Identification and quantification of carbohydrates in the black spruce

Identification and quantification of carbohydrates in the black spruce

In plants, carbohydrates are synthesized in leaves (the source), translocated to the growing tissues (the sinks) by the phloem in form of sucrose and stored as sucrose or starch (Roitsch and Gonzalez, 2004; Ainsworth and Bush, 2011). They play an essential role to sustain cell metabolism and growth. In the cambium zone, as well as in differentiating xylem (Uggla et al9 2001; Antonova and Stasova, 2008) enzymatic activities converting sucrose into UDP-glucose, glucose and fructose (Uggla et al, 2001) are observed in order to sustain wood formation. In poplars, the rates of cell division in cambium was positively linked with carbohydrates content (Deslauriers et al, 2009). Metabolism regulation of growth hormones, like auxin (Sairanen et al3 2013), and metabolic signaling pathway (O’Hara et al, 2013; Bolouri Moghaddam and Van den Ende, 2013) also appeared to be drive by carbohydrates.

In stressing events, carbohydrates are one of the most utilised compounds in the plants. They are known to play a role in maintaining hydraulic function during severe drought (Sala et al, 2012) and to increase freezing tolerance during cold season (Chen et ai, 2012) to cite only few examples. But carbohydrates can also be affected by biotic and abiotic stresses (see Smith and Stitt (2007) and Ericsson et al. (1996) reviews). For example, in general, a decrease of carbohydrates is observed when photosynthesis is less efficient or stopped due to temperature increase and/or water deficit. However, in case of higher night temperatures, amplification of respiration rate can bring an acceleration of starch consuming. Because of the lack of carbon energy reserve in the morning a signal is sent to produce carbohydrates in high quantity when photosynthesis begin, leading to a rapid but not sustained sucrose rise (Turnbull et al, 2002; Turnbull et al, 2004). Also, in conifers, starch was found to increases with altitude (Hoch and Kôrner, 2012) but no effect of artificial warming was detected in the sugars and starch availability at high altitude (Streit et al, 2013).

On the plant metabolism point of view, changes in temperature and water availability have been studied in many species from different age or growth (see review of Kozlowski and Parllardy (2002) and Kranensky and Jonak (2012) for more details). For black spruce (Picea mariana (Mill.) B.S.P.), the most important species in the boreal forest of North America, increased temperature and drought are both supposed to affect photosynthesis and growth : a rise of 8°C during day and night led to a growth reduction and a decrease of the root/shoot ratio on young seedlings (Way and Sage, 2008a). Moreover, such a temperature increase, negatively affected the needle morphology of black spruce by producing thinner and less dense needles with higher mesophyll cells per foliar surface, and the gas exchange with a decrease of CO2 assimilation and photosynthesis inducing higher respiration rate (Way and Sage, 2008a, b). Therefore, these effects on the leaf morphology and on the gas exchange could decrease the carbon gain (both sugar and starch) leading to growth reduction. An increase of 5°C on ten-years-old black spruce had no effect on photosynthesis and respiration and allowed higher production of new shoots but a lower production of fine roots (Bronson and Gower, 2010). On a seasonal scale, a warming of 3°C was supposed to increases the period of cambium and xylem growth from 125 to 160 days (Rossi et al, 2011), which means that more carbon will be needed to sustain growth for a longer period.

Study area and experimental design 

The study took place in a greenhouse complex located at the Université du Québec à Chicoutimi5 QC, Canada (48° 25′ N, 71° 04′ W, 150m above sea level). The mean annual temperature in 2010 and 2011 were 5.2°C and 2.2°C respectively. The higher mean temperature in 2010 was caused by a particularly hot winter and spring with a mean January-May temperature of -0.2°C compared with -4.5°C in 2011. The temperature in the summer of 2010 and 2011 were about the same with a mean of 18.1°C and 17.6°C respectively.

