proteomics has become an advanced technique for comprehensive analysis of protein datasets in various aspects of plant biology. Proteomics is used at a large scale for protein analysis in different domains of plant research (Bi et al. 2011; Prassinos et al. 2011). The proteomic analysis possibly computes and categorizes the differentially expressed proteins based on variations in expression patterns at various development stages (Chibani et al. 2006). However, limited studies are available about proteome expression patterns during maintenance and release of dormancy in perennial fruit plants, especially in grapevine. Protein profile varies during bud dormancy release that may facilitate to comprehend the mechanism of dormancy maintenance and its release in grapevine. In this study, the tandem mass tag (TMT)-based proteomic analysis was performed to critically examine the changes in protein expression patterns, which were produced before and after the application of HC. These findings will provide a strong molecular basis for a comprehensive understanding of HC induced dormancy release in grapevine.

Materials and methods

Plant material

Four-year-old grapevines cv. Shine Muscat’ (Vitis labruscana Bailey × V. vinifera L), grown under sheltered tunnels at an experimental vineyard located in Hojiatang, Nanjing, P.R China, were used as plant material. Canes were collected from the vineyard and immediately transported to the laboratory. The single bud cuttings were prepared from the excised canes and mixed. The prepared cuttings were distributed into two groups (1) HC treated, and (2) control. The HC-treated group was sprayed with a solution containing 5% v/v HC (‘Dormex’ SKW, Trostberg, Germany), whereas the control was only sprayed with distilled water. After spray, all the cuttings were placed in water-filled glass bottles in a growth chamber under the following conditions; temperature 23 ± 2 °C, light/dark cycles 14/10 h, relative humidity ≥ 70%. The bud break was monitored from each group on day 0, day 6, day 12, day 18, and day 21. A total of 120 buds were assessed for bud break at each time interval. The buds collected on the 18th day from the control and HC-treated cuttings were used for proteomic analysis. Two biological with three technical replicates were used for protein analysis.

Protein extraction

The bud samples were ground in liquid nitrogen and then transferred to a 5-mL centrifuge tube and sonicated with a high-power ultrasonic processor (20 kHz, 195w, Scientz) in lysis buffer (8 M urea, 2 mM EDTA, 10 mM DTT, and 1% protease inhibitor cocktail). The residual debris was removed by centrifugation at 4 °C for 10 min at 20,000 g. Finally, the proteins were precipitated by adding 4 mL of chilled 15% TCA into the supernatant and allowed to settle down for 2 h at − 20 °C. After centrifugation at 4 °C for 10 min, the supernatant was discarded, and the precipitates were washed thrice with cold acetone. The proteins were re-dissolved in a buffer (8 M urea, 100 mM TEAB, pH 8.0) and the protein concentration was determined with the 2-D Quant kit following the manufacturer’s guidelines.

Trypsin digestion and TMT labeling

The protein digestion was performed according to the methodology proposed by Du et al. (2019) with minor modifications. The peptides were desalted before vacuum drying by the Phenomenex C18 SPE column. The samples were liquefied separately in 0.5 M triethylammonium bicarbonate (TEAB) and processed according to the TMT kit manual (Thermo Scientific). After fraternization, the peptide mixture was incubated for 2 h at 25 °C. The tryptic peptides (100 µg of protein) were labeled using TMT 6-plex with 127-label (control 1), 128-label (control 2), 129-label (HC-treated 1), and 130-label (HC-treated 2) (Fig. S1). After observing the label assimilation, the four labeled samples from each replicate group were pooled according to the method described by Li et al. (2016).

