Xylogenesis in black spruce subjected to rain exclusion in the field

Material and methods

Study area and experimental design

The study was carried out in four permanent plots [Simoncouche (abbreviated as SIM), Bernatchez (BER), Mistassibi (MIS) and Camp Daniel (DAN)] located along a latitudinal and altitudinal gradient, in the boreal forest of the Saguenay-Lac-Saint-Jean region, Quebec, Canada (Table 1).
The sites consisted of mature, even-aged black spruce stands characterized by a typical boreal climate, with cold winters and cool summers (Table 1). The mean annual temperature in the years preceding the experiment at the four sites was between -0.9 and 2.0 °C. May-September rainfall ranged between 402 and 532mm and increased with latitude. The soils are podzolic with different depths among sites. The organic layer in SIM ranged between 10 and 20 cm, with the maximum rooting depth limited by a shallow bedrock. In the other sites, the organic layer was deeper and attained 20-40 cm.
In each site, ten dominant or co-dominant trees with upright stems and similar growth rates were chosen, five control and five treated trees. Trees with polycormic stems, partially dead crowns, reaction wood or evident damage due to parasites were avoided. The selection was based on the proximity among the treated trees to allow the installation of the equipment for rain exclusion on the same cluster of individuals. In 2010, plastic transparent under-canopy roofs were installed during late May-early June to exclude the treated trees from precipitation. The majority of the root system of black spruce is localized at a distance of 90-200 cm from the stem collar (Despont et al., août 2007). Accordingly, the plastic roofs extended for at least 3 m from the stem of each tree and drained the rain in sinking points of the soil to avoid water flowing back towards the stem collar.
The plastic roofs were removed in September to ensure the survival of trees. The five control trees per site were left untreated as a control.
The volumetric water content (VWC) of the soil was measured weekly in four points at a distance of 1 m from the stem of each tree and at a depth between 12-20 cm with a time domain reflectometry soil moisture meter (TDR). All VWC measurements were divided by the maximum observed value to standardize results and differences in VWC between the treatments were calculated as the percentage difference between treated and control plots. Rainfall data was recorded every 15 minutes on all sites and stored as hourly sums by means of CR10X dataloggers (Campbell Scientific Corporation). Daily sums of precipitation were later calculated with the time series obtained from the 24 measurements per day.

Tree physiology

The physiology of the selected trees was monitored weekly throughout the growing season in SIM and BER, the two sites experiencing the most extreme growth conditions (Casabon and Pothier, 2007, Cernusak et al., 2009). Short canopy branches were collected at 8-10 m height on the tree using a telescopic branch pruner and photosynthetic rate was measured using the LI-6400XT Portable Photosynthesis System (Li-Cor Biosciences, Lincoln, Nebraska) with the 6400-22LLighted Conifer Chamber. Because Li-Cor uses a standard leaf area, the actual leaf area of each sample was adjusted based on the dry weight of samples collected at the start of each season using a regression according to Flower-Ellis and Olsson (2011). Mid-day (Ψmd) and pre-dawn (Ψpd) water potentials were measured with a pressure chamber (Model 610 Pressure Chamber, PMS instruments). For logistical reasons, the pre-dawn water potential was simulated in the morning by wrapping the sampled branches in aluminum foil for one hour before taking measurements (Farquhar et al., 1989).