Two experiments were performed in a greenhouse divided in three independent sections and automatically controlled with a misting and windows opening systems for the cooling. About three hundreds black spruce seedlings were installed every year in each section for the two irrigation regime treatments (one hundred fifty plants per irrigation regime). Plants consisted of four years old seedlings transplanted in 4.5 L plastic pots filled with a peat moss, perlite and vermiculite mix, and left in an open field for the whole previous growing season and during winter. In April of each year, the seedlings were taken inside the greenhouse for the experiment and plant were fertilized with 1 g-1″1 of NPK (20-20-20) fertilizer dissolved in 500 ml of water. Only the healthier trees were selected for the experiment, while the other ones were used in the buffer zone at the borders. Overall, the seedlings were 48.9 ± 4.7 cm in height, with a diameter at the collar of 8.0 ± 2.0 mm. Each seedling was equipped with drip trickles to perform the irrigation. In each section, different irrigation and temperature regimes were applied (table 1). Control (named TO) corresponded with outside temperature, while the two other sections were subjected to a specific thermal condition in respect to control. In 2010, T2 and T5 experienced a temperature of 2 and 5 K higher than TO, respectively. In 2011, T6D and T6N were warmer of 6 K than TO during the day or during the night, respectively (figure 1). For irrigation, control consisted in maintaining the soil water content over 80% of field capacity, while the other seedlings were submitted to a water deficit from mid-May to mid-June, when cambium was vigorously differentiating (Rossi et al, 2009b; Rossi et al, 2009a).

Xylem growth 

From May to September, stem disks were weekly collected 2 cm above the root collar from 36 randomly-selected seedlings (6 seedlings x 3 thermal conditions x 2 irrigation regimes) from 8:00 AM to midday (Balducci et al, 2013). The samples were dehydrated with successive immersions in ethanol and D-limonene, embedded in paraffin and transverse sections of 8-10 jam thickness were cut with a rotary microtome (Rossi et al, 2006a). In each sampled seedling, the length of apical shoot was measured.

The sections were stained with cresyl violet acetate (0.16% in water) and examined within 10-25 minutes with visible and polarized light at magnifications of 400-500* to distinguish the developing xylem cells. For each section, the radial number of (i) cambial, (ii) enlarging, (iii) cell-wall thickening, and (iv) mature cells were counted along three radial files according to Rossi et al (2006b). In cross section, cambial cells were characterized by thin cell walls and small radial diameters. During cell enlargement, the tracheids still showed thin primary walls but radial diameters were at least twice those of the cambial cells. Observations under polarized light discriminated between enlarging and cell wall thickening tracheids. Because of the arrangement of the cellulose microfibrils, the developing secondary walls glistened when observed under polarized light, whereas no glistening was observed in enlargement zones where the cells were still just composed of primary wall (Abe et al, 1997). The progress of cell wall lignification was detected with cresyl violet acetate reacting with the lignin (Antonova and Shebeko, 1981). Lignification appeared as a colour change from violet to blue. A homogeneous blue colour over the whole cell wall revealed the end of lignification and the reaching of tracheid maturity (Gricare/a/.,2005).

Secondary growth 

In the cambial zone, similar annual trends and amounts of cambial cells were observed between the two treatments (figure 2). In May, on average 6 closely spaced cells were observed in the cambial zone. From DOY 125 to 251, the number of cambial cells fluctuated with values ranging between 5 and 10, with maximum observed on DOY 187- 194 in 2010 and 139-147 in 2011. Once annual activity had ended and the cambium stopped dividing, the number of cells in the cambial zone gradually decreased to the minimum value, corresponding to quiescence conditions of the meristems. Between 4 and 6 cambial cells were observed in autumn, a lower number than at the beginning of the season . The only dissimilarities that could be observed were the 1 to 3 more cells in the irrigated plants than the non-irrigated ones just after the water deficit period in the control temperature and +2K in 2010.

Cell enlargement also showed similar trend between the treatments (figure 2). As for cambium, the maximum of cell enlargement (4 to 5 cells) occurred in the middle of the season in 2010 and at the beginning in 2011, with some difference: in 2010 the maximum occurred at DOY 215, 208 and 201 for the control temperature, +2K and +6K respectively. The difference of 1 or 2 cells along the growing season in 2010 didn’t seem to be related to irrigation or temperature treatments. Despite that the cell enlargement began before the experiment in 2010, the end of enlargement was the same in both year between DOY 251 and 258, irrespective of the treatments.

