Introduction

Grafting, where tissues from one plant genotype are inserted into those of another genotype, so that the two sets of vascular tissues form a unified set (i.e., viable grafted plant), is widely used for breeding (DongKum et al. 2013), variety renewal (Sabbatini and Howell 2013), germplasm conservation (Benelli et al. 2013), as well as determining the genetic stability of plants (Jaganath et al. 2014). It is also employed in plant propagation, including trees (Sanou et al. 2004; Mencuccini et al. 2007), vegetables (Kubota et al. 2008), and flowers (Ginova et al. 2012). Moreover, it is an important method applied in studies addressing shoot–root physiological relationships (Sakamoto and Nohara 2009; Han et al. 2013), resistance mechanisms (Sugawara et al. 2013), material transport (Lin et al. 2007; Flaishman et al. 2008; Zhang et al. 2012), flowering regulation (Yoo et al. 2013), and long-distance signal transmission mechanisms (Chen et al. 2006; Banerjee et al. 2009).

The nurse seedling grafting is a shoot grafting method characterized by the use of young seedlings, with or without leaves, as the rootstock onto which half-lignified branches (scions) are grafted (Moore 1963). Young shoot tissue is particularly well suited for grafting due to its high cell division activity and the high affinity with which several functional phloem and xylem tissues of the scion-rootstock can connect across the graft surface (Gökbayrak et al. 2007). Nurse grafting can make the grafting body easier to survive, thus suitable for propagation of endangered plants and commercial breeding (Sui 2006; Fuentes et al. 2014). The shoot grafting technique has been applied to a number of plant species, such as camellia (Moore 1963), avocado (Whiley et al. 2007), chestnut (Duman and Serdar 2006), ginkgo, and oak (Park 1968), with camellia as the most extensively studied species.

Successful grafting starts by the healing process between the rootstock and the scion. Healing is capable of initiating such cellular responses by triggering various intracellular signaling events (León et al. 2001; Minibayeva et al. 2015; Sophors et al. 2016). Grafting generates an impulse to elicit healing mechanism that generates biological response (isolation layer formation, callus differentiation, callus proliferation and connection, cambium differentiation and connection, and conducting tissue differentiation and connection) (Estrada-Luna et al. 2002; Fan et al. 2015). Studies conducted on graft-healing mechanism focused on determining the quantities of various biochemical substances in the scion (Pina and Errea 2005; Aloni et al. 2008; Muneer et al. 2016), enzyme activity (Zarrouk et al. 2010), and endogenous hormone content (Aloni et al. 2008; van Hooijdonk et al. 2011; Yin et al. 2012), all of which vary with the healing stage; however, signaling pathways of grafting healing was seldom reached.

MAPK signaling pathways are known to play a central role in cell proliferation, differentiation, apoptosis, and development (Weihs et al. 2014), and belong to the extracellular signal-regulated kinase (ERK) subfamily (Wang et al. 2011). Multiple MAPK pathways exist in one cell, and each pathway is linked to different upstream signals and downstream substrates. These pathways function independently and interlink to form a complex signal transduction network (Wang et al. 2015). Calcium is also an important second messenger in plant signaling networks (Shi et al. 2014), to response developmental and environmental stimuli representing signal information to distinct biological responses (Ranty et al. 2016; Wang et al. 2016). The calcium signaling occurs by crosstalk of calcium sensitivity, calcium sensors, and downstream target proteins, and interacts with MAPK signaling pathways (Chuderland and Seger 2008; Liu et al. 2014b).

Recently, evidence for MAPK cascade pathways and calcium signal has been found in the same environmental stimuli, such as drought, cold, wounding, and so on (Cheong and Kim 2010; Shi et al. 2014). However, the knowledge of whether and to what extent MAPK cascade pathways and calcium signaling are involved in graft-healing remains unclear. In the present study, we used camellia (Camellia oleifera) young shoots as grafting material to study MAPK cascade pathways and calcium signaling underlying the nurse seedling graft-healing processes. In doing so, we focused on changes in anatomy, physiology, biochemistry, and protein expression with the ultimate objective of improving the shoot grafting method for its use in both production and research applications.