HPLC fractionation, LC–MS/MS and database analysis

The peptides were liquefied in 6% ACN containing 0.1% FA (v/v) after labeling and separated by high-performance liquid chromatography (HPLC) into fractions through Agilent 300 with C18 column (250 mm length, 5 µm particles, 4.6 mm) with a gradient of 2% to 60% acetonitrile (pH 10) for 80 min, and were then combined into 18 fractions. For mass spectrometry (MS), the coupled peptides were dried and liquefied in 0.1% formic acid by vacuum centrifugation. LC–MS/MS analysis was carried out using EASY-nLC 1000 UPLC system combined with Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, USA). The methodology of gradient elution was followed as described by Du et al. (2019). The MS/MS data were analyzed by coupling MaxQuant with the Andromeda database (v. 1.5.2.8). Tandem mass spectra were analyzed to compare transcriptome databank with mass spectrometry contaminants database and reverse decoy database. The MaxQuant software was set to allow up to two missed cleavages. The identification and quantitation of the proteins by at least one unique peptide were examined with the median ratio of their identical peptides and normalized using the medians of all the calculated proteins. TMT 6-plex was selected for quantification. In Maxquant, all the other parameters were set to default values.

Enzyme activity assay

The antioxidant enzymatic activities like peroxidase (POD) and superoxide dismutase (SOD) were examined following the protocol described by Wang et al. (2009). Three biological replicates for each treatment were assayed.

RT-qPCR analysis

We used Beacon designer (7.0) to design gene-specific primers (Table S1). Reverse transcription quantitative polymerase chain reaction was carried out as described by Khalil-Ur-Rehman et al. (2017a). Vv actin was used as an internal control for data normalization. The $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\text{t}}}$ method was used for relative quantification. For each treatment, two biological and three technical replicates were performed.

Data analysis

The data analysis was carried out using one-way ANOVA (Tukey’s HSD post hoc test, α < 0.05) by SPSS software (v.19.0.0.0), while Sigma plot 10.0 software was used to plot the graphs. The subcellular localization prediction of the DEPs was carried out by online bioinformatics tool WOLF PSORT (https://wolfpsort.hgc.jp/). Functional enrichment analysis was carried out using Fisher’s exact test.

Results

Induction of bud break in grapevine buds after HC treatment

HC treatment induced a significant increase in bud break in comparison with water-treated samples (Fig. 1). The bud break was 64.33% on the 18th day, followed by 37% on the 12th day in the HC-treated samples. The maximum bud break (73.33%)was observed on the 21st day after HC application.

Fig. 1.

Effect of HC treatment on bud break percentage. Bud break was evaluated at 0, 6, 12, 18, and 21 days post-treatment. Vertical lines above the mean bars indicate standard error (n = 3). The lowercase alphabets represent significant difference between the treatments using Tukey’s HSD post hoc test (p < 0.05)

Proteome profiling of grapevine buds after HC treatment

The analysis quantified 6225 proteins out of 7135 detected proteins. The paired t test comparison between the HC-treated and the control bud samples of all the detected proteins was carried out, and fold change (FC) cut-off was expressed in ratios (HC treated vs. control buds) of > 2 or < 0.5. The proteins, which were greater than 2 FC or smaller than 0.5 (p < 0.05), were termed as significantly differentially expressed proteins (DEPs). The proteins with differential expression patterns were shown in Fig. 2. Most of the proteins exhibited higher expression; however, few proteins exhibited lower expression levels.

Fig. 2.

Expression pattern of differentially expressed proteins in HC-treated and control bud samples. Number of up- and down-regulated proteins were represented in red and black colored bars, respectively. Quantitative ratio over 2 was considered up-regulation while quantitative ratio less than 0.5 was considered as down-regulation (p < 0.05)

Prediction of subcellular localization

To comprehend the function of particular proteins, an online subcellular localization tool, WOLF PSORT, was used to detect the location of each DEP in the cell. The results depicted that all the detected DEPs were localized in 13 cell organelles, viz. chloroplast (419), cytoplasm (347), nucleus (262), plasma membrane (88), mitochondria (37), vacuolar membrane (20), cytoskeleton (19), endoplasmic reticulum (12), and peroxisome (7) (Fig. 3 and Table S2). It was noteworthy that about 82% of the total DEPs were found in three organelles of the cell, i.e. chloroplast, cytoplasm, and nucleus. Among these organelles, the maximum number of DEPs, i.e. about 33% of the total detected DEPs resided in the chloroplast, followed by 28% in the cytoplasm, and 21% in the nucleus.

Fig. 3.

