Trees (2011) 25:711–723 DOI 10.1007/s00468-011-0549-7
ORIGINAL PAPER
Changes of carbon and nitrogen metabolites in white spruce (Picea glauca [Moench] Voss) of contrasted growth phenotypes Catherine Dhont • Annick Bertrand Yves Castonguay • Nathalie Isabel
•
Received: 31 August 2010 / Revised: 2 February 2011 / Accepted: 10 February 2011 / Published online: 26 February 2011 Ó Her Majesty the Queen in Rights of Canada 2011
Abstract The present study documents the changes occurring at the biochemical level in white spruce trees (Picea glauca [Moench] Voss) with contrasted growth phenotypes during the summer period. Full-siblings of tall versus small spruces were grown under controlled conditions at constant day/night temperatures (24/15°C) and exposed to a decreasing photoperiod (15.7–12.2 h) simulating natural photoperiod reduction during the summer in eastern Canada. Growth parameters (stem height and tree biomass) were determined and non structural carbohydrates, soluble proteins and amino acids were quantified in current-year needles and stem, oldest stem and roots from mid-July until the end of September 2006. Sucrose was the main soluble sugar found in all organs, but its concentrations did not significantly change during the summer. In contrast, starch concentrations rapidly declined by the end
of the experiment, especially in needles and stems. Both sucrose and starch did not generally differ between growth phenotypes. Total soluble protein significantly accumulated by mid-August (14.4 h of photoperiod) in small trees. Arginine and glutamine were the most abundant amino acids found in spruce organs, and their concentrations strongly increased at 14.4 h of photoperiod, especially in small trees. Our results highlight marked differences in nitrogen metabolism in late summer between contrasted growth phenotypes, especially for arginine, an amino acid typically associated with growth arrest and nitrogen reserve in perennial species. They also reveal that old stems and roots are important storage organs of organic reserves.
Communicated by F. M. Ca´novas.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s00468-011-0549-7) contains supplementary material, which is available to authorized users.
The annual growth cycle in woody plants is characterized by alternating periods of active growth and dormancy. Morphological and developmental changes that occur throughout the annual growth cycle include alterations in source-sink relationships within the tree, involving major changes in carbohydrate and nitrogen metabolism. Comprehensive studies conducted with Scots pine (Pinus sylvestris L.) and Norway spruce documented the presence of starch and soluble sugars in many plant parts including old and new roots, stem and needles at the beginning of the growing season (Domisch et al. 2001). When growth resumes in early spring, starch reserves initially increase in needles of Scots pine (Mandre et al. 2002) and Norway spruce (Repo et al. 2004) to meet the subsequent carbon requirements of bursting buds. Conversely, an accumulation
C. Dhont De´partement des Sciences du Bois et de la Foreˆt, Faculte´ de Foresterie, de Ge´ographie et de Ge´omatique Universite´ Laval, Quebec, QC G1V 0A6, Canada A. Bertrand (&) Y. Castonguay Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec, QC G1V 2J3, Canada e-mail:
[email protected] N. Isabel Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 PEPS St, Quebec, QC G1V 4C7, Canada
Keywords Amino acids Carbohydrates Growth Picea glauca Soluble proteins
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of starch and soluble sugars in Scots pine needles at the end of the growing period was associated with a lower sink strength caused by reduced stem elongation (Iivonen et al. 2001). Following the induction of dormancy, soluble sugars increased in needles of Norway spruce with a concomitant depletion of starch (Repo et al. 2004), a response typically associated with the cold hardening process (Greer et al. 2000). Starch depletion in Scots pine needles was shown to be correlated with the latitudinal effect on growth duration, further highlighting the impact of day length on carbohydrate metabolism (Oleksyn et al. 2000). A close relationship between the amount of soluble sugars that accumulate in Scots pine needles during the autumn and the number of developing buds in subsequent late summer (Mandre et al. 2002) stressed the importance of the seasonal carbohydrate dynamics on conifer development and productivity. However, studies undertaken in conifers mainly focused on carbohydrate changes in needles without paying as much attention to their accumulation in other reserve organs or to their relationship with growth phenotypes. In woody plants, seasonal nitrogen cycling also goes hand-in-hand with the alternation between growth and dormancy periods and reflects metabolic requirements during developmental changes (Cooke and Weih 2005; O’Kennedy et al. 1975; Tromp 1970). Amino acids such as asparagine, glutamine, arginine and proline, as well as specific soluble proteins harboring characteristics of vegetative storage proteins accumulate during the over wintering period and are readily mobilized during spring regrowth in herbaceaous perennials (Dhont et al. 2006). Together with glutamine, arginine was found to be the major storage form of non proteinous nitrogen in bilberry (Vaccinillum myrtillus) rhizome (La¨hdesma¨ki et al. 1990) and in needles of Scots pine (Gezelius 1986; Nordin et al. 2001). Durzan (1968) also documented that arginine was a major storage form of nitrogen in white spruce needles and buds. In white birch (Betula pendula) (Millard et al. 1998), Scots pine (Gezelius 1986) and Norway spruce (Manderscheid and Ja¨ger 1993), nitrogen is stored in older stems and needles and mobilized to sustain new growth. Nitrogen resorption occurs from senescent leaves and nitrogen is sequestered in over wintering organs (Moreno and Garcı´aMartı´nez 1984). The seasonal cycling of amino acids in white spruce revealed that the accumulation of arginine in needles, as stem elongation ceased, preceded its accumulation in newly formed buds (Durzan 1968). Later in the autumn, arginine was incorporated into newly synthesized proteins in the buds. Considering the photoperiodic control of bud set, it is noteworthy that short day length also induces the accumulation of arginine in shoots of black spruce (Picea mariana) (Odlum et al. 1993) and of soluble storage proteins in bark of poplar (Populus sp.) species (Coleman et al. 1991).