For cell wall formation, the trend was essentially similar to that of enlargement. Its maximum appeared one to four weeks after the culmination of cell enlargement with 6 to 10 cells. The difference of 2 to 5 cells observed after the stress period between the control and the water deficit treatment was not correlated to the treatment. The end of cell enlargement and wall formation always occurred on DOY 272, irrespective of the year and treatment.

Total carbohydrates in the cambium and the xylem had similar trends within the temperature and water treatments. In the cambium, sucrose was 2 to 30 times more abundant than the other sugars. Therefore, total available sugar in the cambium was represented mostly by the variation in sucrose (figure 2). In the xylem (figure 2), significant differences between the water deficit treatments were discernible and were mostly influenced by fructose in 2010 and by sucrose and pinitol in 2011. No trend was observed between the water deficit and the control treatment, before, during or after the water deficit period.

The sum of soluble carbohydrates in cambium and xylem (figure 2) showed similar variations with wood formation. When cambial and needle growth started, between DOY 120 and 130, the amount of carbohydrate was high in the cambium. A decline was observed between DOY 150 and 170, which was most pronounced in 2010 with amount near 0. A second decline was also observed in the middle of July (DOY 208 in 2010 and 196 in 2011). Xylem pattern was mostly symmetric of the cambium one. This aspect will be fully analysed in the last section of the results.

DISCUSSION

The carbohydrates found in the stem of the black spruce seedlings during the growing season consisted of sucrose, pinitol, fructose, glucose, raffinose and starch. Concentrations in the xylem only accounted for about 7% of the sugars in the cambium. Sucrose represented 54% of the total sugars in the cambium while the sucrose, pinitol and glucose each represented about 23% of the total sugars in the xylem, with a higher abundance of fructose, corresponding to 28% of the total carbohydrate. Water deficit and the increase of temperature affected soluble sugars content in different ways and time. Except for sucrose and starch, no clear pattern along the growing season was found. Correlations between cell development and selected carbohydrates were presented.

Identification and quantification of carbohydrates 

The NSC found in the black spruce corresponded with those described in the literature for trees (Giovannelli et al, 2011 ; Bertrand and Bigras, 2006). No unknown compound was identified on the HPLC chromatogram and all NSC were separated enough to avoid merging peaks. Only raffinose could have been miscalculated because of its low quantity, near the detection limit. HPLC chromatogram showed that the amount of carbohydrates found in the stem was low, especially for the xylem. Xylem and cambium had a mean of total NSC of 1.61 mg/gdw and 24.62 mg/gdW respectively from May to September, the demand in energy of growing cells of cambium explaining here the larger amount of carbohydrates then in the xylem, a mostly non active part if the stem. In contrast, needles of white spruce contained about 32 mg/gdw while the entire stem contained about 21 mg/gdW of soluble sugars from mid-July to the end of September (Dhont et al, 2011). However, stems are known to have lower amount of carbohydrates than branches and leaves (Hoch et al, 2003).

CONCLUSION

The aim of this study was to establish the nature and the variation of the non-structural carbohydrates in black spruce seedlings and their behaviors under warming and water deficit along the growing season. We also wanted to demonstrate that carbohydrates are directly related to xylogenesis, from the cambial production to the mature cells. We hypothesised that temperature warming and water deficit stresses would result in diminishing carbohydrate availability for xylogenesis and thus, diminishing cambial cell development and growth. Results of the study, merged with cell growth observations of the same trees allowed us to understand a little more about those links between sugars and secondary growth.

Table des matières

INTRODUCTION 
METHODS 
Study area and experimental design
Xylem growth
NSC extraction and assessment
Statistical analysis
RESULTS 
Secondary growth
Identification and quantification of carbohydrates in the black spruce
Variation of carbohydrates in cambium and xylem along the growing season
Cambium
Xylem
Effects of temperature and water deficit treatments on soluble sugars
Relation between carbohydrates and growth
Canonical correlations
DISCUSSION 
Identification and quantification of carbohydrates
Trends and correlation
Treatments effects.
Canonical correlation in the cambium
Canonical correlation in the xylem
CONCLUSION

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