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Tree Physiology Advance Access originally published online on December 3, 2008
Tree Physiology 2009 29(2):183-190; doi:10.1093/treephys/tpn009
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dynamic changes in concentrations of auxin, cytokinin, ABA and selected metabolites in multiple genotypes of Douglas-fir (Pseudotsuga menziesii) during a growing season

Lisheng Kong1,2, Suzanne R. Abrams3, Stacey J. Owen3, Annette Van Niejenhuis4 and Patrick Von Aderkas1

1 Centre for Forest Biology, Department of Biology, University of Victoria, 3800 Finnerty Road, Victoria, BC V8W 3N5, Canada
2 Corresponding author (lkong{at}uvic.ca)
3 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada
4 Western Forest Products Ltd., 8067 East Saanich Road, Saanichton, BC V8M 1K1, Canada


   Abstract
Top
 Notes
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in concentrations of several endogenous phytohormones and metabolites were analyzed in the long shoots of nine genotypes of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) at five developmental stages: (1) closed buds, (2) flushing buds, (3) rapidly elongating shoots, (4) growing shoots and (5) near full-length shoots during one growing season. When averaged across genotypes, indole-3-acetic acid (IAA) concentration was high at stages 1 and 3. The only pattern that correlated with cone productivity was the one that was unique to IAA, in which high concentrations at stages 3 and 4 were found in all genotypes with high female cone productivity. Concentrations of isopentenyl adenosine (iPA) decreased and zeatin riboside (ZR) concentrations increased as the buds initiated and differentiated; ZR was 30 and 28 ng g–1 dry weight (DW) at stages 1 and 4, respectively, before increasing to 166 ng g–1 DW at stage 5. Isopentenyl adenosine peaked at 92 ng g–1 DW at stage 2 and declined to low concentrations at stages 4 and 5. Zeatin-O-glucoside was 30 ng g–1 DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5. High abscisic acid (ABA) concentrations were positively correlated with rapid shoot elongation (stages 1 and 2), but as growth slowed and terminated, ABA concentrations decreased. Abscisic acid was 7 µg g–1 DW at stage 1, increased to 13 µg g–1 DW at stage 2 and then declined. The glucosyl ester (GE) of ABA decreased rapidly in early summer, and increased inversely with an increase in ABA. Between stages 1 and 2, ABA-GE decreased from 10 to 0.2 µg g–1 DW and then increased. Of the ABA catabolites studied, 7'-hydroxy-ABA was about 2 µg g–1 DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5; phaseic acid concentrations were low at all stages, whereas dihydrophaseic acid was detected only at stages 4 and 5.

Keywords: long shoot, plant hormones

Received April 21, 2008; Accepted September 2, 2008


   Introduction
Top
 Notes
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
In conifers, both reproductive and vegetative buds are generated from long shoots. During the first growing season, after the long shoot bud bursts, new lateral buds are initiated early in the growing season and then differentiated over an extended period. The physiological status of the long shoot is thus important for bud formation as well as subsequent bud growth and differentiation.

The major methods for increasing cone production in seed orchards include plant growth regulator (PGR) application, physical treatments (e.g., girdling, root pruning and shoot pruning), temperature treatments, water stress and nutrient control. The most common method is PGR application, with or without other treatments. The PGR of choice is gibberellic acid (GA, usually as a mixture of GA4 and GA7) that has been successfully used to induce cone bud formation in many conifers, including Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) (McMullan 1980, Pharis et al. 1980, Ross 1983, Pharis 1991, Ross and Bower 1991). Other plant hormones applied with a GA treatment can also affect cone bud induction. An auxin, when applied with GA4+7, mainly induces male cones (Pharis et al. 1980, Sheng and Wang 1990), whereas an exogenously applied cytokinin (CK) favors female cone bud formation (Wakushima et al. 1996, 1997, Wakushima 2004). At least one plant hormone, abscisic acid (ABA), plays an important role in plant responses to several environmental stresses, including water stress and extreme temperature stress (Sauter et al. 2001, Bray 2002, Finkelstein et al. 2002). These and other stresses can enhance cone bud production in conifers (Stock and Silvester 1994).