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The metabolic changes occurring in white spruce during the growth cessation period in late summer remain largely unknown. This information could shed light on the biochemical determinants of the large variability that has been reported in growth phenotypes. We therefore hypothesize that growth potential in white spruce is associated with differential accumulation of key carbon and nitrogen reserves. The main objective of this study was to document morphological and biochemical changes occurring throughout the summer, in organs of full-siblings of white spruce with contrasted growth phenotypes (tall versus small), under controlled conditions simulating natural photoperiod decline. Since this research belongs to a functional genomic project, the secondary objective was to determine which combination of metabolite/organ should be targeted for large-scale QTL mapping of genomic regions that affect seasonal productivity.
Materials and methods Plant material In 2002, root cuttings of white spruce (Picea glauca [Moench] Voss) belonging to a QTL mapping population and deriving from a controlled cross (6 cuttings 9 500 progenies; C-94-2516; $ 77111 9 # 2388; Canadian Forest Service) were established under natural outdoor conditions at the Valcartier experimental station of the Canadian Forest Service (46°570 0300 North; 71°290 5000 West; elevation 202 m) in 19-cm diameter, 18-cm deep plastic pots filled with a mixture (250 L: 8.6 kg: 30 mL) of peat moss (Berger Baie-Escouminac, New-Brunswick, Canada), vermiculite (Holiday, Normiska Corporation, Montre´al-Toronto, Canada) and surfactant (Aqua-Gro L with PsiMatricTM Technology, The Scotts Cie, Marysville, OH, USA). At the summer solstice of 2006 (photoperiod of 16 h; Table 1), a subset of the progeny (4-year-old cuttings) was transferred from natural outdoor conditions of Valcartier to set buds under environmentally controlled conditions at Agriculture and Agri-Food Canada (Que´bec, QC, Canada) in a large growth room set to the following conditions: day-time temperature, 24°C; night-time temperature 15°C; photosynthetic photon flux density, of 600–800 lmol photons m-2 s-1 provided by a mixture of high-pressure sodium and metal halide 400 W lamps (PL light Systems, Beamsville, ON, Canada). To simulate natural photoperiod changes occurring at the establishment site of Valcartier, the photoperiod inside the growth room was adjusted weekly from the summer solstice to the end of September, i.e. from 16.0 h to 12.2 h (Table 1). Photoperiod was obtained from an online sunrise/sunset calculator from the National Research Council of Canada
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713
Table 1 Simulation of natural declining photoperiod in growth rooms set to a day-time temperature of 24°C and to a night-time temperature of 15°C
30
Date
Beginning (Hours)
End (Hours)
20
10
16.0
7:00
23:00
30-Jun-06
15.8
7:00
22:48
07-Jul-06
15.7
7:00
22:42
14-Jul-06
15.5
7:00
22:30
21-Jul-06
15.3
7:00
22:18
28-Jul-06
15.1
7:00
22:06
04-Aug-06
14.8
7:00
21:48
11-Aug-06
14.4
7:00
21:24
18-Aug-06
14.1
7:00
21:06
25-Aug-06
13.7
7:00
20:42
01-Sept-06
13.4
7:00
20:24
08-Sept-06
13.0
7:00
20:00
15-Sept-06
12.6
7:00
19:36
22 Sept-06
12.2
7:00
19:12
a
16
15
LS =4.519
0 30
13
Two year old stem
b
12 16 15
20 14 LSD=3.742
10
13 0 30
Oldest stem
c
12 16 15
20
Photoperiod (Hours)
23-Jun-06
Small Tall Photoperiod
14
Height (cm)
Photoperiod
Current year stem
14
(http://www.hia-iha.nrc-cnrc.gc.ca/sunrise_e.html). During the summer 2006, trees were fertigated with a 1 g L-1 solution of a mix 3:1 (w:w) of 20-08-20: 20-20-20 fertilizers. Fertilization was stopped during the first three weeks of August to promote bud set and growth cessation, but trees were kept well watered. Fertigation was subsequently resumed with a 1 g L-1 solution of fertilizer 08-20-30 until the end of the experiment. The solutions for fertigation were prepared with commercial fertilizers (Outdoor conditions: Solutech, Coope´rative Fe´de´re´e, QC, Canada; Controlled conditions: Plant-Prod, Brampton, ON, Canada). White spruce growth phenotypes White spruces showing contrasting growth phenotypes year after year were selected among root cuttings of the 500 progenies from the QTL mapping population (C-94-2516). Twenty-four small and 24 tall trees (one root cutting per each 48 different genotypes) were selected once the bud flush was completed on the basis of previous growth, i.e. the height of the older stem sections. The mean cumulative height of the two-, three- and four- year old stem sections was about 29 cm in small trees versus 48.3 cm in tall trees (Fig. 1b, c). The corresponding average height for the family was about 36.4 cm. Apical bud phenology was recorded according to Dhont et al. (2010). Mean of apical bud stage was 3.3 and 4.0 for small and tall trees, respectively, at the beginning of the experiment, and reached 5.0 for both small and tall trees at the end (Stage 3: apical bud with turning brown scales—needle fan still enclose the
10
LSD=4.182
13 0 60
Cumulative
dD
12 16 15
50 LSD=5.862
14 40 13 30 12 10-Jul
24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct
Fig. 1 Individual height of current-year stem (a), two-year-old stem (b), oldest stems (c) and cumulative height of the stems (d) of small (closed circle) and tall (open circle) white spruce seedlings from midJuly to the end of September under a simulated natural photoperiod decline (Dash line; 15.7–12.2 h). Each mean is the average of three replicates and least significant differences (LSD) at P \ 0.05 are indicated as vertical bars with their corresponding numeric values. Significance of any pairwise comparison of means is available in the Supplementary Data 1 file
