In forest production systems, vegetative propagation of elite clones through adventitious rooting is a common practice. In Chile, adventitious rooting is the main methodology for vegetative reproduction of Pinus radiata . However, the capability of produce adventitious roots in gymnosperms decreases with aging. While it is true that some efforts have been made to identify markers or/and regulators of the aging process and adventitious rooting, molecular mechanisms that regulate both processes are scarcely known, especially at protein level. This research evaluated qualitative and quantitative changes in protein accumulation during the adventitious rooting process of P. radiata stem cuttings, with different rooting capabilities. Beside, an analysis of morpho-anatomical changes was performed in stem cuttings with high and low rooting capabilities, during the adventitious rooting process. It was observed that juvenile 1-year-old stem cuttings rooted in a 100%, while aged stem cuttings (3 - year-old) presented only a 20% of rooting. According to the results of differential protein accumulation, univariate and multivariate analysis indicated that in total, 114 and 89 proteins were differentially accumulated in juvenile and aged cuttings, respectively. Also, identification of such proteins showed the presence of proteins related to cell wall organization and the presence of a protein related with proper distribution of auxin PIN transporter, both key in the new meristem formation process during adventitious rooting.
It is widely known that vegetative reproduction in plants is highly successful in early stages of plant development. In forestry species such as P. radiata, decrease in organ regeneration capability from differentiated somatic cells, is highly related to tree age and maturation stage [
The negative effects of aging on rooting have created the need to describe and characterize physiological markers for both processes. For aging, studies have been focused mainly in the relation between the loss of morphogenic competence and plant hormones. Endogenous content of indol-acetic acid (IAA) and abscisic acid (ABA) has been analyzed mainly in relation to different stages of the rooting process [
On the other hand, molecular markers associated to adventitious rooting and aging are scarce, especially in relation to changes in morphogenic competence. However, some genes have been linked to the process of rooting in gymnosperms hypocotyls. Specifically, genes of the GRAS proteins family, such as SCARECROW-LIKE (SCL) [
Plant material was obtained from Proplantas nursery S.A located in Bio-Bío region in central Chile (36˚37'25.87''S and 72˚21'23.80''W). Plant material corresponds to rootstock plants of 1- and 3-year-old from the same full-sib family, and cultivated under the same conditions of fertilization and watering. In brief, watering was applied daily through nebulized watering, fertilization consisted on 400 mg∙L−1 of N (with NaNO3, (NH4)2HPO4, CO(NH2)2 and (NH4)2SO4 as sources), 150 mg∙L−1 of P (with KH2PO4 and Ca(H2PO4)2 as sources), 100 mg∙L−1 of K (with K2SO4, K2CO3 and KH2PO4 as sources), 40 mg∙L−1 of Mg with MgSO4 as source, 60 mg∙L−1 of S (with MgSO4, K2SO4 and (NH4)2SO2 as sources) and 80 mgL−1 of Ca (with CaCO3 and Ca(H2PO4)2 as sources). Stem cuttings were rooted on the nursery of the Forestry Science Faculty of the Universidad de Concepción according to Proplantas S.A nursery protocol for container rooting. In brief: cuttings were washed with 0.5 g∙L−1 of benomilo® solution to avoid fungal contamination. Then, cuttings were placed in containers with 88 cavities of 130 cm3 with pine bark compost as a substrate. Cuttings were irrigated three times a day to maintain foliage and substrate wet at field capacity. Rooting percentage was evaluated until younger cuttings reached 100% rooting.
The collection of plant material for protein extraction and anatomy analysis was performed at serial time points: 0 (T0), 5 (T1) and 15 days (T2) after cutting preparation. Plant material for protein extraction corresponds to 250 mg of fresh stem from the base of the cuttings. The base of the stem cuttings were washed with distilled water and frozen in liquid nitrogen. Plant material was stored at −80˚C until protein extraction. For the anatomical analysis, the base of the stem cuttings were cut and soaked in formaldehyde-alcohol-acetic acid (FAA) until the histological cuts were performed.