Flowering in conifers is a complex process that is under the control of multiple factors, among which plant hormones play an important role (Bernier et al. 1993, King et al. 2006). Profiling plant hormones and their major metabolites during the period of cone bud initiation and differentiation can provide information on their function. Several studies have analyzed endogenous GAs, and occasionally other hormones, in vegetative and potentially reproductive buds of a variety of pinaceous conifers (Odén et al. 1987, 1994, 1995, Moritz et al. 1990, Wang et al. 1992, 1996, Zhang et al. 2001, 2003), including Douglas-fir (McMullan 1980, Meyer et al. 1986, Imbault et al. 1988, Morris et al. 1990, Pilate et al. 1990, Doumas et al. 1992). Our first objective was to survey the IAA, CK and ABA hormone groups in long shoots of Douglas-fir during development.

Genotype diversity in plant metabolism is known to occur in higher plants (Krabel and Petercord 2000, Krzakowa and Matras 2005). We tested the hypothesis that in Douglas-fir there are metabolic differences attributable to genotype. We also examined if such phytohormone differences are correlated with cone induction. We used a tandem mass spectrometry (MS) with multiple reaction monitoring (MRM) approach in which multiple plant hormones (IAA, CKs, ABA and their metabolites) were quantified in the same plant sample using deuterated internal standards for each metabolite. This approach was previously proved to be successful in the studies of conifer seed development and seed parasitism (Feurtado et al. 2004, 2007, Chiwocha et al. 2007).


   Materials and methods
Top
 Notes
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental plants
Nine genotypes were selected from the seed orchard of Western Forest Products Ltd. located in Saanichton, BC (48°35'39'' N and 123°24'51'' W). Seven of the nine genotypes were selected randomly. Two additional genotypes were included: one is high in female cone production and the other is low. These two genotypes were chosen on the basis of the data collected in the past 8 years. Ramets of each selected genotype were 15 years old.

Sample collection, processing and storage
Tissue samples were collected from one ramet of each selected genotype. Five to 25 long shoot buds or elongating shoots were collected from each ramet at each sampling time, the number depending on the size of the bud or shoot. Samples were collected at five developmental stages during long shoot growth, between May and September (Table 1). Stage 1 samples included the carefully dissected stems (needles removed) of long shoot buds before bud flushing. Stage 2 samples consisted of long shoot stems that were collected shortly after bud flushing; again, needles were removed. Stage 3 samples consisted of long shoot stems (no needles) that were in a rapid elongation phase. Stage 4 samples consisted of long shoot stems that were more than half their final length; here, the newly formed lateral buds were visible on such long shoots, and buds and stems were kept together as samples. Stage 5 samples were full-length long shoot stems and well-formed new buds were obvious on the long shoots.


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  		Table 1. Long shoot developmental stages, morphology and sample collection dates. Nine P. menziesii genotypes were selected at the seed orchard of Western Forest Products Inc., Saanichton, BC. Samples were collected at five developmental stages for analysis of several endogenous hormones.

 
After bud scales or needles, or both, were removed from the stems, the stem samples were wrapped in an aluminum foil, labeled and quickly submerged in liquid nitrogen. The samples were kept frozen at –20 °C for 2 to 3 days and subsequently lyophilized for 48 h. Lyophilized samples were sealed in plastic bags and stored at –20 °C.

Measurement of water content
Tissue masses of the samples were determined before (fresh) and after (dry) lyophilization. Water content of each sample was expressed as a percentage of fresh mass.