bud—significant increase in bud volume; Stage 4: brown bud with brown scales—needle fan starts to spread outwards; Stage 5: brown bud with developed concave scales—needle fan completely opened towards the outside). Organ samplings, height and biomass determinations Trees were sampled on eight occasions throughout the experiment, from mid-July to the end of September 2006 (Table 2). Each tree was dissected into six different organs:
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Table 2 Sampling timetable including sampling dates, corresponding photoperiods, and sampling intervals
Date
Intervals Days between samplings
Days from summer solstice
Controlled conditions
18-Jun-06
16.0
–
0
Sampling 1
13-Jul-06
15.7
0
25
Sampling 2
26-Jul-06
15.3
12
38
Sampling 3
09-Aug-06
14.8
14
52
Sampling 4
17-Aug-06
14.4
8
60
Sampling 5
24-Aug-06
14.1
7
67
Sampling 6 Sampling 7
31-Aug-06 14-Sep-06
13.7 13.0
7 13
74 88
Sampling 8
29-Sep-06
12.2
15
103
apical bud, current-year needles, current-year stem, 2-year old stem, oldest stems (3- and 4-years old) and roots. The height of each stem section was measured and cumulative height was obtained by adding them. Entire stem sections (without needles) and roots were weighed for total biomass determination, and then cut into small segments (about 2–3 mm) for sub sampling. Entire apical bud (20–160 mg) and two sub samples of each other organs (300–600 mg for current-year needles; 100–500 mg for current-year stems; 400–700 mg for 2-year old stems; 500–700 mg for oldest stems; 1 g for roots) were weighed, frozen in liquid nitrogen and stored at -80°C until further extractions. A sub sample of each organ (except apical bud) was weighed and oven dried for 48 h at 55°C for dry matter determination. Percent dry weight was used to calculate the concentrations of organic components and to determine stem sections and root total biomasses on a dry weight basis. Soluble sugar and amino acid extraction Soluble sugars and amino acids were extracted by the method described by Dhont et al. (2002, 2003) and adapted to the small size of the samples. Frozen samples were ground with liquid nitrogen in a mortar with a pestle to obtain a fine powder. Extraction buffer containing methanol-chloroform-water (MCW) (12:5:3; v:v:v:) was added at a 1:10 (w:v) ratio to ground samples, and mixtures were immediately incubated at 65°C for 30 min to inhibit enzymatic activities. The phase separation occurred in micro tubes by adding 250 lL of distilled water to an aliquot of 1 mL of the MCW extract and by centrifuging at 13,000 rpm for 3 min. Part of the aqueous phase (750 lL) was collected and evaporated to dryness in a vacuum evaporator (Savant SpeedvacÒ Plus, SC210A, Thermoquest, Holbrook, NY, USA). The dried extract was then solubilized with 750 lL of ethylene-diaminetetraacetic acid buffer (EDTA; Na?, Ca2?; 0.13 mM). These extracts were kept frozen at -80°C until HPLC analyses. The non-
123
Photoperiod (Hours)
soluble residues left after extraction were washed twice with 10 mL of methanol and used for starch determination. Carbohydrate determination Mono-, di-, tri- and tetrasaccharides were separated and quantified on a Waters HPLC analytical system controlled by the Empower software (WATERS, Milford, MA, USA) as described by Dhont et al. (2002). Peak identity and sugar quantity were determined by comparison to standards. Starch was quantified as glucose equivalents with the p-hydroxybenzoic acid hydrazide method of Blakeney and Mutton (1980) after gelatinization at 100°C and digestion for 90 min with amyloglucosidase (Sigma A7255, Sigma Chemical Co., St-Louis, MO). Starch amounts were determined spectrophotometrically by reference to a glucose standard curve. Only sucrose and starch are presented and discussed with regard to the tree size and sampling date treatments, but complete carbohydrate data and values of least significant differences for each variable are available in Supplementary Table 1. Soluble amino acid determination Nineteen amino acids (aspartatic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, c-amino butyric acid, a-amino butyric acid, methionine, valine, phenylalanine, leucine, isoleucine, lysine) were separated and quantified on a Waters HPLC analytical system controlled by the Empower software (WATERS, Milford, MA, USA) using pre-column derivatization with o-phtaldehyde as described by Dhont et al. (2003). Peak identity and amino acid quantity were determined by comparison with a standard mix containing the 19 amino acids. Because of their relative abundance compared with the other amino acids, only the results of arginine and glutamine are presented and discussed in regard to the tree size and photoperiod
Trees (2011) 25:711–723
715
Table 3 Analyses of variance of tree size, sampling date main effects and corresponding interaction for phenotypic and metabolic data from white spruce organs Organs
Effects
Height
Biomass
Sucrose
Apical bud
Size
–
–
Date
–
–
Size 9 date Size
– –
Date Size 9 date
Needles
Current-year stem
Two-year old stem
Oldest stem
Roots
Starch
Total amino acids
Arginine
Glutamine
Total soluble proteins
ns
ns
ns
ns
0.059
–
ns
\0.001
ns
0.003
ns
–
– –
ns ns
ns ns
ns 0.002
0.044 \0.001
0.067 0.066
– 0.062
–
–
\0.001
\0.001
\0.001
\0.001
ns
0.050
–
–
ns
ns
ns
ns
ns
ns 0.049
Size
ns
ns
ns
ns
ns
ns
0.021
Date
ns
ns
0.023
\0.001
0.020
\0.001
ns
ns
Size 9 date
ns
ns
ns
ns
ns
ns
ns
ns \0.001
Size
\0.001
\0.001
ns