Protein isolation was performed with a tris-glycerol-SDS protocol, with a phenol purification step. In brief: 250 mg were grounded in liquid nitrogen with a mortar and pestle. Aliquots of 250 mg of frozen powder were placed in 400 uL of extraction buffer (100 mM Tris-HCl pH 8.0, 5% SDS, 10% glycerol, 2 mM PMSF and 10 mM DTT) vortexed and incubated at 95˚C for 5 minutes. Then, samples were incubated in ice 5 min and vortexed. Four hundred microliters of extraction buffer with 1.5 M sacarose plus 400 uL of saturated phenol were added to the samples. Samples were homogenized in a vortex and incubated at room temperature for 10 minutes. The homogenate was centrifuged 5 min at maximum speed (14.000 rpm). The supernatant was stored, while the pellet was re-extracted as described above. Both phenolic phases were mixed. Two volumes of 0.1 M ammonium acetate in methanol were added to the homogenate and stored at −20˚C overnight, to allow protein precipitation. Homogenate was centrifuged at 5000 g, 4˚C for 5 minutes and the supernatant was eliminated. Pellet was washed twice with cold 100% and 90% acetone respectively; pellet was sonicated to dissolve proteins. Finally, the pellet was allowed to air-dry and resuspended in rehydration buffer (8 M urea, 2% CHAPS and 0.5% ampholites), samples were centrifuged at maximum speed for 5 min and supernatant was saved. Total soluble protein concentration was measured with the bicinchoninic acid (BCA) method. After protein quantification, DTT was added to reach a final concentration of 8 mM.
For each stem cutting age and rooting time, 100 ug of protein were loaded onto precast IPG strips (pH 5 - 8 linear gradient, 7 cm; Bio-Rad, Hercules, USA), and four biological replicates were done for each time point and cutting age. Isoelectric focusing (Ettan-IPGphor isoelectric focusing system, Amersham Biosciences) was performed under the following conditions: passive rehydration for 12 h, followed by a 1 h and 10 min at 150 V, a gradual increase to 250 V for 20 min and finally 10,000 Vh at 4000 V. The focused strips were stored at −20˚C. Before second separation step, IEF strips were incubated twice for 15 min each time in equilibrium buffer (6 M urea, 30 % w/v glycerol, 2% w/v SDS in 0.05 M Tris-HCl buffer pH 8.8) containing 1% DTT in the first equilibration step and 4% iodoacetamide in the second step.
In the second dimension, proteins were separated on 13.5% SDS-PAGE using a Mini-Protean Tetra Cell electrophoresis system (Bio-Rad, Hercules, USA) operating at 30 V for 30 min and 90 V until the front dye reached the end of the gel. Following 2-DE, gels were stained with flamingo fluorescent stain (Bio-Rad, Hercules, USA) and imaged with a Typhoon Trio scanner (Amersham Biosciences) for fluorescent samples.
Digitalized gel images were analyzed with PDQuest 8 software (Bio-Rad, Hercules, USA). Spot-by-spot visual validation of automated analysis was done to increase the reliability of the matching [
Missing spot volumes were estimated from the data set employing a sequential K-Nearest Neighbor (KNN) algorithm using the R 2.14.1 environment [
Selection of differentially abundant protein spots was performed through a repeated measurement model; the best variance and co-variance structure was selected by selecting the lower Akaike index between the different structures tested. Both time and rootstock plant age were set as fixed effects, SAS 9.1 software was used for this analysis. Also, multivariate analysis was done. A partial least square discriminant analysis (PLS-DA) was applied to the data set corresponding to 1-year-old and 3-year-old rootstock plants, scores and loading plots were obtained using the mixOmics [
According to the results, 16 spots were selected for identification, which were manually excised from the gel. Spots were digested following the protocol described by Shervchenko et al. [
Peptides were deposited in a MALDI plate using the dry drop method (ProMS, Genomic Solutions, Chelmsford, MA, USA) and CHCA as matrix at mg/mL in 70% ACN, 0,1% TFA. Samples were analyzed in a mass spectrometer analyzer MALDI-TOF-TOF 4700 (Applied Biosystems, Foster City, CA, USA) in a 800 - 4000 m/z range, with an acceleration voltage of 20 k V, in replectron mode with a delayed extraction of 120 ns. The specter was internally calibrated with trypsin auto-lysis peptides. The three most abundant ions were subjected to spectrometry analysis in mass tandem (MS/MS). An identification search was performed through peptidic fingerprint (PMF) (MS plus MS/MS) in non-redundant data base NCBI, using the GPS Explorer v 3.5 software (Applied Biosystems) plus the MASCOT (Matrix Science, London, UK) search engine. The following parameters were allowed: taxonomy restriction at Viridiplantae, cleavage allowed, mass tolerance at 100 ppm in MS and 0.5 Da for MS/MS data, fixed modification of cysteine carbamidomethilation and methionine oxydation as variable modification. PMF matches coincidences was based in MOWSE score (Molecular Weight SEarch) and confirmed by precise superposition of matching peptides with higher peaks from the mass spectra and protein score with a P value lower than 0.05. Combination between PMF and MS/MS ion scores allowed the coincidence of significant peptides for 8 of the selected spots. Access numbers are referred according to SWISS-Prot or NCBI while theorical Mr (in kDa) and the pI of homologous proteins were calculated through the Mr/pI tool available at Expasy, http://www.expasy.ch/tools/pi_tool.html). Molecular function was inferred from Gene and Genome Encyclopedia (KEGG).