Chemicals
We have created calibration curves and quality controls (QCs) for dihydrophaseic acid (DPA), abscisic acid glucose ester (ABA-GE), phaseic acid (PA), 7'-hydroxy-ABA (7'-OH-ABA), neo-phaseic acid (neoPA) and indole-3-acetic acid glutamate (IAA-Glu) from the Plant Biotechnology Institute of the National Research Council of Canada (PBI-NRC Saskatoon, SK, Canada); ABA, indole-3-acetic acid aspartate (IAA-Asp), IAA, zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine (iPA) and isopentenyl adenine (2iP) were purchased from Sigma–Aldrich; and dihydrozeatin (dhZ), dihydrozeatin riboside (dhZR) and zeatin-O-glucoside (Z-O-Glu) were purchased from Olchemim Ltd. (Olomouc, Czech Republic). Bulk amounts of the deuterated forms of the hormones, used as internal standards, were obtained: d3-DPA, d5-ABA-GE, d3-PA, d4-7'-OH-ABA, d3-neoPA, d4-ABA, d3-IAA-Asp and d3-IAA-Glu from PBI-NRC; d5-IAA from Cambridge Isotope Laboratories (Andover, MA); and d3-dhZ, d3-dhZR, d5-Z-O-Glu, d6-iPA and d6-2iP from Olchemim Ltd. Bulk amounts of the deuterated forms of selected hormones, used as recovery standards, were obtained: d6-ABA and d2-ABA-GE from PBI-NRC.

Extraction and purification
Lyophilized stem tissue was homogenized in a bead mill for 2–6 min. A 100-µl aliquot containing all the internal standards, each at a concentration of 0.2 pg µl–1, was added to about 50 mg of homogenized stem tissue; 3 ml of isopropanol:water:glacial acetic acid (80:19:1, v/v) was then added, and the samples were agitated for 24 h at 4 °C. Samples were then centrifuged and the supernatant was isolated and dried on a Büchi Syncore Polyvap (Büchi, Switzerland). Samples were reconstituted in 100 µl acidified methanol, adjusted to 1 ml with acidified water, and then partitioned against 2 ml hexane. After 30 min, the hexane layer was removed and the hexane partitioning was repeated. The aqueous layer was then isolated and dried. Dry samples were reconstituted in 800 µl acidified methanol and adjusted to 1 ml with acidified water. The reconstituted samples were passed through equilibrated Sep-Pak C18 cartridges (Waters, Mississauga, ON, Canada), the eluate being dried on a centrifugal evaporator. An internal standard blank was prepared with 100 µl mixture of the deuterated internal standards. A QC standard was prepared by adding 20 ng of each analyte to 100 µl of the internal standard. Finally, all samples, blanks and QCs were reconstituted in a solution of 40% methanol (v/v), containing 0.5% acetic acid and 100 pg µl–1 of each of the recovery standards.

Hormone quantification by HPLC-ESI-MS/MS
The procedure for quantification of multiple hormones and metabolites, including auxins (IAA, IAA-Asp and IAA-Glu), abscisic acid and metabolites (ABA, PA, DPA, 7'-OH-ABA, neoPA and ABA-GE), and CKs (2iP, iPA, Z, ZR, dhZ, dhZR and Z-O-Glu), has been described in detail by Chiwocha et al. (2003, 2005). Samples were injected onto a Genesis C18 HPLC column (100 x 2.1 mm, 4 µm, Chromatographic Specialties, Brockville, ON, Canada) and separated by a gradient elution of water against an increasing percentage of acetonitrile that contained 0.04% acetic acid. Calibration curves were generated from the MRM signals obtained from standard solutions based on the ratio of the chromatographic peak area for each analyte to that of the corresponding internal standard, as described by Ross et al. (2004). The QC samples, internal standard blanks and solvent blanks were also prepared and analyzed along each batch of tissue samples. Mean minimum limits of quantification (LOQ) for each analyte were: 8 ng g–1 dry weight (DW) for Z, dhZ, Z-O-Glu, 2iP, iPA and ABA; 60 ng g–1 DW for ZR, dhZR, IAA-asp, IAA-Glu, IAA and 7'-OH-ABA; 118 ng g–1 DW for DPA; 78 ng g–1 DW for PA; 56 ng g–1 DW for ABA-GE; and 30 ng g–1 DW for neoPA.

Evaluation of cone production
Female cone production was assessed in 2007, the year following sample collection during the growing season in 2006. Female cone production was evaluated and ranked based on mean cone yield per tree derived from measurements of multiple ramets. Historic cone production data were used only in the initial selection of two of the genotypes (high and low cone producers), and were not used to categorize cone productivity. Cone yields presented for the nine genotypes are exclusively based on 2007 cone collections.