Xylem dynamics

Cambium phenology was studied from May to October on control and treated trees at weekly time resolution. Microcores were collected from the stem following a spiral trajectory between 90 and 170 cm above ground using Trephor (Rossi et al., 2006a). The wood samples were embedded in paraffin, cut into sections of 7 μm with a rotary microtome, fixed on slides and staine  with cresyl violet acetate (0.15% in water). The phenology of xylem was followed under visible and polarized light at 400-500× by counting the number of cells (i) in the cambium, (ii) in radial enlargement, (iii) in cell wall thickening and lignification, and (iv) the number of mature cells along three radial files (Deslauriers et al., 2003a, Rossi et al., 2006b). Rows with cells with large tangential sizes were selected, to ensure that only cells cut at their middle longitudinal part were considered. Cambial cells were characterized by thin cell walls and small radial diameters. During cell enlargement, the primary cell walls were still thin, but the radial diameter was at least twice that of a cambial cell. Observation under polarized light discriminated between the phases of enlargement and cell wall thickening. Because of the arrangement of cellulose microfibrills, the developing secondary walls glisten when observed under polarized light, whereas no glistening is observed in enlargement zones, where the cells are still composed of just primary wall (Abe et al., 1997). The progress of cell wall lignification was detected with cresyl violet acetate reacting with the lignin (Rossi et al., 2006b). Lignification appeared as a color change from violet to blue. A homogeneous blue cell wall revealed the end of lignification and the tracheid reaching maturity (Gricar et al., 2005). The number of cells in each developmental stage was averaged for each tree.
One sample per tree, collected in October, at the end of the growing season when wood formation was finished, was stained with safranin to measure cell features. A camera fixed on an optical microscope was used to record numerical images at a magnification of 20x. Lumen area and cell wall thickness were measured in the tree rings produced in 2009, the year before the treatment, and 2010 on three radial files per section using WincellTm (Deslauriers et al., 2003a) and were averaged for each tree and year. Since the measurements were repeated on the same experimental unit in 2009 and 2010, a repeated measures analysis of variance was used to compare the results from the two years. A general linear model (GLM) was used with sampling time in the repeated statement and treatment and site as model factors to test for the effect of treatment and year (before and during the treatment) on cell number and cell characteristics. To test if the proportion of latewood was affected by the treatment a generalized linear mixed model (GLIMMIX) was used with year as a repeated measure in the random statement. All analyses were performed using SAS

(SAS institute Inc., Cary, NC).

Stem radius variations

Ten automatic point dendrometers (Agricultural Electronics Corp., Tucson, AZ,) per site were installed on treated and control trees at a height of 2 m on the stem to monitor radius variations during and after the period of rain exclusion. Dendrometers were based on a precision linear variable differential transducer (LVDT) enclosed in an aluminum housing and fixed to the tree with stainless steel rods having a thermal linear expansion coefficient of 17 μm m−1 C−1. With this equipment, the percentage of metal expansion was less than 1% of stem variation. A sensing rod held against the surface of the bark measured the radius variations, which in our monitoring represented the overall variation in size of xylem and phloem together. The sensitivity of dendrometers to temperature and humidity was negligible due to the use of dimensionally stable compounds in their manufacture and the dead bark was partially removed where the sensing rod touched the tree to minimize error due to hygroscopic thickness variations. As the stem changed in size, the core of the LVDT moved and translated the displacement in an electrical signal. Measurements were taken every fifteen minutes and stored in CR-1000 dataloggers (Campbell Scientific Corporation) providing precise and high-resolution data of radius variation during the growing season.

Results

Soil water content and tree physiology

In general, the two northern sites had higher VWC, and control plots fluctuated according to the rainy periods (Fig. 1). When measurements started, between DOY (day of the year) 173 and 179, VWC in treated plots of BER was already slightly lower than that of the control. The VWC was substantially lower in the treated plots during the period of treatment, the average reduction in VWC ranged from 46.8% of reduction in DAN, to 67.9% in MIS. After removal of the undercanopy roofs, the difference in VWC was maintained for one and three weeks in SIM and BER, respectively. During the last measurements on DOY 285, DAN and MIS still showed a reduction of 25-39% in VWC between treated and control plots (Fig. 1). Lower Ψpd and Ψmd were observed in the treated trees during the whole period of rain exclusion in SIM, and from DOY 174 until the end of the treatment in BER (Fig. 2). During the treatment, the difference in water potential between treated and control trees was larger in SIM than in BER, and was 0.16-0.17 MPa for Ψpd and 0.26-0.33 MPa for Ψmd. In both sites, the difference in water potential decreased rapidly after the removal of the under-canopy roofs. The photosynthetic rate of trees ranged between 1.6 and 6.2 μmol CO2m-2s-1, was highly variable on both sites. Higher values were regularly observed from the beginning of August in treated trees in BER. In SIM, the photosynthetic rate in September diverged markedly between treatments, but the difference disappeared with the ending of rain exclusion (Fig. 2).