Juvenile (1-year-old) and aged (3-year-old) P. radiata stem cuttings were evaluated through adventitious rooting process. While almost 100% of juvenile stem cuttings developed roots within two month of the rooting process, only 18.8% of aged cuttings showed this response within the same period of time (
Anatomical Characteristics | Rootstock plants age | |
---|---|---|
1-year-old | 3-year-old | |
Total diameter (µm) | 2355.6 ± 209.2 (b) | 3717.5 ± 170.7 (a) |
Xylem diameter (µm) | 461.7 ± 74.2 (b) | 811.4 ± 13.4 (a) |
Phloem diameter (µm) | 119.6 ± 10.2 (a) | 149.1 ± 12.0 (a) |
Periderm width (µm) | 81.5 ± 8.5 (b) | 117.5 ± 1.3 (a) |
Rooting capability | ||
% of rooting | 96.7 ± 1.4 (a) | 18.8 ± 0.9 (b) |
Values ± standard deviation (n = 5), different letters correspond to significant differences according to Student’s t test (P < 0.05).
1-year-old stem cuttings (
According to the differential protein accumulation analysis, 205 total spots were detected including gels from 1- and 3-year-old cuttings.
Univariate and multivariate analysis were performed in the data set to select the differentially abundant proteins. For the univariate analysis 33 and 20 protein spots were differentially accumulated in 1- and 3-year-old cuttings, respectively. Due to the fact that univariate statistical tools treat each spots as an independent variable, it was important to perform a multivariate analysis, which consider a group of variables together rather than one variable at a time. For this reason, we performed a partial least square discriminant analysis (PLS-DA) on 1- and 3-year-old cuttings data set, separately. As
Plant material | Number of spots | Consistent spots | |||
---|---|---|---|---|---|
Min/Max | Average | Total | Common | Qualitative differences | |
A1T0 | 107/139 | 124.2 ± 3.6 | 199 | 184 | 15 |
A1T1 | 86/119 | 105.7 ± 3.8 | |||
A1T2 | 84/131 | 112.2 ± 4.9 | |||
A3T0 | 95/156 | 127.7 ± 6.3 | 190 | 184 | 6 |
A3T1 | 110/151 | 125.6 ± 4.5 | |||
A3T2 | 122/141 | 127.5 ± 2.3 |
observed in
From differential spots, 13 spots were extracted and sequenced, within these, 8 were successfully identified (
SSP | Experimental | Theorical | Protein | Action pathway | Reference organism | Access N° | Sequence coverage (%) | Score | ||
---|---|---|---|---|---|---|---|---|---|---|
Mr | pI | Mr | pI | |||||||
6306 | 22.9 | 6.7 | 1.2 | 6.0 | Oxygen-evolving enhancer protein 2 (Fragment) | Photosynthesis | Pinus pinaster | PSBP_PINPS | 100 | 83 |
6207 | 20.5 | 6.6 | 15.6 | 11.3 | Histone H3-like 5 | Choromosome and other proteins | Arabidopsis thaliana | H3L5_ARATH | 5 | 26 |
Protein ROOT HAIR DEFECTIVE 3 homolog 2 | Oryza japonica | RHD32_ORYSJ | 1 | 16 | ||||||
8401 | 24.3 | 7.2 | 193.2 | 5.3 | Clathrin heavy chain 1 | Endocytosis | Arabidopsis thaliana | CLAH1_ARATH | 1 | 39 |