Experimental design and statistical analysis
Nine genotypes were used as replicates for general hormone profiling. The same nine genotypes were also assessed for cone bud production in the spring. The genotypes were divided into three groups (high, moderate or low) based on cone yield data collected in 2007. Data were subjected to one-way analysis of variance (ANOVA) using Minitab Statistical Software, Version 13.1 (Minitab, State College, PA). The variance was analyzed using Tukey’s significant difference with the level of significance set to P < 0.05. Correlation analysis of ABA and ABA-GE was carried out using the same software.


   Results
Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Changes in tissue mass and water content
Both fresh and dry mass of the long shoot increased as the season advanced (Figure 1). Fresh mass increased between stages 2 and 3, when the stem grew quickly. Increase in fresh mass slowed at stages 4 and 5. Stem dry mass increased steadily from stage 2 to 5. In contrast, water content increased from stage 1 to 3 (Figure 1), but decreased sharply from stage 3 to 5.


Figure 1
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  		Figure 1. Changes in fresh mass (FM), dry mass (DM) and water content (WC) in long shoots of P. menziesii during the 2006 growing season. Mean values of nine independent replicates (genotypes) with standard errors.

 
General patterns of concentration changes in IAA
Before bud flushing, IAA concentration in the dissected stem was as high as 219.5 ng g–1 DW (Figure 2). The IAA concentration decreased by 66% at stage 2 (bud flushing). During the period of rapid elongation at stage 3, IAA concentration peaked at 256.9 ng g–1 DW. As growth slowed (stages 4 and 5), IAA concentrations decreased. The IAA metabolites, IAA-Asp and IAA-Glu, were generally below quantifiable values, though we found IAA-Asp at 11.2 ng g–1 DW in stage 1 samples.


Figure 2
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  		Figure 2. Changes in IAA concentrations in long shoots of P. menziesii during the 2006 growing season. Mean ± SE, n = 9 genotypes.

 
General patterns of concentration changes in CKs and CK metabolites
The Z-O-Glu concentration was 29.8 ng g–1 DW at stage 1. It then declined and remained low at stages 2 and 3 (Figure 3). The Z-O-Glu concentration increased from stage 4 and peaked at 32.4 ng g–1 DW (a sixfold increase relative to stage 3) at stage 5. Zeatin riboside concentration was quantifiable at stages 1, 4 and 5, and peaked at 166.3 ng g–1 DW (a sixfold increase relative to stage 4) at stage 5. Other zeatin-related CKs and metabolites, Z, dhZ and dhZR, were below LOQ.


Figure 3
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  		Figure 3. Changes in concentrations of CKs and metabolites in long shoots of P. menziesii during the 2006 growing season. Mean ± SE, n = 9 genotypes.

 
The concentration of iPA was 69.1 ng g–1 DW at stage 1, peaked at 92.1 ng g–1 DW at stage 2, and then declined. Concentrations of 2iP were below the quantifiable limits at stages 1 to 3, becoming quantifiable in a few samples only at stages 4 and 5 (data not shown).

General patterns of concentration changes in ABA and its metabolites
Concentrations of both ABA and ABA-GE were initially high at 6.7 and 10.0 µg g–1 DW, respectively (Figure 4). By stage 2, ABA concentration almost doubled, and this was accompanied by a 13-fold decrease in ABA-GE. The ABA concentration then decreased fivefold in a continuous manner as the season advanced. In contrast, ABA-GE underwent a 17-fold increase between stages 2 and 5 (Figure 4). Correlation analysis revealed a significant inverse correlation between ABA and ABA-GE (r = –0.496; P = 0.002).


Figure 4
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  		Figure 4. Changes in concentrations of ABA and ABA metabolites in long shoots of P. menziesii during the 2006 growing season. Mean ± SE, n = 9 genotypes. Significant differences at the P < 0.05 level are indicated by different letters between developmental stages for either ABA (capital letters) or ABA-GE (lowercase letters).