Materials and methods

Experimental materials

Camellia oleifera fruits were collected from a single superior clone (Min48) growing at the Minhou Tongkou State Forest Farm, Fujian Province, China (26°09′, 119°14′) and were placed in a ventilated room until they naturally opened. Large, plump, shiny seeds were selected and stored indoors in clean, dry river sand until use (the following spring (March)). Seeds were germinated in wet sand that was previously treated with a 1000–2000× thiophanate solution. The sand and seeds were placed in alternating single layers (≈10 cm thick), and they were watered at 4–5-day intervals. The germinated seeds were used as grafting rootstock only after reaching a height of ≈3 cm (Fig. 1a). The germinated shoot tips were cut, and the stems, which were ≈1.5 cm long, were used as rootstock (Fig. 1b, c). The seedling roots were trimmed to approximately 6 cm in length. Robust semi-woody branches from the same plants (i.e., homograft) were employed as scions (≈2–3 cm long) for cleft grafting (Fig. 1d, e). The seedling nursery site was fertilized with farmyard manure at rate of 1000 kg per acre. The seedbed was 1.0 m wide and 0.15 m high with a surface layer of yellow soil that was covered with a plastic film (Fig. 1f). A sun shelter was installed before grafting with a height of 2 m to provide shading rate of 70–80%.

Fig. 1
figure 1

Camellia oleifera nurse seedling grafting steps: a rootstock cultivation, b cutting off part of the shoot and root in rootstock, c splitting the stem of rootstock, d prepared scion, e grafted plants, and f transplanting to seedbed

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Experimental design

In total, 10,000 seedlings were homo-grafted, and planted in the seedbed at a spacing of 2–3 and 10–14 cm within and between rows, respectively. The soil was compacted and soaked with water during planting, and the seed case (autotrophic nutrition source) was exposed on the surface before being covered with a transparent plastic film. The temperature inside the greenhouse was maintained at 28–30 °C.

After the grafted plants were transplanted, random samples were collected at 2-day intervals (days 0–26), after which sampling commenced at 3-day intervals (days 29, 32, and 35), followed by 5-day intervals (days 40 and 45), and finally one additional sample after 10 days (day 55), representing a total of 20 samples. Each sampling date included a total of 300 grafted seedlings. The grafted seedling samples were washed under clean running water, and 1–1.5 cm sections of the stems, including the graft junction, were cut and stored in Ziploc® bags. Sampled seedlings were used for: (1) measurement of enzyme activity, hormone levels, and the abundance of various proteins (frozen in liquid nitrogen and stored at −70 °C), (2) measuring soluble sugar content, cellulose, the chlorogenic acid content, and the relative conductivity rate (stored at −20 °C), and (3) anatomy assays (fixed with a formalin/acetic acid/alcohol (FAA) solution less than 48 h after collection).

Measurement methods

Before each sampling event, withered leaves or scions were discarded. The mortality rate of the grafted seedlings was calculated using the following formula:

$$ p_{i} = n_{i} /\left( {N - \sum {n_{i - 1} - \sum {A_{i - 1} } } } \right) \times 100, $$

where p i is the dynamic mortality rate of grafted seedlings; N is the total number of graft recruits (N = 10,000); and i is the times of sampling (i = 1–20); A i is the number of recruits in each grafted sampling (i = 1–20); and n i is the times of grafted seedling with withered leaves or scions (i = 1–20). After each sampling event, 50 randomly selected grafted seedlings were weighed, and the weight gain was denoted as seedling growth.

Soluble sugar and cellulose contents were determined using the anthrone method (Abidi et al. 2010). This is done by 0.5-mL 2% anthrone solution in 5-mL concentrated sulfuric acid to an aqueous solution of grafting union. The absorbance of the green color of the solution is measured using a UV–Vis spectrophotometer SP-756 (Shanghai Spectrum Instruments Company, China) at 620 and 630 nm and it is proportional to the cellulose content and soluble sugar of the sample, respectively. Microcrystalline cellulose and sugar were used as standards for the calibration. All the measurements were repeated three times.

Chlorogenic acid content was measured with a spectrophotometer (Prigent et al. 2003) using ten graft unions. The samples were weighed, ground in liquid nitrogen, transferred to 10-mL centrifuge tube containing 5 mL 80% methanol, placed in refrigerator at 4 °C after shaking extraction for about 4 h, and centrifuged at 3500 r/min at 4 °C for 8 min, after which the supernatant passed through C-18 solid-phase extraction column (column Steps were 80% methanol, 100% methanol, 100% diethyl ether, and 100% methanol cycle) using 80% methanol as a control and absorbance was measured using 756 UV–visible spectrophotometer at 324 nm wavelength. The absorbance measure is proportional to the chlorogenic acid concentration in the sample after using chlorogenic acid as a standard for calibration. All the measurements were repeated three times.