Xylem dynamics

The radial rows of cells had a clear pattern of variation in the number of tracheids in the different developmental stages during the year, which resulted in a decreasing curve of cambial cells, two delayed bell-shaped curves of radial enlarging and wall thickening and lignifying cells, and a rising curve of mature cells (Fig. 3). All patterns of variation are similar between treatments and among sites. However, the number of cells in control trees in SIM was always markedly higher than that observed in treated trees and in the other sites. Cell enlargement started before the application of the under-canopy roofs, except for control trees in BER, where the first enlarging cells were detected one week after the treatment began. During late summer, the number of enlarging cells decreased to zero between DOY 207 and 243. The earliest and latest endings of cell enlargement were observed in DAN and SIM, respectively (Fig. 3). The first cells in wall thickening and lignification were detected between mid-May and the end of May, at the beginning of rain exclusion. Mature cells were observed from DOY 151 in SIM and DOY 172 in MIS and DAN. In the northern sites, the ending of xylogenesis occurred between DOY 242 and 270, on average 10 days earlier than in the southern sites, where a higher number of cambium cells were produced (Fig. 3).

Xylem characteristics

Fig. 4 shows the number of xylem cells and their size in control and treated trees for 2009, the year before the treatment, and 2010, the year of the treatment. Cell production along a radial row varied between 10 and 47, with significant differences observed among sites (F=7.43, p<0.01, Table 2). No significant interaction year×treatment was found (p>0.05), indicating that the number of cells produced by the cambium was not affected by the treatment. This was particularly clear in SIM, where both control and treated trees reduced cell production in 2010, but with the same intensity (Fig. 4). Lumen area ranged from 307 μm2 in DAN to 394 μm2 in BER and cell wall thickness varied from 2.3 μm to 3.9 μm. No significant interaction was found for cell wall thickness (p>0.05, Table 2), there was a difference in cell wall thickness between the treated and control plots (F=8.90, p<0.01), but this difference was already present in the year before the experiment (Fig.4). For lumen area, significant differences were observed between 2009 and 2010 (F= 6.13, p<0.05) but variations differed between treatments, as shown by the significant interaction year×treatment (F= 4.27, p<0.05, Table 2). The GLM demonstrated that rain exclusion reduced lumen area of xylem, but did not affect the number of cells produced by the cambium (Fig. 4). The proportion of latewood ranged from 0.21 in BER to 0.37 in DAN, there was a decrease during the second year in the treated trees in SIM and in the control trees of BER and DAN. The proportion of latewood increased in the other plots (Table 2, Fig. 4). The interaction year×treatment did not influence the proportion of latewood (F=0.08, p>0.05).

Stem radius variations

Stem diameter varied according to the diurnal rhythms of water storage depletion and replenishment and during precipitation events (Fig. 5). Greater variations were observed at the beginning of the growing season, between mid-May and the end of June. After that, stem increase was markedly reduced, finally attaining a plateau from the beginning of August. During and after the treatment, no difference in stem radius variation between treated and control trees was observed in DAN, the northern site. In the three other sites, stem radius variations of the treated trees were lower than those of control. In BER and SIM, a difference between the treatments occurred soon after the installation of the experiment, indicating that stem radius of treated trees increased more slowly and to lesser extent than that of the control. The pattern of treated trees was similar to that of the control, as shown by the low variations in the difference between treatments. In MIS, the differences between control and treated trees occurred later, at the beginning of July (DOY 185).
The pattern of the treated trees differed from that of the control, this is clear from the higher variations in the difference. After removal of the under-canopy roofs, the difference between both treatments decreased, but treated and control trees only attained similar final values of stem radius variation in BER.