Iron-sulfur assembly protein IscA-like 3. mitocondrial | Arabidopsis thaliana | ISAM3_ARATH | 13 | 22 | ||||||
8711 | 47.3 | 7.7 | 26.3 | 8.3 | Agamous-like MADS-box protein AGL17 | Arabidopsis thaliana | AGL17_ARATH | 5 | 26 | |
Probable glycerophosphoryl diester phosphodiesterase 2 | Glycerophospholipids methabolism | Arabidopsis thaliana | GLPQ2_ARATH | 2 | 24 | |||||
Putative pectate lyase 11 | Interconversion of pentose and glucoranate | Arabidopsis thaliana | PLY11_ARATH | 4 | 23 | |||||
2512 | 31.2 | 5.5 | 1.4 | 5.8 | Unknown protein 1 (Fragment) | Vitis rotundifolia | UP01_VITRO | 100 | 41 | |
5403 | 29.3 | 6.1 | 48.1 | 9.9 | UDP-glucuronate 4-epimerase 2 | Sugar and sucrose methabolism | Arabidopsis thaliana | GAE2_ARATH | 3 | 15 |
Taxadiene synthase | Diterpenoid biosynthesis | Taxus baccata | TASY_TAXBA | 3 | 33 | |||||
1505 | 34.5 | 5.4 | 26.7 | 5.6 | (DL)-glycerol-3-phosphatase 2 | Riboflavin methabolism | Arabidopsis thaliana | GPP2_ARATH | 5 | 26 |
3304 | 20.5 | 5.6 | 84.2 | 6.6 | Probable RNA-dependent RNA polymerase 1 | Oryza sativa | RDR1_ORYSJ | 1 | 18 | |
2601 | 40.5 | 5.6 | - | - | No hit | - | - | - | - | |
3303 | 21.8 | 5.6 | - | - | No hit | - | - | - | - | |
1702 | 47.5 | 5.3 | - | - | No hit | - | - | - | - | |
3601 | 41.5 | 5.7 | - | - | No hit | - | - | - | - |
the spot 6207, which corresponds to a Histone H3-like and a ROOT HAIR DEFECTIVE 3 proteins, increased their level during the first 5 days for juvenile and aged cuttings (
SPP | Normalized spot intensity | |||||
---|---|---|---|---|---|---|
A1T0 | A1T1 | A1T2 | A3T0 | A3T1 | A3T2 | |
6306 | 0.69 ± 0.28 | 0.10 ± 0.05 | 0.0 ± 0.0 | 0.46 ± 0.19 | 0.64 ± 0.11 | 0.07 ±0.04 |
6207 | 0.41 ± 0.24 | 4.58 ± 0.75 | 3.22 ± 0.47 | 0.90 ± 0.29 | 2.97 ± 1.48 | 2.06 ± 1.03 |
8401 | 0.23 ± 0.08 | 0.16 ± 0.11 | 0.0 ± 0.0 | 0.14 ± 0.05 | 0.46 ± 0.07 | 0.05 ±0.05 |
8711 | 0.34 ± 0.07 | 0.0 ± 0.0 | 0.08 ± 0.05 | 0.50 ± 0.14 | 0.29 ± 0.15 | 0.31 ± 0.14 |
2512 | 7.39 ± 3.69 | 2.97 ± 1.48 | 0.35 ± 0.17 | 8.62 ± 1.84 | 3.10 ± 1.04 | 0.18 ± 0.11 |
5403 | 0.61 ± 0.24 | 0.55 ± 0.19 | 1.33 ± 0.15 | 0.96 ± 0.31 | 1.38 ± 0.08 | 1.12 ± 0.14 |
1505 | 3.58 ± 0.64 | 0.0 ± 0.0 | 2.82 ± 0.66 | 3.71 ± 0.77 | 3.88 ± 0.87 | 0.0 ± 0.0 |
3304 | 0.16 ± 0.08 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.20 ± 0.08 | 0.19 ± 0.01 |
2601 | 1.12 ± 0.42 | 0.26 ± 0.11 | 0.0 ± 0.0 | 0.22 ± 0.01 | 0.65 ± 0.19 | 0.18 ± 0.07 |
3303 | 0.51 ± 0.24 | 0.27 ± 0.18 | 0.49 ± 0.31 | 0.18 ± 0.10 | 0.54 ± 0.12 | 0.17 ± 0.10 |
1702 | 0.92 ± 0.25 | 0.54 ± 0.23 | 0.51 ± 0.34 | 0.18 ±0.11 | 1.36 ± 0.37 | 0.0 ± 0.0 |
3601 | 0.35 ± 0.01 | 0.27 ± 0.05 | 0.44 ± 0.06 | 0.21 ± 0.01 | 0.28 ± 0.05 | 0.30 ± 0.11 |
sented in juvenile cuttings a decrease in accumulation until be undetected at day 15, while in 3-year-old cuttings a 3-fold initial increase was observed, during the adventitious rooting process.