Electrical conductivity of the collected samples (1.0 g fresh weight) was determined with a Mettler Toledo Electric Conductivity Meter (Delta 326, precision ±0.5%), Samples were cut to equal size pieces, immersed in test tubes with 5-mL distilled water, vacuumed for 20 min, set aside for 1 h at room temperature, during which the tubes were shaken several times. Measurements were recorded as S1 data, and then, the tubes were immediately immersed in boiling water at 100 °C for 10 min, followed by cooling until reaching room temperature. The electrical conductivity was re-assayed and recorded S2 data. All the measurements were repeated three times. The relative electrical conductivity rate was calculated by dividing S1 by S2.

Anatomy assay samples were transferred to a 50% acetone solution for 24 days for tissue softening. Sections of 0.5 cm in length were immersed in an improved Kano fixative (70% alcohol/glacial acetic acid, v/v, 3:1); subjected to vacuum for 20 min; fixed for 24 h; washed with water; dyed with 20% ammonium ferrous sulfate for 30 min; rinsed with water; dyed with hematoxylin, eosin, and 2% fuchsin basic for 24 h; washed with water; treated with ammonia for 5 min for contrast; and then washed with water. The materials were then cut into 10-μm slices via the conventional paraffin slice method. An Optec BDS200-FL inverted biological microscope was used to observe the slices, and photographs were taken with a Canon A650 camera.

For enzyme activity determination, a sample (1.0 g fresh weight) which contained about ten graft unions was placed in a precooled mortar, mixed with a small amount of quartz sand and 5 mL of phosphate buffer solution, and ground to a slurry in an ice bath. The extract was centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was transferred to a test tube and stored in a refrigerator. Superoxide dismutase (SOD) activity was determined using the tetrazolium (NBT) photoreduction method with phosphate buffer solution (0.05 mol/L, pH 7.5); catalase (CAT) activity was calculated using the H2O2 method with phosphate buffer solution (0.05 mol/L, pH 7.0) (Basha and Rani 2003); peroxidase (POD) activity was measured using the guaiacol method with phosphate buffer solution (0.05 mol/L, pH 7.0) (Zhang et al. 2005); and l-phenylalanine ammonia-lyase (PAL) activity was determined via the production of cinnamate with phosphate buffer solution (0.1 mol/L, pH 8.8) (Cheng and Breen 1991). Polyphenol oxidase (PPO) activity was assayed according to the pyrocatechin method with phosphate buffer solution (0.1 mol/L, pH 6.8) (Ziyan and Pekyardimci 2004). All measurements parameters were performed in triplicate.

To determine hormone levels, a sample (1.0 g fresh weight) was ground to a slurry in a mortar with 2 mL of sample extraction solution (80% methanol, 1 mmol/L butylated hydroxytoluene, BHT), homogenized in an ice bath, and transferred to a 10 mL test tube. The mortar was then washed with an additional 2 mL of the sample extraction solution, which was added to the sample in the test tube, mixed, and leached at 4 °C for 4 h. The extract was subsequently centrifuged at 3500 rpm for 8 min, and the supernatant was collected. The precipitate was mixed with 1 mL of the sample extraction solution and leached at 4 °C for 1 h. After centrifugation at 3500 rpm for 8 min, the supernatant was combined with the previously collected supernatant, after which the total volume was recorded, and the residue was discarded. The supernatant was then purified using a C18 solid-phase extraction column. Hormone levels were measured using an enzyme-linked immunosorbent assay (ELISA) (Bai et al. 2011). Experiments were carried out in triplicate.

The trichloroacetic acid (TCA)/acetone method was employed to extract proteins from the different developmental stages of grafted unions which were collected on 4, 8, 16, 22, 29, and 35 days after grafting, and protein concentrations were determined using the Bradford method (Wei et al. 2009). Proteins were analyzed via two-dimensional electrophoresis (2-DE) (Larsen et al. 2001), and the resultant 2-DE protein maps were analyzed with Image Master TM 2D Platinum software v 7.0. Experiments were carried out in duplicate. Spots of the levels of the differentially expressed proteins were mapped back to and picked from the duplicate 2D gel, and analyzed using a flight tandem mass spectrometer (4700 Proteomics Analyzer; Applied Biosystems, USA). The laser source was an Nd:YAG laser with a wavelength of 355 nm and an acceleration voltage of 20 kV. The data were sampled using the positive ion and automatic data acquisition modes in the analysis. The peptide mass fingerprinting (PMF) scan range was 700–3500 Da, and the five highest intensity peaks were analyzed via tandem mass spectrometry (MS/MS). The spectra were calibrated with external standard calibration digested myoglobin peptides. Database searches were performed to identify differentially expressed proteins using GPS (Applied Biosystems, USA) and MASCOT (Matrix Science, UK) software. The applied search parameters were as follows: database NCBInr; the retrieval species were Viridiplantae (Green Plants); the data retrieval method was combined; the maximum allowable leakage cut locus was 1; the enzyme was trypsin; the quality error range setting was PMF 100 ppm; MS/MS was 0.6 Da; and peaks of trypsin degradation products and pollutants were removed manually during database retrieval. The protein function was determined using the freely accessible NCBInr database associated with proteins in a proteomics results ID list.