 
The concentration of 7'-OH-ABA was 2.4 µg g–1 DW at stage 1. It had declined threefold at stage 3 and then increased (Figure 4). Phaseic acid was present in all samples at low concentrations. Its concentration was 0.3 µg g–1 DW initially, but then declined (Figure 4). Dihydrophaseic acid was quantifiable only at stages 4 and 5 at low concentrations of 60.2 and 50.7 ng g–1 DW, respectively (data not shown). The concentration of neoPA was below quantifiable limits in all samples.

Genotypic differences in cone production relative to endogenous hormone concentrations in the stem
Cone yields of the studied genotypes are categorized into three groups: high, moderate and low (Table 2). A significant difference (P < 0.05) existed between the groups based on the mean cone yield per tree.


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  		Table 2. Female cone yield in three groups of P. menziesii genotypes. The genotypes were divided into high-, moderate- and low-yield groups based on cone production in 2007. Significant differences at P < 0.05 are indicated by different letters within a row. Values are mean ± SE, n = 3.

 
Changes in IAA concentrations in 2006 stems of the three genotypes in the 2007 high cone-yield group shared a distinct pattern, i.e., there was a low concentration of IAA at stage 2, followed by high concentrations at stages 3 and 4 (Figure 5). Similar IAA patterns to this distinctive IAA pattern were observed for only two of the six genotypes in the moderate and low cone-yield groups. Other phytohormones, typical examples of which are iPA and ABA (Figure 5), did not share a characteristic pattern across genotypes.


Figure 5
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  		Figure 5. Changes in concentrations of endogenous IAA, iPA and ABA in developing long shoots of P. menziesii as a function of developmental stage, with the genotypes divided into high- (high), moderate- (moderate) and low-yield (low) groups based on female cone production in 2007 (see Table 2). Values below the horizontal line are below the limits of detection and do not represent real quantities.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
This study provides the first precise and accurate examination of IAA, ABA and several CKs (and their metabolites) in long shoots of Douglas-fir over a single growing season. There were dynamic changes in IAA concentrations during the growing season, with higher IAA concentrations at stages 1 and 3 (bud burst and rapid stem growth, respectively). Involvement of IAA in vegetative stem growth, by regulation of cambial activity, has been well documented (Sundberg et al. 1991, Uggla et al. 1998, 2001, Mwange et al. 2005), with the highest IAA concentration being observed in the fastest-growing cambia in Scots pine (Uggla et al. 1998).

We found that IAA had a consistent trend among high cone producing genotypes: high IAA concentrations at stages 3 and 4 in stem and bud tissues of long shoots were noted in all three genotypes with high cone productivity. This is the time when the cone buds of next season will be undergoing initiation and differentiation. Indole-3-acetic acid was the only phytohormone with a trend that correlated with cone productivity. Similarly, distinctive IAA patterns were absent for genotypes of lower cone productivity. These findings imply that elevated concentrations of IAA are important in cone bud differentiation. In the previous studies, endogenous IAA concentrations were shown to be elevated in the cambial region (Wang et al. 1992) and in long shoots (Kong et al. 2008) after gibberellins (GA4+7) were exogenously applied. Cambial activity can also be associated with changing IAA:ABA ratios (Mwange et al. 2005).

In our study, concentrations of iPA and ZR changed markedly over the course of the season, with iPA decreasing steadily and ZR increasing as buds initiated and differentiated. Concentrations of zeatin-type (Z-type) CKs were generally lower than the concentrations of isopentenyl-type (iP-type) CKs until stage 5. The sharp increase in t-ZR at stage 5 effectively elevated the ratio of Z-type CKs to iP-type CKs. The dynamics of the ratio changes may explain the contradictory reports that the concentration of Z-type CKs was either lower (Pilate et al. 1990) or higher (Morris et al. 1990) than that of the iP-type CKs in the vegetative shoots of Douglas-fir. As different methodologies, enzyme immunoassay and MS methods have been used widely in hormone analysis, method limitations may underlie some of the discrepancies among the previously published data. The MRM analytic methodology that we had used should reduce the potential errors and increase the analytic efficiency, because of the simplified process and the simultaneous analysis of multiple compounds.