Discussion

This paper presents a manipulative experiment of rain exclusion on mature black spruce growing in four sites of the boreal forest of Quebec, Canada, with the aim of studying the effects of summer drought on xylem phenology and anatomy in trees growing in their natural environment.
Drought was expected to cause a decrease in water potential, with modifications of the activity of sources and sinks within the tree. The results showed significant changes in the size of the xylem cells, but no substantial change was observed in photosynthesis and cell production, thus only partly confirming the expectations.
An important consequence of drought is cavitation, which is caused by air-seeding and occurs when air is pulled through the pit membranes and fills the entire conduit (Cruiziat et al., 2002). More and larger pits per conduit increase the susceptibility to cavitation spreading between conduits, following the rare pit hypothesis (Christman et al., 2009). In general, smaller conduits are more resistant to cavitation, because they contain fewer pits and have better mechanical strength (Sperry et al., 2006, Fortin et al., 2008). Cell expansion is physically sensitive to changes in hydrostatic pressure during the early stages of a water deficit (Abe et al., 2003, Bouchard and Pothier, 2011). The turgor of cells declines proportionally with xylem water potential (Chertov et al., 2009), resulting in a decrease of cell expansion (Lockhart, 1965). According to the Hagen- Poiseuille law, hydraulic conductivity increases with the number or size of conduits, therefore reduced lumen dimension leads to a smaller area being available for water transport (Cruiziat et al., 2002, Anfodillo et al., 2006, Anfodillo et al., 2011). For a long time, cavitation was thought to be irreversible, but it has been shown that embolisms can be reversed, even under negative water potentials (Sobrado et al., 1992). Even though the reduced hydraulic conductivity can be recuperated by rebuilding the water conduits, repeated drought may lead to a reduced growth and the formation of smaller vessels in the following year, because the anatomical modifications require larger supplies of cellulose and lignin (Cannell et al., 1976, Pizzolato, 2008, Galle et al., 2010).
Often a higher wood density due to drought can be explained by a higher amount of latewood (Domec and Gartner, 2002). However, in this study the proportion of latewood was not affected by the treatment (Fig. 4, Table 2). Since the proportion of latewood and total number of cells did not change, the decrease in the overall lumen area can be ascribed to the applied drought treatment. Other studies have reported a reduction in lumen diameter under dry conditions for Scots pine [Pinus sylvestris] (Sterck et al., 2008, Heijari et al., 2010). However, Eilmann et al. (2009) found that an uncommon drought event caused the formation of larger conduits for Scots pine, a species that is well adapted to a wide range of hydrological conditions (Poyatos et al., 2007).
Different levels of stress have different impacts on tree physiology. Photosynthesis of white spruce was not affected by drought until a severe stress of -2.0 MPa was reached (Bradley et al., 2001). In our experiment, simulated Ψpd hardly fell below -1.5 MPa, and photosynthesis was only slightly inhibited, which demonstrated that the water stress was not sufficient to cause a physiological reaction of the trees. Consequently, cell production also remained unchanged.
Accordingly, there is evidence that the timings of the water stress are particularly important for producing a marked effect on the secondary meristem. As shown in Fig. 3, cell division and even cell enlargement had already started at the end of May, when snowmelt was just finished and the sites were accessible to set up the experiment. Trees are most susceptible to environmental signals
in the first period of cell division which is during cambium reactivation (Frankenstein et al., 2005).
The expected increase in winter precipitation will provide abundant water during snowmelt (Walsh et al., 2011). Even though there is more snow, the melting will not be delayed, photosynthesis will thus not be inhibited and cambium reactivation is expected to occur earlier due to the increasing temperatures (Casabon and Pothier, 2007, Chang et al., 2009, Frechette et al., 2011). Adequate conditions for secondary growth in spring are thus assumed. Since the number of cambium cells is determining for the total number of cells produced (Rossi et al., 2008a), the start under more favorable conditions at the beginning of the growing season may compensate for the consequences of a severe drought during summer. This is supported by the results, which showed that the applied drought during summer had no influence on the total number of cells produced.
In SIM, the number of cambium and mature cells was markedly higher in the control plot. As shown in Fig. 4, control trees also produced more cells than treated trees in 2009. This indicated that there was an initial difference in growth between treatments before the experiment, and that the lower cell production in treated trees was not due to drought. Such a conclusion was confirmed by the lack in significant interaction yearxtreatment found by GLM (Table 2).
The results clearly showed that the treated trees of southern sites (SIM and BER) were less able to rehydrate at night and during rainfall events. Nevertheless, after removing the plastic roofs from the stem, the treated trees, especially in BER, were able to rehydrate within 23 days and finally showed no difference from the control. The trees in MIS and SIM also showed a rehydration, although not complete. In DAN, there was no difference between treated and control trees, which can be explained by the differences in soil characteristics. In the northern sites, soils are deeper, with steady accumulations of organic matter producing thicker organic layers (Rossi et al., 2009b), that need more time to dry out or rehydrate.
Despite the lower variations in stem radius, there was evidence that treated trees shrunk and swelled according to the circadian cycle (Downes et al., 1999). As was shown by Giovannelli et al. (2007) in poplar, a higher stem shrinkage may take place during the early stages of drought, when tree water potential begins to decrease. In SIM and BER the treated trees followed the same pattern of de- and re-hydration, but with lower amplitudes (Fig. 5). This means that they were still influenced by rainfall events, nocturnal rehydration and changes in sapflow (Sevanto et al., 2008), indicating that sources of water were either still accessible or not completely cut off by the undercanopy roofs. However, the treatment appeared more effective and the treated trees were less able to rehydrate in MIS. These results suggest that site characteristics play an important role in the responses of trees to drought.