Data analyses

The differences in physiological and biochemical parameters in grafting unions were analyzed with one-way analysis of variances (ANOVAs), with time as the independent factor. Comparisons among mean parameter were further determined using Duncan HSD post hoc tests. In addition, correlations between all physiological and biochemical parameters were explored using Pearson product-moment correlations. The correlation between different proteins was also explored by the same method. A principal component analysis (PCA) was also performed for mean physiological and biochemical parameters and different proteins using the following steps: (1) input data were standardized, (2) correlation coefficient matrix R was determined, (3) eigen value of the matrix R was determined, (4) principal component loading matrix was determined based on the standard criteria of an eigen value ≥1 and a cumulative contribution rate ≥80% (Pizzeghello et al. 2011; Reed et al. 2013) to select the relevant principal components, (5) calculate the weight of times, and (6) main parameters of each principal component based on factor loading ≥0.20 were selected and ranked from high to low, and ended at variance contribution rate ≥70% (the total of selected variable variance contribution/the total of all variable variance contribution × 100%). All data were analyzed using the statistical software SPSS (version 18.0).

Results

Developmental stages of camellia shoot graft-healing anatomy

The shoot graft-healing anatomy showed five distinct developmental stages that characterized by: (1) the formation of a dark necrotic tissue layer caused by the mechanical damage during surface cutting which acted as an isolation layer at the wound surface (4 days from grafting; Fig. 2a), (2) the parenchyma cells of the rootstock near the wound, cambium, and pericycle had dedifferentiated and recovered the ability to divide resulting in the formation of a callus (Ci) (8 days after grafting; Fig. 2b), (3) the parenchyma cells under the isolation layer at the scion wound had undergone differentiation into a callus (Ci) that was divided and expanded (Fig. 2c; 16 days after grafting), (4) the callus tissue continued proliferation and filled the junction and creating a callus bridge between the rootstock and the scion (22 days from grafting; Fig. 2d). This progress ensued by the formation of new vascular cambium cells at the edge of the newly formed callus that were differentiated from the parenchyma cells adjacent to vascular cambium of both the scion and the rootstock, crossed the callus between the scion and the rootstock, and fused (29 days from grafting; Fig. 2e), and (5) finally, the cambium or parenchyma cells differentiated into new vessels and sieve tubes, which reconnected the vessels and sieve tubes damaged by grafting, resulting in the formation of vascular bundles and successful completion of the graft-healing process (35 days from grafting; Fig. 2f).

Fig. 2
figure 2

Anatomical analysis of graft union development in C. oleifera. a Isolation layer (IL) of dark necrotic tissue formed at the wound surface production stage (4 days after grafting), b rootstock callus differentiation stage with parenchyma cells near the rootstock (St) wound dedifferentiated to form a callus (Ci) (8 days after grafting), c scion callus (Sc) differentiation stage with parenchyma cells at the scion wound differentiated to form a callus (Ci) (16 days after grafting), d callus proliferation and connection stage with a callus (Ci) bridge formed between the rootstock and scion (22 days after grafting), e vascular cambium (Vc) differentiation and connection stage at the edge of the newly formed callus, 3–4 layers of long, flat vascular cambium cells differentiated from the parenchyma cells (29 days after grafting), and f conducting tissue [vascular bundle (Vb)] differentiation and connection stage with the cambium differentiated into a new vessel and sieve tube and reconnected the vessel and sieve tube damaged by grafting, resulting in healing of the grafting union (35 days after grafting)

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Physiological and biochemical profiles of grafting union development

The analysis of variance identified 15 physiological and biochemical parameters during the development of nurse seedling grafting, all of which were significantly affected by grafting union development (P < 0.001). Similarly, the mortality rate also substantially varied and ranged from 0.0 to 5.1% (Table S1). There were peak physiological, biochemical, and mortality rate values at different times after grafting; weight (0 day), ZR (8 day), cellulose, PAL, PPO, and mortality rate (16 day), POD (18 day), CAT (20 day), relative conductivity (22 day), soluble sugar (24 day), SOD (26 day), IAA (29 day), chlorogenic acid content and GA (32 day), and ABA (35 day) (Table S1).