ns
0.010
0.057
0.028
Date
ns
0.008
0.004
\0.001
\0.001
\0.001
ns
ns
Size 9 date
ns
ns
ns
ns
ns
ns
ns
ns
Size
\0.001
\0.001
ns
ns
\0.001
\0.001
0.005
0.002
Date
ns
0.025
ns
\0.001
0.002
\0.001
0.072
0.004
Size 9 date
ns
ns
ns
ns
ns
ns
ns
ns
Size
–
\0.001
ns
ns
0.007
0.004
0.010
\0.001
Date
–
\0.001
ns
ns
0.051
0.001
ns
0.004
Size 9 date
–
ns
ns
ns
ns
ns
ns
ns
Significance levels are indicated ns non significant
treatments. However, complete amino acid data and values of least significant differences for each variable are available in Supplementary Table 2. Total soluble proteins Total soluble protein extraction was optimized for white spruce organs by Dr. J. Cooke et al. (University of Alberta; Personal communication) and adapted to our sample size. Frozen samples were ground in liquid nitrogen with a mortar and a pestle to obtain a fine powder. Extraction buffer contained 100 mM of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 10 mM dithiothreithiol (DTT), 10% glycerol, 7.5% polyvinylpolypyrrolidone (PVPP), 0.3% sodium diethyldithiuocarbamate (DIECA) and a protease inhibitor cocktail (Bioshop Canada, Burlington, ON, Canada; working concentrations of 0.5 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 lM aprotinin, 10 lM bestatin, 10 lM E64 and 10 lM leupeptin). Extraction buffer was added to ground samples with a ratio of 1:5 (w:v) and mixture was homogenized by alternation of vortexing and sonication in a BransonicÒ ultrasonic cleaner 2210 (Branson Ultrasonics Corporation, Danbury, CT, USA). Homogenates were then centrifuged
at 4,000 rpm at 5°C for 30 min. Proteins within the supernatant were precipitated with four parts of ice-cold acetone at -20°C for 60 min. Protein pellets were air dried and solubilized in NaOH 0.1 N before the quantification using the Folin-Phenol reagents (Lowry et al. 1951) with reference to a standard curve of bovine serum albumin. Data and statistical analyses Metabolite profiling data for the 2-year-old stem were similar to those obtained from oldest stem. For that reason, only old stem data are actually presented with regard to the metabolites. The experiment was a factorial combination of two sizes of white spruce 9 eight sampling dates arranged in a randomized complete block design with three replicates. Experimental units were single trees. Analyses of variance were performed on tree size and sampling date main effects, and their corresponding interactions for each variable and each organ (Table 3). To establish the significance of differences between each isolated dates for both types of phenotypes, pairwise comparisons of means were performed for all biomass and biochemical variables measured. Pairwise comparisons of means are based on the least significant differences calculated as twice standard error of the mean. Significance of any pairwise comparisons of means, which
123
716
Trees (2011) 25:711–723
was established at P \ 0.05, is available in the Supplementary Data 1 file. Standard Deviations are also provided in Supplementary Table 3. Overall statistical significance was postulated at P \ 0.05. Statistical analyses were performed by SAS statistical procedures (Statistical Analysis System Institute Inc. 1999–2001).
1.0
Current year stem
0.8 0.6
Small Tall Photoperiod
a
16
15
LSD=0.190
14
0.4 13
0.2 0.0 5
Results
4
White spruce growth
Two year old stem
b
12 16 15
LSD=0.812
Carbohydrate components in white spruce during the summer period Sucrose was the most abundant carbohydrate found in the different spruce organs, representing 30–70% of total non structural carbohydrates (Supplementary Table 1). Seasonal evolution of sucrose concentrations markedly vary among organs during the summer (Fig. 3a–d; Table 3). In needles, sucrose concentrations did not significantly differ between tall and small spruces (Fig. 3a). Those concentrations sharply dropped from 30 to 16 mg g-1 DW between 13 and 26 of July. Sucrose concentrations subsequently recovered and reached a peak near their initial values by the end of August and significantly declined afterwards until the end of the experiment (Fig. 3a). Fluctuations of sucrose concentrations were observed in the
123
Biomasses (g DW)
14 2 13
1 0 10 8
Oldest stem
c
12 16 15
LSD 2.084
6
Photoperiod (Hours)
3
The elongation of the current-year stem did not differ between tall and small spruces (Fig. 1a; Table 3). The height of the 2-year-old and oldest stems remained stable throughout the summer period and was significantly higher in tall spruces than in small growth phenotypes (Fig. 1b, c). Overall, the cumulative height was stable throughout the experiment and was significantly higher in tall spruces (Fig. 1d). Current-year stem biomass remained stable throughout the experiment and did not noticeably differ between small and tall seedlings (Fig. 2a; Table 3). In contrast, biomasses of 2-year-old and oldest stems and roots significantly increased throughout the summer period and were significantly different between growth phenotypes, being two- to three-times higher in tall seedlings as compared with the small ones (Fig. 2b, c and d). The magnitude of the difference in old stem and root biomasses between growth phenotypes was similar at the beginning and the end of the experiment. In tall seedlings, biomass increase for 2-yearold stems and roots mainly took place between 26th of July and 9th of August (Fig. 2b, d). Biomass increase was more progressive and sustained until the end of August in the oldest stems of tall seedlings (Fig. 2c). Oldest stem and root biomasses of small seedlings increased until midAugust (Fig. 2c, d).