Morris et al. (1990) reported that Z-type CK concentration was relatively higher in female cone buds, whereas the concentration of iPA was higher in male cone buds. In two conifers, gender determination was affected by an applied CK-like PGR, N6-benzylaminopurine (Wakushima et al. 1996, 1997, Wakushima 2004). In our study, the increasing concentration of ZR (Figure 3; stage 5) implies that biologically active CKs function in cone bud differentiation or in cambial region growth and differentiation during late summer. The importance of CKs in cone bud differentiation is also supported by the previous studies (Imbault et al. 1988, Pilate et al. 1990, Zhang et al. 2003). The concentration of zeatin was generally low, below quantifiable levels, implying that there is a small pool of zeatin or a rapid turnover of Z to ZR, or both, in the Douglas-fir shoot.

We assessed ABA, ABA-GE and PA concentrations in the shoot over a single growing season. Concentrations of ABA increased as the shoot elongated during stages 1 and 2. As shoot elongation slowed, which is also the period when buds are initiated and differentiated, ABA concentrations decreased. The three major ABA catabolic pathways include: (1) the 8'-hydroxylation pathway leading to the formation of PA and DPA, (2) the 7'-hydroxylation pathway leading to the formation of 7'-OH-ABA and (3) the catabolic pathway through the conjugation of ABA with glucose to form ABA-GE (Nambara and Marion-Poll 2005). Abscisic acid glucose ester that appears to be physiologically inactive (Cutler and Krochko 1999) accumulates in vacuoles of living cells, such as mesophyll cells, during growth and maturation (Bray and Zeevaart 1985, Lehmann and Glund 1986), and has been generally viewed as one of the end products of ABA catabolism. If true, ABA-GE does not represent a storage form of ABA that can be hydrolyzed in vegetative tissues of plants to release free ABA. There is, however, some evidence that ABA can be released from ABA-GE (see the subsequent text). The kinetics of PA and ABA-GE accumulation during long shoot growth in our study indicate that glucose-conjugation is the major catabolic pathway, and may regulate the changes in ABA concentration (Figure 5). The increase in free ABA concentration at stage 2 was proportional to the decrease in ABA-GE and a significant inverse correlation existed between these compounds. Dietz et al. (2000) have reported that an apoplastic β-glucosidase in barley is able to release ABA from ABA-GE, and more importantly it has been demonstrated that, in response to stress, free ABA can be released to the cytosol via hydrolysis of glucose-conjugated ABA by the polymerization of a β-glucosidase enzyme in microsomes (Lee et al. 2006). Our results imply that a reversible pathway exists between ABA and ABA-GE in rapidly elongating shoots of Douglas-fir (see Figure 5; stages 1 and 2). However, this would need to be validated by labeled ABA-GE feeds, metabolite tracing and perhaps an assessment of β-glucosidase activities in the elongating shoot tissue. The presence of 7'-OH-ABA in Douglas-fir long shoots demonstrates that a minor ABA catabolic pathway is also present, although 7'-OH-ABA concentrations were much lower than those of ABA-GE.

An MRM approach provides simultaneous profiling that can be used to establish genotypic variations in some plant hormones and their metabolites when multiple genotypes are used for analysis. A characteristic dynamic pattern was found for endogenous IAA in genotypes that exhibited high cone-yield; however, cone-yield capability may depend on multiple factors. A complete metabolomic analysis including more types of plant hormones, such as gibberellins, could provide increased understanding of hormonal regulation during cone initiation and differentiation.


   Acknowledgements
 
The cone-induction project was funded through the British Columbia Forest Investment Account, Tree Improvement Program under the Business Plan of the Forest Genetics Council of BC (FGCBC). The authors express their appreciation to the project steering committee; Jack Woods (FGCBC and SelectSeed Co. Ltd.); Cathy Cook and Mal Kirk (Western Forest Products Inc.); Samir Demdoum, Sébastien Bonthoux and Genoa Barchet (University of Victoria); and Monika Lafond, Vera Cekic and Irina Zaharia (NRC-PBI) for their help in this research.


   Notes
Top
 Notes
Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This version contains the full author list, including S.J. Owen. Back


   References
Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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