Principal components analysis (PCA) of physiological and biochemical parameters during camellia shoot graft-healing

The first five principal components (PC1–5) of the 15 studied attributes (physiological, biochemical, and mortality rate) during the camellia shoot graft-healing period accounted for 80.92% of the total variation (Table 1). The main parameters of each PC were selected based on their variance contribution [parameters were identified from high to low with cumulative variance contribution reaching ≥70% of the total variance (cumulative variance contribution of the selected variable/the total of all variable variance contribution × 100%)]. PC-1 was mainly influenced by POD, soluble sugars, cellulose content, relative conductivity, chlorogenic acid content, and mortality rate with positive contribution, while seedling weight had negative contribution (Table 1). The cumulative variance contribution of these parameters amounted to 75.35% and accounted for 34.51% of the total variation (Table 1). PC-2 was mainly influenced by GA, ABA, and soluble sugars with positive contribution, and ZR and cellulose content with negative contribution, cumulatively accounting for 70.74% and representing 18.23% of the original data variation (Table 1). PC-3 accounted for 11.80% of the original variation, and was positively and negatively affected by PPO and chlorogenic acid contents, and CAT and SOD, respectively, with cumulative variance contribution of 79.63% (Table 1). PC-4 accounted for 10.05% of the original variation and was influenced by IAA, SOD, and relative conductivity with positive contribution and PAL and mortality with negative contribution, all with cumulative variance of 89.19% (Table 1). Finally, while PC-5 had an eigen value of <1.0 (0.949), the cumulative variance contribution of PC1–5 accounted for 80.92 of which 6.33% are attributable to PC-5, and thus, it is an important element for the studied 15 attributes detected during the camellia shoot graft-healing period. PC-5 was affected by IAA, morality rate, and cellulose content with positive contribution and ABA and chlorogenic acid content with negative contribution, all representing a cumulative variance contribution of 72.47% (Table 1).

Table 1 PCA (PC1–5) of physiological and biochemical parameters during the healing process of camellia shoot grafts
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Protein profile and PCA results during the camellia shoot graft-healing process

Using two-dimensional electrophoresis (2DE) and MALDI-TOF-TOF/MS method, a total of 38 differentially expressed proteins were detected from six total proteins at six development stages (4, 8, 16, 22, 29, and 35 days after grafting) during graft-healing process, and identified functions by compared with NCBInr database (Table 2).

Table 2 Relative abundance changes of differentially protein during nurse seedling grafting development (protein function obtained from NCBInr database)
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PCA analysis conducted on the 38 different proteins measured during the six developmental stages of the shoot graft-healing process and based on the standard criteria of an eigen value ≥1 and a cumulative contribution rate ≥80%, identified the first three principal components (PC1–3) as essential and accounted for 85.87% the total variation (Table 3). Main parameters of each PC were selected based on their variance contribution and ranked from high to low with a cut-off factor loading of <0.20, and no-function proteins. PC-1 accounted for 39.59% of the total variation and was positively influenced by calcium-dependent protein kinase (CDPK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), triosephosphate isomerase (TPI), chalcone isomerase (CHI), and fructose-1,6-diphosphate aldolase (ALD), while glutathione transferase (GST), bromodomain protein (BRD), aldehyde reductase (AR), and phenylcoumaran benzylic ether reductase (PCBER) contributed negatively, all with cumulative variance contribution of 63.93% (Table 3). PC-2 accounted for 25.63% of the total variation and was positively affected by mitochondrial ribosomal protein (MRPs), kinesin (centromeric protein)-like protein (CENP), polyubiquitin, heat shock proteins (HSP), membrane-binding steroid protein (MARRS), and negatively by peptidyl-prolyl cis–trans isomerase-1 (PPI-1), selenium proteins, germination-like protein, auxin-induced proteins, and oxidative steroid-binding proteins (OSBP), all with cumulative variance contribution of 69.18% (Table 3). PC-3 was associated with 20.65% of the total variation and was positively influenced by cytokinin synthase, copper/zinc superoxide dismutase (Cu–Zn-SOD), translationally controlled tumor protein (TCTP), MARRS, NAD kinase, annexin, peptidyl-prolyl cis–trans isomerase-2 (PPI-2), and retrotransposon protein, while PI3K contributed negatively, all with cumulative variance contribution of 59.08% (Table 3).