Prediction of subcellular localization of differentially expressed protein in HC-treated and control bud samples. Different cell organelles were divided with different colors and percentage. Maximum number of differentially expressed proteins were localized in chloroplast (33%) while minimum number were found in vacuolar membrane (2%)

Functional enrichment analysis

The identified DEPs were categorized into three main groups, i.e. molecular function, biological process, and cellular component (Fig. 4). The results revealed that the biological process group comprised of DEPs related to response to stress (124, 19.52%), microtubule-based process (41, 6.45%), abiotic stimulus (18, 1.99%), and external stimulus (6, 1.89%). Moreover, the DEPs in the cellular component group were mainly associated with ribosome (178, 89%), cytoskeletal part (35, 17.50%), and supramolecular complex (16, 8%). Lastly, the molecular function group contained the DEPs mostly related to oxidoreductase activity (84, 9.85%), transferase activity (129, 15.14%), heme binding (102, 11.97%), and tetrapyrrole binding (104, 12.20%). In KEGG analysis, it was identified that five pathways were enriched, including “ribosomes,” “starch and sucrose metabolism,” “pyruvate metabolism,” “phenylpropanoid metabolism”, and “circadian rhythm plant”. Furthermore, the metabolic pathway-related proteins were also found to be enriched in the pathway enrichment analysis (Fig. S2).

Fig. 4.

Gene ontology functional enrichment analysis of proteins in HC-treated and control bud samples

Proteins related to stress, hormone signaling, and metabolism

Based on the functional and metabolic classification, the proteins were characterized into different categories: stress and hormone signaling, metabolism, and oxidoreductase activity. In HC-treated vs control buds, a total of 26 proteins exhibited differential expression pattern, among which 12 proteins were related to hormone signaling, 3 for carbohydrate metabolism, 5 for oxidation–reduction and energy metabolism, while 6 for stress conditions (Table 1).

Table 1.