Material and methods

Study area and experimental design

The study was carried out in four permanent plots [Simoncouche (abbreviated as SIM), Bernatchez (BER), Mistassibi (MIS) and Camp Daniel (DAN)] located along a latitudinal and altitudinal gradient, in the boreal forest of the Saguenay-Lac-Saint-Jean region, Quebec, Canada (Table 1).
The sites consisted of mature, even-aged black spruce stands characterized by a typical boreal climate, with cold winters and cool summers (Table 1). The mean annual temperature in the years preceding the experiment at the four sites was between -0.9 and 2.0 °C. May-September rainfall ranged between 402 and 532mm and increased with latitude. The soils are podzolic with different depths among sites. The organic layer in SIM ranged between 10 and 20 cm, with the maximum rooting depth limited by a shallow bedrock. In the other sites, the organic layer was deeper and attained 20-40 cm.
In each site, ten dominant or co-dominant trees with upright stems and similar growth rates were chosen, five control and five treated trees. Trees with polycormic stems, partially dead crowns, reaction wood or evident damage due to parasites were avoided. The selection was based on the proximity among the treated trees to allow the installation of the equipment for rain exclusion on the same cluster of individuals. In 2010, plastic transparent under-canopy roofs were installed during late May-early June to exclude the treated trees from precipitation. The majority of the root system of black spruce is localized at a distance of 90-200 cm from the stem collar (Despont et al., août 2007). Accordingly, the plastic roofs extended for at least 3 m from the stem of each tree and drained the rain in sinking points of the soil to avoid water flowing back towards the stem collar.
The plastic roofs were removed in September to ensure the survival of trees. The five control trees per site were left untreated as a control.
The volumetric water content (VWC) of the soil was measured weekly in four points at a distance of 1 m from the stem of each tree and at a depth between 12-20 cm with a time domain reflectometry soil moisture meter (TDR). All VWC measurements were divided by the maximum observed value to standardize results and differences in VWC between the treatments were calculated as the percentage difference between treated and control plots. Rainfall data was recorded every 15 minutes on all sites and stored as hourly sums by means of CR10X dataloggers (Campbell Scientific Corporation). Daily sums of precipitation were later calculated with the time series obtained from the 24 measurements per day.

Tree physiology

The physiology of the selected trees was monitored weekly throughout the growing season in SIM and BER, the two sites experiencing the most extreme growth conditions (Casabon and Pothier, 2007, Cernusak et al., 2009). Short canopy branches were collected at 8-10 m height on the tree using a telescopic branch pruner and photosynthetic rate was measured using the LI-6400XT Portable Photosynthesis System (Li-Cor Biosciences, Lincoln, Nebraska) with the 6400-22L Lighted Conifer Chamber. Because Li-Cor uses a standard leaf area, the actual leaf area of each sample was adjusted based on the dry weight of samples collected at the start of each season using a regression according to Flower-Ellis and Olsson (2011). Mid-day (Ψmd) and pre-dawn (Ψpd) water potentials were measured with a pressure chamber (Model 610 Pressure Chamber, PMS instruments). For logistical reasons, the pre-dawn water potential was simulated in the morning by wrapping the sampled branches in aluminum foil for one hour before taking measurements (Farquhar et al., 1989).