Table 3 PCA (PC1–3) of the contents of different proteins during the camellia shoot grafts healing process (protein function obtained from NCBInr database)
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Discussion

Calcium signal in the vascular bundle development during the camellia shoot graft-healing process

The PC-1 (Table 1) scores were positive between day 14 and 35 and associated with rootstock and scion callus formation, callus connection, and vascular bundle differentiation stages (Table 4; Fig. 2). The PC-1 (Table 3) scores were positive between days 8 and 33 during this time which the union was in the callus formation and vascular bundle differentiation stage (Figs. 2, 3). These indicated that PC-1 (Table 1) and PC-1 (Table 3) were related to the development of the vascular bundle of grafting union. The 7 main indicators of the 17 main indicators in PC-1 (Table 1) and PC-1 (Table 3) (41%) directly were related with calcium signal [POD (Erinle et al. 2016), cellulose content (Lin et al. 2016), chlorogenic acid content (Ngadze et al. 2014), CDPK (Fu et al. 2013), CHI (Li et al. 2004), GST (Dulhunty et al. 2001), and BRD (Zhou et al. 2004)]. Therefore, after grafting, vascular bundle development at the scion may depend on biological responses in which intracellular calcium acts as a second messenger.

Table 4 PCA scores of physiological and biochemical parameters during the healing process in camellia shoot grafts
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Fig. 3
figure 3

PCA score diagram for different proteins during the healing process of C. oleifera shoot grafts

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In response to grafting, the calcium concentration changed, activating CDPK and the subsequent phosphorylation of the corresponding substrates to cause a signal transduction cascade (Fu et al. 2013). Cell proliferation was promoted by the inhibition of BRD activity in response to calcium signal stimulation (Zhou et al. 2004; Korner and Tibes 2008; Sterner et al. 1999). It is qualified by the presence of two indicators (CDPK, positive contribution; BRD, negative) in PC-1 (Table 3). Then, intracellular calcium can increase the glycolytic pathway (Vaz et al. 2016; Hoque et al. 2012). Glucose (soluble sugars) can be degraded into dihydroxyacetone phosphate (DHAP) and GAPDH in the presence of ALD. At the same time, DHAP is converted into phosphoglycerate (PGA) in the presence of TPI and GAPDH (Ikemoto et al. 2003). PGA is converted into phosphoenolpyruvate (PEP) with entering into second metabolism (Facchinelli and Weber 2011). Calcium can increase the second metabolism, to produce the chlorogenic acid (Ngadze et al. 2014), such as cinnamic acid and coumaric acid (Clifford et al. 2003). Cinnamic acid was dimerized to lignans (Heinonen et al. 2001), and then reduced by PCBER to other secondary metabolites, such as phytoalexins and heartwood-protective substances (Min et al. 2003). In the presence of POD, lignin and other secondary metabolites participate in cell wall formation, thereby contributing to the development of vascular bundles (Kong et al. 2013). In contrast, coumaric acid is converted to flavonoids in the presence of CHI (Dao et al. 2011). These were qualified by four positive indicators (POD, soluble sugars, cellulose content, and chlorogenic acid content) in PC-1 (Table 1), and by five indicators (GAPDH, TPI, ALD, and CHI; positive; PCBER, negative) in PC-1 (Table 3).

When GST activity reduced, flavonoids increased, and the cell differentiation can be promoted (Kampranis et al. 2000; Laborde 2010), with releasing the material that can be used as carbon source to the new cells, but that also can be transformed to the alcohols by AR. Therefore, reducing the AR can benefit the cell proliferation (Middleton et al. 2000). On the other hand, flavonoids reduce intracellular calcium ion levels, thus activating CDPK (Middleton et al. 2000), thereby contributing to sustained cell proliferation and growth. These were certified by GST and AR that were negative contribution in PC-1 (Table 3).