14
4 13
2 0 20 15
Roots
D d
12 16 15
LSD=3.251
10
14
5
13
0 10-Jul
12 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct
Fig. 2 Biomasses of current-year stem (a), two-year-old stem (b), oldest stems (c) and roots (d) of small (closed circle) and tall (open circle) white spruce seedlings from mid-July to the end of September under a simulated natural photoperiod decline (Dash line; 15.7–12.2 h). Each mean is the average of three replicates and least significant differences (LSD) at P \ 0.05 are indicated as vertical bars with their corresponding numeric values. Significance of any pairwise comparison of means is available in the Supplementary Data 1 file
current-year stem but final concentrations were similar to those measured at the beginning of the experiment (Fig. 3b). Sucrose concentrations in oldest stems were significantly different between growth phenotypes, being noticeable by mid-August with higher concentrations in small trees than in tall ones (Fig. 3c). Except for a transient increase in small trees from mid to late August (17th–24th), root sucrose concentrations remained stable at approximately 17 mg g-1 DW (Fig. 3d). Starch content varied significantly between sampling dates, ranging from 5 to up to 55% of total non structural carbohydrates (Supplementary Table 1). Seasonal profiles
Trees (2011) 25:711–723 Sucrose
50
Starch
a
Needles 40
e
16
LSD=4.274
LSD=6.120
15 Small Tall Photoperiod
30
14
20 13
10 0 30
b
Current year stem
f
LSD=5.426
12 16
LSD=3.083
15 20
10
0 20
c
Oldest stem LSD=3.563
15
13
g
12 16
LSD=3.127
15
10
14
5
13
0 30
d
Roots LSD=5.309
h
Photoperiod (Hours)
14
Photoperiod (Hours)
Carbohydrate (mg. g -1 DW)
Fig. 3 Concentrations of sucrose (a–d) and starch (e–h) in current-year needles (a and e), current-year stem (b and f), oldest stems (c and g) and roots (d and h) of small (closed circle) and tall (open circle) white spruce seedlings from mid-July to the end of September under a simulated natural photoperiod decline (Dash line; 15.7–12.2 h). Each mean is the average of three replicates and least significant differences (LSD) at P \ 0.05 are indicated as vertical bars with their corresponding numeric values. Significance of any pairwise comparison of means is available in the Supplementary Data 1 file
717
12 16
LSD=8.191
15 20 14 10 13
0
12
10-Jul 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct 10-Jul 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct
of starch concentrations were very similar in both small and tall spruces, regardless of the organ (Fig. 3e–h; Table 3), with the exception of current-year stem on mid-August sampling where starch concentrations of small seedlings exceeded values observed in tall ones (Fig. 3f). A significant decrease in starch concentrations occurred in needles and youngest stems at the beginning of the experiment (Fig. 3e, f). In needles, concentrations of starch rapidly dropped from about 21 mg g-1 DW to reach baseline as early as 26th of July (Fig. 3e). Starch concentrations progressively decreased from 25 to 7 mg g-1 DW until late August in the current-year stem (Fig. 3f). In oldest stem sections, a more gradual decline of starch concentrations occurred from 13 to 3.5 mg g-1 DW during the summer period (Fig. 3g). No defined trend was observed for root starch concentrations in white spruce (Fig. 3h).