Identification of different proteins in HC treated vs control buds

Protein accessiona	Description hormone signaling	MW(KDa)b	Scorec		Coverage (%)d	Peptidese	HC/control ratio
VIT_01s0011g05230.t01	PREDICTED: protein C2-DOMAIN ABA-RELATED 7 [Vitis vinifera]	18.787	41.35		40.4	5	0.497
VIT_15s0046g01280.t01	PREDICTED: jasmonic acid-amido synthetase JAR1 [Vitis vinifera]	72.474	15.815		5.4	3	0.461
VIT_10s0003g00020.t01	PREDICTED: ABA-inducible protein PHV A1 [Vitis vinifera]	29.676	16.589		15.6	4	0.233
VIT_14s0066g01790.t01	PREDICTED: gibberellin-regulated protein 14 [Vitis vinifera]	31.832	13.165		13.8	4	2.265
VIT_14s0083g00960.t01	PREDICTED: auxin transport protein BIG [Vitis vinifera]	561.92	92.165		6.1	30	0.351
VIT_10s0003g00090.t01	PREDICTED: auxin-repressed 12.5 kDa protein isoform X1 [Vitis vinifera]	13.461	34.558		40.5	4	0.313
VIT_13s0067g01150.t01	PREDICTED: gibberellin 2-beta-dioxygenase 1 [Vitis vinifera]	42.053	11.398		10.2	4	3.576
VIT_07s0031g00710.t01	PREDICTED: ethylene-responsive transcription factor ERF109-like [Vitis vinifera]	20.831	6.2937		7.5	1	0.294
VIT_16s0050g02620.t01	PREDICTED: abscisic acid receptor PYL8 [Vitis vinifera]	20.967	2.9422		13.5	3	0.471
VIT_13s0019g04380.t01	PREDICTED: auxin response factor 18 [Vitis vinifera]	74.823	2.0813		1.8	1	0.292
VIT_19s0014g04690.t01	PREDICTED: indole-3-acetic acid-amido synthetase GH3.6 [Vitis vinifera]	69.415	5.2962		1.8	1	0.413
VIT_13s0067g01940.t01	PREDICTED: abscisic acid receptor PYL4 [Vitis vinifera]	22.97	1.805		4.2	1	0.469
	Carbohydrate metabolism
VIT_16s0098g01780.t01	PREDICTED: starch synthase 1, chloroplastic/amyloplastic [Vitis vinifera]	69.752	12.06		5.5	3	3.511
VIT_03s0038g00370.t01	PREDICTED: fructose-1,6-bisphosphatase, chloroplastic [Vitis vinifera]	39.192	59.039		23.8	6	2.124
VIT_12s0057g00940.t01	Glucose-6-phosphate 1-dehydrogenase	66.321	16.335		19.3	11	0.217
	Stress related proteins
VIT_01s0010g03750.t01	PREDICTED: stress-associated endoplasmic reticulum protein 2 [Vitis vinifera]	7.3915	3.0637		14.7	2	0.451
VIT_08s0007g00130.t01	PREDICTED: heat shock cognate 70 kDa protein 2 [Vitis vinifera]	71.065	87.896		50.4	35	2.043
VIT_16s0050g01150.t01	PREDICTED: heat shock protein 83 [Vitis vinifera]	80.867	22.85		30	23	0.342
VIT_01s0010g00680.t01	PREDICTED: heat shock protein 90–5, chloroplastic [Vitis vinifera]	90.464	323.31		46.6	32	0.222
VIT_07s0005g00170.t01	PREDICTED: stress-related protein [Vitis vinifera]	27.608	38.998		34.7	8	0.227
VIT_12s0134g00430.t01	PREDICTED: universal stress protein PHOS32 [Vitis vinifera]	27.631	47.141		28.5	7	0.486
	Oxidation–reduction and energy metabolism
VIT_18s0001g06360.t01	PREDICTED: alcohol dehydrogenase-like 1 [Vitis vinifera]	42.291	12.555		10.6	4	2.372
VIT_12s0055g00810.t01	PREDICTED: peroxidase 43 [Vitis vinifera]	34.619	16.343		4.6	1	2.425
VIT_06s0004g04310.t01	PREDICTED: alcohol dehydrogenase class-3 [Vitis vinifera]	41.301	4.7435		7.1	3	3.251
VIT_13s0067g02360.t01	PREDICTED: peroxidase 4-like [Vitis vinifera]	34.419	75.835		31.8	8	0.491
VIT_07s0005g00010.t01	PREDICTED: glutathione S-transferase [Vitis vinifera]	23.625	15.13		29.4	5	4.103
	Protein metabolism
VIT_08s0040g02330.t01	Elongation factor 1-alpha	49.335	20.96		43.8	25	2.126

^aProtein accession number of database used for search

^bMolecular weight of protein

^cTo check the significance of the result

^dPercent of identified peptide sequence covering in a protein sequence

^eNumber of identified peptides

Based on these results, we can speculate that all these proteins directly or indirectly play a vital role in dormancy break. Additionally, the proteins related to hormone signaling, energy metabolism, and redox process might cause more impact as compared to other proteins after the HC application.

Cluster analysis of DEPs

The DEPs were further deeply analyzed and categorized into three groups: molecular function, biological process, and cellular component. In the biological process, the proteins associated with the oxidoreductase coenzyme metabolic process were highly expressed but those related to phototransduction exhibited lower expression in HC-treated buds as compared to those of control. In the cellular component category, the ribosome, microtubule and ribonucleoprotein complex-related proteins exhibited down-regulation, while supramolecular complex- and cytoskeleton-related proteins revealed increased activity in HC treated as compared to those of control buds. Finally, in the molecular function category, the proteins related to coenzyme binding and lyase activity showed increased levels in the HC-treated buds as compared to the control (Fig. S3).

Activity of antioxidant enzymes

The results demonstrated that after the HC application, the POD activity was higher after 6 and 12 DPT but repressed at later time points, i.e. 18 and 21 DPT. Overall, the SOD activity remained significantly higher in the HC-treated samples as compared to the control during the whole forcing period. These results indicated that the antioxidant system had relations with the grape bud dormancy release after the HC application (Fig. 5).

Fig. 5.