MAPK signaling pathway in callus proliferation during the camellia shoot graft-healing process

The PC-2 (Table 3) scores were positive between days 8 and 22, a time representing the callus formation stage. The maximum values of PC-2 occurred on days 16 a time associated with rapid callus formation (Figs. 2, 3). The PC-3 (Table 1) scores were positive between day 2 and 4 (coinciding with the isolation layer stage), days 10 and 12 (a rapid callus formation of rootstock stage), and days 20 and 26 (rapid callus formation of scion and rootstock) (Table 4; Fig. 2). The PC-5 (Table 1) score fluctuated dramatically throughout the shoot–rootstock healing development period with high scores corresponding to the five grafting steps (expect vascular bundle differentiation), and with before and after cells growth of callus (Table 4; Fig. 2). Therefore, PC-2 (Table 3), PC-3 (Table 1), and PC-5 (Table 1) were related with callus proliferation during the camellia shoot graft-healing process. The main indicators of them took part in cell proliferation. The 10 main indicators of 18 main indicators in PC-2 (Table 3), PC-3 (Table 1), and PC-5 (Table 1) (56%) directly were related with MAPK signaling pathway [polyubiquitin (Chen et al. 2015), HSP (Svensson et al. 2013), MARRS (Wagatsuma and Sakuma 2014), PPI-1 (Zhimin and Hunter 2014), selenium proteins (Bi et al. 2016), OSBP (Weber-Boyvat et al. 2013), PPO, CAT and IAA (Zheng et al. 2015), and ABA (Wang et al. 2005)]. Therefore, after grafting, callus proliferation may depend on biological responses in MAPK signaling pathway during the camellia shoot graft-healing process.

When cells are stimulated by grafting, OSBP interacts with membrane cholesterol, resulting in increased extracellular signal-regulated kinase (ERK) levels which change in cellular calcium concentration (Weber-Boyvat et al. 2013), corresponding signaling cascade by the ubiquitination process, and regulating auxin-induced protein (Chen and Kao 2012; Dharmasiri and Estelle 2004; Schroepfer 2000). By reducing auxin-induced protein, this can increase the levels of free IAA and the polyubiquitin (Dharmasiri and Estelle 2004). And PPI-1 activity was decreased by the ubiquitylation (Zhimin and Hunter 2014). These were confirmed by three main indicators (polyubiquitin, positive contribution; PPI-1 and auxin-induced proteins, negative) in PC-2 (Table 3), and by IAA as main positive indicator in PC-5 (Table 1).

With the deceasing PPI-1 activity, MARRS activity was increasing (Bettoun et al. 2002; Farach-Carson and Nemere 2003; Zhimin and Hunter 2014). In addition, selenium proteins, as MARRS receptor (Schütze et al. 1998), can promote the cell proliferation (Zeng 2009; Zhang et al. 2013), and, on the other side, regulate the reduction of CENP that mediates mitotic progress (Cardoso et al. 2015), with the heat shock proteins (HSP) acting upon transcription (Kochupillai 2008). Then, MRPs are encoded by nuclear genes, are synthesized in the cytosol, and are then imported into mitochondria for assembly (Zhang et al. 2015). These were qualified by the presence of six main indicators (HSP, MARRS, MRPs, and CENP, positive contribution; PPI-1 and selenium proteins, negative) in PC-2 (Table 3).

Cell proliferation can facilitate a series of biochemical metabolic reactions, along with providing energy and materials for cell growth (Venditti et al. 2013), such as chlorogenic acid contents, PPO, and cellulose content. Germin-like proteins can bind carbohydrates such as cellulose to participate in cell wall modification, and, on the other hand, can prevent cell growth through negative feedback with H2O2 (Dunwell et al. 2008; Caliskan et al. 2004), which can be cleared by SOD and CAT (Liu et al. 2014a). Therefore, reducing SOD and CAT activities result in H2O2 increase, which can decrease OSBP activity to reduce cells proliferation and can increase ABA level to activate the MAPK signaling pathway for cells differentiation (Wang et al. 2005). These can be confirmed by two main indicators (germination-like protein and OSBP, negative contribution) in PC-2 (Table 3), by four main indicators (chlorogenic acid content and PPO, positive; SOD and CAT, negative) in PC-3 (Table 1), and by two main indicators (cellulose content, positive; ABA, negative) in PC-5 (Table 1).