Nitrogen components in white spruce during growth cessation Soluble protein concentrations showed significant differences between spruce growth phenotypes during the summer period (Table 3; Fig. 4a–d). In needles, concentrations of soluble proteins decreased during the first half of the experiment from 16 to 10.5 mg g-1 DW between mid-July and mid-August (Fig. 4a). Thereafter, these concentrations increased significantly until the end of August and remained relatively stable until the end of September. In spite of a clear tendency for small tree concentrations to be higher in late summer (August 31), there was no significant difference between tall and small trees (Fig. 4a). With the exception of the two first samplings in July, current-year stem concentrations of soluble proteins also did not
123
718
20
Total Soluble Proteins Needles
a
15
16
16
15
15
Total Amino Acids
e
150
100
10
Current year stem
13
12 16
12 16
15
15
LSD=33.445
bb
25 20
LSD=8.038
13
5 0 8
Oldest stem
c
12 16
15
6
Photoperiod (Hours)
14
0
100 80
LSD=22.215
15 10
f
60
-1
30
13
50
Small Tall Photoperiod
0
14
14
40 13
20
12 16
g
0 60 50
15
40 4
14
14
13
13
12 16
12 16
15
15
LSD=11.493
30 20
2 0 10
LSD=1.575
Roots
DW)
LSD=3.420
14
Total Amino Acids (µmoles. g
5
Total Soluble Proteins ( mg. g -1 DW)
Fig. 4 Concentrations of total soluble proteins (a–d) and total amino acids (e–h) in currentyear needles (a and e), currentyear stem (b and f), oldest stems (c and g) and roots (d and h) of small (closed circle) and tall (open circle) white spruce seedlings from mid-July to the end of September under a simulated natural photoperiod decline (Dash line; 15.7–12.2 h). Each mean is the average of three replicates and least significant differences (LSD) at P \ 0.05 are indicated as vertical bars with their corresponding numeric values. Significance of any pairwise comparison of means is available in the Supplementary Data 1 file
Trees (2011) 25:711–723
10
d
h
0 60 50
8
40 6
LSD=16.456
14
14
13
13
4 2
20 LSD=1.652
10
12 0 10-Jul 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct
significantly differ between tall and small trees (Fig. 4b). These concentrations showed essentially a profile similar to the one observed for the needles. Proteins profiles in oldest stem also showed a progressive increase that was more pronounced in the small trees by the end of September (Fig. 4c). Soluble protein concentrations in roots increased progressively throughout the summer period (Fig. 4d). At both first and last sampling times, root protein concentrations were significantly higher in the small trees as compared with the tall ones. Concentrations of total amino acids significantly differed between small and tall spruces (Table 3; Fig. 4e–h). In needles, total amino acid concentrations rapidly decreased from 97 to 30 lmoles g-1 DW in both phenotypes between mid and late July (Fig. 4e). Thereafter, total amino acids significantly increased in small trees while remaining stable in the tall ones. In current-year stem, concentrations of total amino acids remained stable in tall
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trees throughout the summer period and showed a transient but significant increase in the small ones by the end of August (Fig. 4f). Similarly in both old stem and roots, amino acid concentrations increased significantly by midAugust in small seedlings, whereas they remained stable in the tall ones throughout the experiment (Fig. 4g, h). Among the 19 amino acids measured in both small and tall seedlings, arginine and glutamine were the two most abundant found in the different spruce organs (Supplementary Table 2). Respectively, they constituted between 11 to 88% and 4 to 38% of total amino acids, depending on growth phenotypes, dates and organs. In all organs, concentrations of arginine differed significantly between small and tall seedlings (Fig. 5a–d; Table 3). In both growth phenotypes, arginine concentrations in needles rapidly decreased from 57 to 16 lmoles g-1 DW between mid and late-July (Fig. 5a). Thereafter, arginine accumulated in needles of small trees, whereas it
Trees (2011) 25:711–723 Arginine 80
Glutamine
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Needles
Small Tall Photoperiod
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e
16
15
14
40
20
13
LSD=4.321 LSD=21.404
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15 LSD=5.065
LSD=16.909
40
c
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20 15
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14
20
0 25
12 16
13
g
12 16
15
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0 80
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Fig. 5 Concentrations of arginine (a–d) and glutamine (e–h) in current-year needles (a and e), current-year stem (b and f), oldest stems (c and g) and roots (d and h) of small (closed circle) and tall (open circle) white spruce seedlings from mid-July to the end of September under a simulated natural photoperiod decline (Dash line; 15.7–12.2 h). Each mean is the average of three replicates and least significant differences (LSD) at P \ 0.05 are indicated as vertical bars with their corresponding numeric values. Significance of any pairwise comparison of means is available in the Supplementary Data 1 file
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LSD=5.021
LSD=5.634
14 10 13
5 0 25
h
d
Roots
20 15
12 16
15 LSD=6.558
LSD=6.547
14 10 13
5 0
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10-Jul 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct 10-Jul 24-Jul 07-Aug 21-Aug 04-Sep 18-Sep 02-Oct
remained stable at a low level in tall trees (Fig. 5a). In the current-year stem, concentrations of arginine increased progressively from 18 to 40 lmoles g-1 DW with a transient peak reaching 75 lmoles g-1 DW in small trees by midSeptember (Fig. 5b). In both old stem and roots, a marked increase in arginine concentrations occurred throughout the summer period (Fig. 5c, d). This increase, that started earlier and was of higher amplitude in small trees, was clearly amplified at mid-August. Concentrations of glutamine in needles and current-year stem were markedly lower than those of arginine in these two organs (Fig. 5e, f; Table 3). Seasonal patterns of glutamine concentrations significantly differed between growth phenotypes in stems and roots (Fig. 5f–h). Concentrations of glutamine remained relatively stable in tall
trees throughout the summer period while it markedly accumulated in small trees, particularly after mid-August in oldest stems and roots. However, by the end of September, glutamine concentrations in tall trees no longer differed from those measured in small trees (Fig. 5f–h).