Antioxidant enzymatic activity assay a POD and b SOD in HC-treated and control bud samples. Vertical lines above the means bars indicate standard deviation (n = 3). The a–b depicts the significant difference between two treatments

Validation of proteomic data by RT-qPCR

To examine the expression pattern of proteins and to verify their proteomic analysis output, the real-time quantitative polymerase chain assay was performed to analyze the expression pattern of nine selected genes related to hormone signaling, energy metabolism, protein metabolism, and redox activity. The gene and protein expression patterns showed similar results, which authenticated the reliability of the proteome data (Fig. S4).

Discussion

Hydrogen cyanamide is commonly used agrochemical that stimulates bud break in deciduous fruit plants. We examined the variations in the expression patterns of proteins before and after HC treatment in grapevine buds using TMT-based proteomic analysis. Various studies proposed that adequate energy is the prerequisite for bud meristem to retain bud growth at the time of bud break. We observed that several detected proteins were linked with sugar metabolism, hormone signaling, oxidative stress, and energy metabolism. Among several proteins, alcohol dehydrogenase class-3 and alcohol dehydrogenase like-1 were specifically related to the dormancy release of grape buds and were part of energy metabolism (Table 1). Previous reports found that alcohol dehydrogenase was stimulated under respiratory stress in HC-treated grape buds (Or et al. 2000). In HC-treated buds, in contrast to those of control, the proteins associated with energy metabolism were differentially expressed. HC influenced the activity of proteins related to energy metabolism that might play an important role in the early bud break of grapevine.

Sugars and carbohydrates are considered as primary energy sources and are widely consumed by living organisms. Our data revealed that proteins related to sugar metabolism including VIT_16s0098g01780.t01 (starch synthase 1, chloroplastic/amyloplastic), VIT_03s0038g00370.t01 (fructose-1,6-bisphosphatase, chloroplastic) and VIT_12s0057g00940.t01 (Glucose-6-phosphate 1-dehydrogenase) exhibited differential expression patterns in HC-treated buds as compared to those of control. Hence, HC stimulated the activity of proteins associated with sugar metabolism that might be involved in early dormancy release in grapevine. Therefore, we can speculate that proteins related to sugar metabolism might be involved in dormancy regulation.

Proteins associated with protein synthesis are required for cell division and can be utilized as a signaling molecule for meristematic tissue state (Chang et al. 2018). In the current study, the elongation factor 1-alpha showed higher expression in the HC-treated bud samples, which indicated the involvement of this protein in the dormancy release of grapevine after the HC application. Elongation factor 1 (EF-1) is essential for cell division and protein synthesis in meristematic tissues (Zhuang et al. 2013). A previous report by Pawłowski et al. (2007) proposed that EF-1 might be associated with cell division, proteins synthesis of root meristem, and dormancy release of beech seeds. Elongation factor 1-alpha exhibited higher expression in the HC-treated buds. In the present study, three heat shock proteins, including VIT_08s0007g00130.t01 (heat shock cognate 70 kDa protein 2), VIT_16s0050g01150.t01 (heat shock protein 83), and VIT_01s0010g00680.t01 (heat shock protein 90-5, chloroplastic), exhibited differential expression pattern in the HC-treated buds as compared to those of control (Table 1). A previously published study illustrated that the heat shock proteins protected against oxidative injury in beech seeds germination (Chang et al. 2018). Therefore, the results of this study depicted that metabolism-related proteins might be involved in grape bud dormancy release after HC application.

A particular type of antioxidant enzyme defense system exists in plants containing superoxide dismutase (SOD), peroxidase (POD), and other enzymes. The scavenging of reactive oxygen species (ROS) is performed by the antioxidant defense system against oxidative stress (Chang et al. 2018). Several studies demonstrated that proteins related to POD and SOD were essential in the dormancy release of many perennial woody plants (Or et al. 2000; Halaly et al. 2008). A previously published study revealed that SOD plays a key role in the antioxidant defense process under adverse environmental conditions, including drought or low temperature (Cai et al. 2019). In the current study, three main proteins (peroxidase 4-like, peroxidase 43, and glutathione S-transferase) were recognized that were associated with H₂O₂. Variations in enzymatic activities were also be observed in the HC-treated and the control buds (Fig. 5). Our findings were in agreement with a previous study (Ben Mohamed et al. 2012). The proteins related to the antioxidant defense system might be essential for the dormancy maintenance and release process.