MAPK signaling pathway in cell differentiation during the camellia shoot graft-healing process

PC-2 scores (Table 4) were positive after day 22 from grafting, a period coincides with the time of cell differentiation stage for vascular bundle development (Table 4; Fig. 2). The PC-3 scores varied dramatically, with 4–22 days after grafting the score decreased to a minimum on 22 days, a period associated with the largest of naive cell, then the cell went into the secondary growth with score rising up to 29 days in which time most of cells completed the secondary growth to form the vascular bundle, and then, the rest of the score decreased again (Figs. 2, 3). These indicated that PC2 (Table 1) and PC-3 (Table 3) were related to cell differentiation. The 7 main indicators of 15 main indicators in PC2 (Table 1) and PC-3 (Table 3) (47%) directly took part in MAPK signaling pathway [GA (Zheng et al. 2015), ABA (Wang et al. 2005), cytokinin synthase (Mishra et al. 2006), MARRS (Wagatsuma and Sakuma 2014), annexin (Shimizu et al. 2012), PPI-2 (Zhimin and Hunter 2014), and PI3K (Manna et al. 2016)]. Therefore, after grafting, cell differentiation may depend on biological responses in MAPK signaling pathway during the camellia shoot graft-healing process.

Negative feedback with cell proliferation increases ABA level (Wang et al. 2005). As the level of ABA increases rapidly in the scion cells, the MAPK signaling pathway is inhibited (Lehto and Olkkonen 2003) and TCTP is activated (Bommer and Thiele 2004), resulting in the arrest of cell division, through regulation of tubulin binding (Liu 2012). These were verified by ABA as main positive indicator in PC-2 (Table 1), and by TCTP as main positive indicator in PC-3 (Table 3).

Decline of MAPK signaling pathway increased PI3K pathway (Manna et al. 2016) and then leads to increased levels of GA, generating secondary messengers (Ca2+) (Day 2008). Increased PPI activity, which specifically catalyzes the phosphorylation of Ser/Thr residues (Ryo et al. 2003), thereby promoting the formation of annexin fibers in the cell wall (Grewal and Enrich 2009), results in cell elongation (Lee et al. 2002). These were confirmed by GA and ABA as positive indicator in PC-2 (Table 1), by annexin and PPI-2 as positive indicator in PC-3 (Table 3).

Increased GA levels cause increased intracellular Ca2+ contents and endonuclease activation induced to protoplast disintegration, and then, the soluble sugar and hydrogen peroxide (H2O2) contents increase (Xie et al. 2014). Soluble sugars participate in cell wall formation. Increased H2O2 activates SOD, which prevents cell necrosis and maintains H2O2 at moderate levels, so that protoplast disintegration can proceed in a controlled manner (Guaragnella et al. 2011). When the GA content reaches a threshold level, retrotransposon protein is induced, which forms a negative feedback loop for the GA signal transduction pathway by PI3K activity reduced. GA induces cytokinin synthase activity, resulting in cytokinin accumulation in the cells and inhibiting transcription acted as another negative feedback loop. Thereby, maintaining the balance between GA and cytokinins can promote the formation of vascular bundles at the scion graft. These were verified by three main indicators (GA and soluble sugars, positive; ZR, negative) in PC-2 (Table 1), by four main indicators (cytokinin synthase, Cu–Zn-SOD and retrotransposon protein, positive; PI3K, negative) in PC-3 (Table 3).

Conclusion

Camellia oleifera nurse seedling graft-healing illustrates the metabolic activities related with calcium singling and MAPK pathway, providing the raw materials and energy required for cell growth and differentiation involved in cell proliferation and differentiation, which can regulate vascular bundle formation and wound healing. The calcium signal transduction pathways were interlinked with metabolic activity providing the basis for grafted union development, and signal transduction pathways directing the development. At the same time, the calcium signal transduction pathways relied on materials and energy metabolism for energy and intermediates. During material and energy metabolism, there are two key points; namely, (1) BRD initiated the glycolytic pathway that provided ATP and cell components for grafted union and (2) maintaining the balance between CHI and PPO ensuring cell proliferation and subsequent cell differentiation.

Finally, MAPK transduction pathway at the scion consisted of two parts, the first is the transduction of: (1) cell proliferation signal and (2) cells differentiation, with H2O2 as the key signal link. In the transduction pathway of the cell proliferation signal, PPI was a key enzyme in the cell proliferation that drove cells from the arrested state into a division cycle. In the cell differentiation signal, GA was the key point, and its level determined the differentiation state.

Author contribution statement

Conceived and designed the experiment: J-LF, HC, and YAK. Performed the experiment: J-LF. Data collection and figures preparation: J-LF and Z-JY. Data analysis: J-LF and C-PC. Wrote the manuscript: J-LF and YAK.