Discussion White spruce growth Tall spruces had significantly higher root and oldest stem biomasses, advantageously conferring these trees a larger capacity for assimilation and transport of photosynthates as well as a larger potential of reserve organs for carbohydrate
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and nitrogen. Most of the current-year stem growth was completed before plants were transferred to environmentally controlled conditions, with current apical growth being similar for tall and small trees. Similar growth of current-year stems was unlikely the result of the transfer to environmentally controlled conditions since other clonal copies of these trees, which remained under natural conditions, showed a similar lack of difference in current-year stem growth between the two growth phenotypes (data not shown). No significant elongation of the current-year stem was observed following tree transfer, indicating that apical growth had already stopped at the time of the first sampling in mid-July (15.7 h of photoperiod). This indication is further reinforced by the observation of advanced stages of apical bud development at 15.7 h in both tall and small trees. As a consequence, subsequent biomass increase in stems would be attributable to the accumulation of organic reserves and to radial growth resulting from secondary meristem activity. The arrest of radial growth resulting from wood formation and cambial activity (Egertsdotter et al. 2004b; Gricˇar et al. 2005) occurred under a shorter photoperiod than the arrest of apical growth in Norway spruce (Heide 1974) and in birch (Betula pubescens Ehrh.) (Ha˚bjørg 1972). Even though the increase in stem biomass occurred progressively throughout the present experiment, it mostly took place before the photoperiod has reached 14.4 h. This could be a reflection of a transition from earlywood to latewood formation, which was positively related to the day length reduction in Norway spruce (Heide 1974) and Scots pine (Pinus sylvestris) (Uggla et al. 2001). Changes in carbohydrate metabolism in white spruce during growth cessation The present study documents major changes occurring in main carbohydrate composition during growth cessation of white spruce. Most organs investigated showed a decrease in starch concentrations in the summer, with current-year needles and stems having the most extensive depletion. Previous observations made with young and mature Scots pine trees under natural declining photoperiod also highlighted a marked decrease of starch concentrations in current-year needles between July and September (Mandre et al. 2002; Oleksyn et al. 2000; Terziev et al. 1997). Soluble sugars are known to constitute most of the carbohydrate pool of the needles and roots of young black spruce (Picea mariana (Mill.) B.S.P.) (Bertrand and Bigras 2006). We observed similar carbohydrate composition in needles and roots of white spruce, as well as in current-year and old stems (Supplementary Table 1). Since starch depletion was not concomitant with a sucrose increase, we questioned the fate
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of carbohydrate reserves after stem elongation has ceased in white spruce. Starch in needles and stems of white spruce could be mobilized to sustain the deposition of cell wall polysaccharides required for radial growth and wood formation (Uggla et al. 2001). This is supported by our observation of starch depletion at the time when stem biomass increased. It is also possible that starch from needles and stems provided C skeletons and energy supply for amino acid biosynthesis, as previously observed in young apple trees (Malus domestica Borkh.) (Cheng et al. 2004). In addition, sucrose and starch remained at high levels in roots as compared with the other organs, suggesting a pivotal role for belowground organs as carbohydrate storage reserve. Scots pine roots were shown to accumulate starch in the spring to sustain new shoot growth, and soluble sugars later in the autumn to withstand subfreezing temperatures (Iivonen et al. 2001; Oleksyn et al. 2000). Non structural carbohydrate concentrations did not generally differ between tall and small spruces, in spite of major biomass differences in roots and oldest stems. This differs from previous reports obtained with Scots pine, showing that decrease in tree productivity was related to lower needle carbohydrate concentrations as well as to lower sink strength of aboveground organs (Oleksyn et al. 2000). The current study rather suggests that carbohydrate availability may not have been a limiting factor for growth under our experimental conditions and that carbohydrates may not be used as indicators of growth potential in white spruce. Changes in nitrogen metabolism in white spruce during growth cessation Nitrogen sources in the form of free amino acids and soluble proteins increased significantly in small trees in late summer, primary evidence of a major transition in nitrogen metabolism during the growth cessation period. Arginine and glutamine were the two most abundant amino acids found in the different spruce organs investigated, especially in old stems and roots (Supplementary Table 2). Arginine was by far the most important nitrogen component in white spruce organs. It is a major form of nitrogen storage in many tree species, including Scots pine (Gezelius 1986; Nordin et al. 2001), loblolly pine (Pinus taeda L.) (King and Gifford 1997), black spruce (Bertrand and Bigras 2006; Kim et al. 1987), Norway spruce (Manderscheid and Ja¨ger 1993) and deciduous sycamore (Acer pseudoplatanus L.) (Millard and Proe 1991). Durzan (1968) previously reported a peak of arginine in buds and needles of white spruce during growth cessation and bud set period. We observed a progressive but significant accumulation of arginine in stems and roots, with a surge occurring by mid-August. Our results showed that a transition seems to occur in nitrogen metabolism when natural day length approaches 14 h. Odlum et al. (1993) concluded that
Trees (2011) 25:711–723