According to the adventitious rooting analysis, it was confirmed that aged stem cuttings (3-year-old) presented a clear decrease in their rooting capability. On the contrary, almost 100% of juvenile P. radiata cuttings rooted during the evaluated period (2 months). This indicates that within a period of three years, it is possible to observe the effects of tree aging, which translates mainly in the loss of rooting capability [
In relation to the anatomic analysis, it is possible to observe that, in 1-year-old cuttings, the initial formation of the radicular meristem that will generate the adventitious root can be identified within the first 15 days of the rooting process (
From the analysis of differential protein accumulation, it was possible to observe that from the 205 detected spots for both types of cuttings, 114 and 89 spots were differentially accumulated in 1- and 3-year-old cuttings, respectively. These results show that juvenile cuttings possess a higher amount of proteins that can modify their accumulation during the adventitious rooting process. This could indicate that 1-year-old cuttings can generate greater changes in protein accumulation and adjust to the demands of the new forming meristem. Also, this could indicate that juvenile cuttings have a higher number of active metabolic pathways [
According to the analysis of spots present in both kinds of cuttings, it is observed that 1-year-old cuttings have 15 proteins that are exclusive to this type of cutting, while the 3-year-old ones only have 6 spots (
Likewise, the RHD3 protein presented the same behavior than histone H3. This gen could have a function in cell enlargement during growth of radicular hairs in Arabidopsis, because rhd3 mutants displayed an increase in the proportion of cytoplasm and reduction in the vacuole size, particularly affecting the radicular hair expansion phase [
Otherwise, a clathrin heavy chain (CHC) protein was also identified, and this protein presented the same behavior as GPD in both types of cuttings. Clathrin is a complex of proteins in the shape of a trisquel consisting on heavy chains (CHC) and light chains (CLC) forming a lattice. Clathrin plays a major role in endocytosis, vesicle formation, protein abundance in plasmatic membrane and in the trans-Golgi network during signaling events [
According to the results, both juvenile and aged cuttings showed a decrease in the accumulation of agamous-like MADS-box protein (AGL17), identified in the spot 194. Genes that belong to the MADS-box family play a role during floral development, so their expression in restricted to floral organs [
Finally, this research provides a characterization of proteins involved in the formation of adventitious roots on P. radiata stem cuttings and how this process is influenced by aging of rootstock plants. According to the results obtained in this research, in comparison to 1-year-old juvenile cuttings, a delay in 3-year-old cuttings rooting process was observed; concomitant with changes at anatomical level and in the protein accumulation pattern,. Besides, proteins involved in the formation and organization of cell wall were identified, indicating that this protein could be essential for the formation of adventitious roots.
The authors of this manuscript would like to thank to Proplantas Nursery S.A, INNOVA BIOBIO research project n˚ 12.121 and CONICYT scholarships.
Álvarez, C., Valledor, L., Sáez, P., Sánchez-Olate, M. and Ríos, D. (2016) Proteomic Analysis through Adventitious Rooting of Pinus radiata Stem Cuttings with Different Rooting Capabilities. American Journal of Plant Sciences, 7, 1888-1904. http://dx.doi.org/10.4236/ajps.2016.714174