Xylem dynamics

Cambium phenology was studied from May to October on control and treated trees at weekly time resolution. Microcores were collected from the stem following a spiral trajectory between 90 and 170 cm above ground using Trephor (Rossi et al., 2006a). The wood samples were embedded in paraffin, cut into sections of 7 μm with a rotary microtome, fixed on slides and stained with cresyl violet acetate (0.15% in water). The phenology of xylem was followed under visible and polarized light at 400-500× by counting the number of cells (i) in the cambium, (ii) in radial enlargement, (iii) in cell wall thickening and lignification, and (iv) the number of mature cells along three radial files (Deslauriers et al., 2003a, Rossi et al., 2006b). Rows with cells with large tangential sizes were selected, to ensure that only cells cut at their middle longitudinal part were considered. Cambial cells were characterized by thin cell walls and small radial diameters. During cell enlargement, the primary cell walls were still thin, but the radial diameter was at least twice that of a cambial cell. Observation under polarized light discriminated between the phases of enlargement and cell wall thickening. Because of the arrangement of cellulose microfibrills, the developing secondary walls glisten when observed under polarized light, whereas no glistening is observed in enlargement zones, where the cells are still composed of just primary wall (Abe et al., 1997). The progress of cell wall lignification was detected with cresyl violet acetate reacting with the lignin (Rossi et al., 2006b). Lignification appeared as a color change from violet to blue. A homogeneous blue cell wall revealed the end of lignification and the tracheid reaching maturity (Gricar et al., 2005). The number of cells in each developmental stage was averaged for each tree. One sample per tree, collected in October, at the end of the growing season when wood formation was finished, was stained with safranin to measure cell features. A camera fixed on an optical microscope was used to record numerical images at a magnification of 20x. Lumen area and cell wall thickness were measured in the tree rings produced in 2009, the year before the treatment, and 2010 on three radial files per section using WincellTm (Deslauriers et al., 2003a) and were averaged for each tree and year. Since the measurements were repeated on the same experimental unit in 2009 and 2010, a repeated measures analysis of variance was used to compare the results from the two years. A general linear model (GLM) was used with sampling time in the repeated statement and treatment and site as model factors to test for the effect of treatment and year (before and during the treatment) on cell number and cell characteristics. To test if the proportion of latewood was affected by the treatment a generalized linear mixed model (GLIMMIX) was used with year as a repeated measure in the random statement. All analyses were performed using SAS.

(SAS institute Inc., Cary, NC).

Stem radius variations

Ten automatic point dendrometers (Agricultural Electronics Corp., Tucson, AZ,) per site were installed on treated and control trees at a height of 2 m on the stem to monitor radius variations during and after the period of rain exclusion. Dendrometers were based on a precision linear variable differential transducer (LVDT) enclosed in an aluminum housing and fixed to the tree with stainless steel rods having a thermal linear expansion coefficient of 17 μm m−1 C−1. With this equipment, the percentage of metal expansion was less than 1% of stem variation. A sensing rod held against the surface of the bark measured the radius variations, which in our monitoring represented the overall variation in size of xylem and phloem together. The sensitivity of dendrometers to temperature and humidity was negligible due to the use of dimensionally stable compounds in their manufacture and the dead bark was partially removed where the sensing rod touched the tree to minimize error due to hygroscopic thickness variations. As the stem changed in size, the core of the LVDT moved and translated the displacement in an electrical signal. Measurements were taken every fifteen minutes and stored in CR-1000 dataloggers (Campbell Scientific Corporation) providing precise and high-resolution data of radius variation during the growing season.

Results

Soil water content and tree physiology

In general, the two northern sites had higher VWC, and control plots fluctuated according to the rainy periods (Fig. 1). When measurements started, between DOY (day of the year) 173 and 179, VWC in treated plots of BER was already slightly lower than that of the control. The VWC was substantially lower in the treated plots during the period of treatment, the average reduction in VWC ranged from 46.8% of reduction in DAN, to 67.9% in MIS. After removal of the undercanopy roofs, the difference in VWC was maintained for one and three weeks in SIM and BER, respectively. During the last measurements on DOY 285, DAN and MIS still showed a reduction of 25-39% in VWC between treated and control plots (Fig. 1).
Lower Ψpd and Ψmd were observed in the treated trees during the whole period of rain exclusion in SIM, and from DOY 174 until the end of the treatment in BER (Fig. 2). During the treatment, the difference in water potential between treated and control trees was larger in SIM than in BER, and was 0.16-0.17 MPa for Ψpd and 0.26-0.33 MPa for Ψmd. In both sites, the difference in water potential decreased rapidly after the removal of the under-canopy roofs. The photosynthetic rate of trees ranged between 1.6 and 6.2 μmol CO2m-2s-1, was highly variable on both sites. Higher values were regularly observed from the beginning of August in treated trees in BER. In SIM, the photosynthetic rate in September diverged markedly between treatments, but the difference disappeared with the ending of rain exclusion (Fig. 2).