Oxidative stress is considered as an essential element for the dormancy release process. Antioxidant activities that comprise the POD family, SOD, and ascorbate peroxidase (APx) family proteins play a crucial role in the dormancy release (Mazzitelli et al. 2007; Halaly et al. 2008; Zhuang et al. 2013). This study revealed that the proteins related to the POD family (VIT_12s0055g00810.t01), (VIT_13s0067g02360.t01), and (VIT_18s0072g00160.t01) showed differential expression pattern in the HC-treated buds, as compared to those of control (Table 1). A previously published report identified that bud break corresponds with the elevated levels of the antioxidant defense system (Mazzitelli et al. 2007). H₂O₂ is generated by NADH as a result of POD activity, which exhibited higher expression in peach buds (Prassinos et al. 2011). The results of this study also showed that the proteins related to oxidoreductase activity were highly expressed in the HC-treated buds, as compared with those of control, which were in agreement with the aforementioned studies. A previous study noticed a transitory expansion in the levels of H₂O₂ before dormancy release in the HC-treated grapevine buds (Pérez and Lira 2005). Another earlier published study (Prassinos et al. 2011) proposed that a transient increase in the levels of H₂O₂ prior to dormancy break might act as signaling molecules to intricate the shift from dormant to an active state. In the current study, three proteins related to oxidoreductase activity showed differential expression patterns in the HC-treated buds, as compared to those of control. So, it is suggested that oxidative stress might be associated with dormancy release in deciduous fruit plants. During the transition from a dormant to an active state, the expression levels of stress signaling proteins showed down-regulation, especially in the HC-treated buds, as compared to those of control. Moreover, the majority of the heat shock proteins exhibited lower expression levels in the HC-treated buds, in comparison with those of control. A previous study (Ueno et al. 2013) illustrated that the heat shock proteins showed higher expression levels in oak trees. Such stress signaling proteins might be involved in environmental adaptation and dormancy regulation in perennial plants.

Hormone signaling is associated with transitioning from a dormant to an active phase, involving the effect of low temperature, under natural conditions (Druart et al. 2007). Literature review revealed that ABA involvement in bud development is yet to be classified (Rohde and Bhalerao 2007; Rohde et al. 2007). According to our results, the expression patterns of ABA signaling proteins (VIT_01s0011g05230.t01; VIT_10s0003g00020.t01; VIT_16s0050g02620.t01, and VIT_13s0067g01940.t01) showed downregulations in the HC-treated buds, in contrast to the control. The results in Table 1 demonstrated that the proteins associated with the gibberellin pathway exhibited higher expression, while those related to IAA/Auxin biosynthesis pathway showed downregulation in the HC-treated buds as compared with those of control. Taken together, these results suggested that the hormone signaling pathways might link with dormancy release after HC treatment in grapevine buds. Further validation of proteins linked with dormancy release after HC application is required in future studies.

Conclusion

The alteration of grapevine buds from a dormant to an active state stimulated by HC was associated with numerous signaling and metabolic processes. Moreover, the majority of differentially expressed proteins were associated with hormone signaling, sugar metabolism, stress, and energy metabolism. Furthermore, the hormone signaling and stress-related proteins showed notable variations during dormancy release after HC treatment. Finally, the diversified metabolic changes took place during bud break after HC application in grapevine buds. However, functional validation of crucial proteins is yet to be investigated.

Electronic supplementary material

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Abbreviations

HC

Hydrogen cyanamide

TMT

Tandem mass tag

DEPs

Differentially expressed proteins

DPT

Days post-treatment

POD

Peroxidase

SOD

Speroxidedismutase

ABA

Abscisic acid

HPLC

High-performance liquid chromatography

FC

Fold change

EF

Elongation factor

Author contributions

MKR and JMT designed the study. MKR performed the experiment and wrote the manuscript. WW and SI collected the samples. MKR, ZGS, and HZ analyzed the data. MF provided technical discussion and edited the manuscript draft. All authors approved the final version of manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.