the accumulation of arginine in black spruce shoot was likely induced by exposure to short days. The surge of arginine was of higher amplitude in small trees, indicating differential arginine metabolism between growth phenotypes. N-rich amino acids such as arginine have been proposed to reflect nitrogen status in woody plants (Ca´novas et al. 2007). In the context of growth cessation, higher accumulation of arginine could indicate a higher level of nitrogen recycling and storage in small spruces. Arginine has also been related to the regulation of plant development and stress tolerance as a precursor of polyamines (Bouchereau et al. 1999; Sua´rez et al. 2002) and to the acquisition of cold tolerance (Coker 1991). Recently, Durzan (2010) reported that constant shading stress resulted in an accumulation of arginine and reduced growth of white spruce. Preferential accumulation of arginine in white spruce organs could be a part of a global adaptive response to cope with reduced growth, biotic and abiotic stresses. Regardless of its physiological role, arginine accumulation during the summer appears as a useful indicator for growth phenotypes in white spruce. Further investigation of changes in key enzyme activities and corresponding gene expression of arginine metabolism during the growth cessation period would help to unravel the physiological and molecular basis of differential growth phenotypes. Screening QTL mapping populations for arginine contents could also help to define genome regions affecting white spruce productivity. Glutamine showed different profiles between growth phenotypes, increasing only in the small trees by midAugust. These results support the occurrence of a transition in nitrogen metabolism in the small spruces when photoperiod neared 14 h. Glutamine is a key amino acid involved in nitrogen assimilation in plants (Forde and Lea 2007), and because of its uncharged properties, it has been hypothesized to be an efficient form of nitrogen transport in Scots pine (Gezelius 1986; Nordin et al. 2001). Recent reviews on nitrogen assimilation in woody plants stressed the importance of glutamine as a precursor of tyrosine and phenylalanine, which are involved in the phenylpropanoid pathway leading to the biosynthesis of lignin (Gallardo et al. 2003; Sua´rez et al. 2002). This pathway is of major importance in the context of wood tracheid formation initiated in late summer and usually completed within the autumn or next spring (Donaldson 1992; Egertsdotter et al. 2004a; Gricˇar et al. 2005). In the perspective of a stem diameter increase and radial growth of spruce with declining photoperiod, the transient differential accumulation of glutamine in small trees could result from a delay in the lignin synthesis and deposition. On the other hand, transient accumulation of glutamine could also reflect more efficient nitrogen recycling during the period of active diameter growth, which could be of major importance in terms of nitrogen economy for the small trees (Ca´novas
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et al. 2007; Forde and Lea 2007). Recent reviews highlighted the role of glutamine and its biosynthesis via glutamine synthetase in the regulation of nitrogen assimilation (Miller et al. 2007), tree growth and development (Forde and Lea 2007), and response to environmental stresses (Bernard and Habash 2009). Further analyses of glutamine profile in relationship with lignin deposition might help to understand the contrasting responses of small and tall spruces with regard to growth cessation and nitrogen metabolism. Patterns of soluble protein accumulation in old stems and roots were very similar to those observed for arginine and glutamine, with concentrations increasing progressively during the summer as photoperiod declined. These results suggest that increases in free amino acids did not result from protein breakdown, but rather from de novo synthesis. Significant accumulation of nitrogen in old stems and roots of white spruce pointed out a storage role for those organs, as previously reported by Millard and Proe (1991) in over wintering deciduous sycamore. Since amino acids and protein accumulation are known to occur at the expense of non structural carbohydrates (Cheng et al. 2004), nitrogen storage in old stems and roots must likely be a strong sink for carbohydrates. Several woody species are known to accumulate specific proteins under short days. For instance, the accumulation of storage protein in terminal buds of interior spruce (Picea glauca (Moench) Voss 9 Picea engelmanni Parry) exposed to an 8-h photoperiod was considered as an integral part of fall acclimation (Binnie et al. 1994). Renaut et al. (2008) recently reported that major modifications in peach (Prunus persica L. Batsch.) bark proteome induced by short days (8 h) were associated with growth cessation and terminal bud set. Interestingly, poplar (Populus deltoı¨des) bark storage proteins (bsp) started to accumulate after a photoperiod of 14.1 h (Coleman et al. 1991), and bsp gene regulation appeared to be not only phytochromemediated, but also influenced by primary metabolites like sucrose and glutamine (Zhu and Coleman 2001). Even though an earlier study by Wetzel and Greenwood (1989) did not reveal the presence of storage protein in bark of Picea spp., it is likely, based on our results, that proteins responsive to photoperiod accumulate in stems and roots of white spruce. Research is currently underway to characterize qualitative profiles of soluble proteins during the transition from active growth to fall dormancy in white spruce.
Conclusion Organic reserve profiling of white spruce in late summer revealed significant changes in central nitrogen metabolites
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when photoperiod neared 14 h, suggesting a major transition in nitrogen metabolism at that time. The use of full-siblings with contrasting phenotypes enabled the discrimination of metabolic changes related to growth. This approach revealed marked differences in levels of nitrogen components between small and tall spruces, with a higher accumulation of arginine and glutamine occurring in small trees. The use of full-siblings also warrants that variations in arginine and glutamine contents are endorsed by segregating genetic background. Therefore, arginine and glutamine contents are good candidate traits for QTL mapping of genomic regions associated with growth potential of white spruce (Pelgas et al. 2011). Our results also indicated that old stems and roots are major pools of organic reserves in white spruce. Investigation of enzymes and genes of arginine metabolism and lignin biosynthesis, as well as a more detailed analysis of white spruce proteome in late summer in these organs would help to understand the molecular determinant of growth in white spruce. Acknowledgments The authors gratefully acknowledge Patricia Sylvestre from the Laurentian Forestry Centre of the Canadian Forest Service (Natural Resource Canada, Que´bec), Jose´e Bourassa and Lucette Chouinard from Agriculture and Agri-Food Canada (Que´bec) for their technical assistance during sample collection. Thanks are extended to students: Ste´phanie Labbe´-Gigue`re, Charles-Olivier Laporte and Joannie Normandin, for their contribution in sample preparation and analyses. This research was undertaken on behalf of the Arborea project (http://www.arborea.ulaval.ca/en/) and supported by a research grant from Genome Quebec, Genome Canada and Canadian Genome Initiative to J. Mackay, J. Bousquet and N. Isabel.
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