Xylem dynamics

The radial rows of cells had a clear pattern of variation in the number of tracheids in the different developmental stages during the year, which resulted in a decreasing curve of cambial cells, two delayed bell-shaped curves of radial enlarging and wall thickening and lignifying cells, and a rising curve of mature cells (Fig. 3). All patterns of variation are similar between treatments and among sites. However, the number of cells in control trees in SIM was always markedly higher than that observed in treated trees and in the other sites. Cell enlargement started before the application of the under-canopy roofs, except for control trees in BER, where the first enlarging cells were detected one week after the treatment began. During late summer, the number of enlarging cells decreased to zero between DOY 207 and 243. The earliest and latest endings of cell enlargement were observed in DAN and SIM, respectively (Fig. 3). The first cells in wall thickening and lignification were detected between mid-May and the end of May, at the beginning of rain exclusion. Mature cells were observed from DOY 151 in SIM and DOY 172 in MIS and DAN. In the northern sites, the ending of xylogenesis occurred between DOY 242 and 270, on average 10 days earlier than in the southern sites, where a higher number of cambium cells were produced (Fig. 3).

Xylem characteristics

Fig. 4 shows the number of xylem cells and their size in control and treated trees for 2009, the year before the treatment, and 2010, the year of the treatment. Cell production along a radial row varied between 10 and 47, with significant differences observed among sites (F=7.43, p<0.01, Table 2). No significant interaction year×treatment was found (p>0.05), indicating that the number of cells produced by the cambium was not affected by the treatment. This was particularly clear in SIM, where both control and treated trees reduced cell production in 2010, but with the same intensity (Fig. 4). Lumen area ranged from 307 μm2 in DAN to 394 μm2 in BER and cell wall thickness varied from 2.3 μm to 3.9 μm. No significant interaction was found for cell wall thickness (p>0.05, Table 2), there was a difference in cell wall thickness between the treated and control plots (F=8.90, p<0.01), but this difference was already present in the year before the experiment (Fig.4).
For lumen area, significant differences were observed between 2009 and 2010 (F= 6.13, p<0.05) but variations differed between treatments, as shown by the significant interaction year×treatment (F= 4.27, p<0.05, Table 2). The GLM demonstrated that rain exclusion reduced lumen area of xylem, but did not affect the number of cells produced by the cambium (Fig. 4). The proportion of latewood ranged from 0.21 in BER to 0.37 in DAN, there was a decrease during the second year in the treated trees in SIM and in the control trees of BER and DAN. The proportion of latewood increased in the other plots (Table 2, Fig. 4). The interaction year×treatment did not influence the proportion of latewood (F=0.08, p>0.05).

Stem radius variations

Stem diameter varied according to the diurnal rhythms of water storage depletion and replenishment and during precipitation events (Fig. 5). Greater variations were observed at the beginning of the growing season, between mid-May and the end of June. After that, stem increase was markedly reduced, finally attaining a plateau from the beginning of August. During and after the treatment, no difference in stem radius variation between treated and control trees was observed in DAN, the northern site. In the three other sites, stem radius variations of the treated trees were lower than those of control. In BER and SIM, a difference between the treatments occurred soon after the installation of the experiment, indicating that stem radius of treated trees increased more slowly and to lesser extent than that of the control. The pattern of treated trees was similar to that of the control, as shown by the low variations in the difference between treatments. In MIS, the differences between control and treated trees occurred later, at the beginning of July (DOY 185). The pattern of the treated trees differed from that of the control, this is clear from the higher variations in the difference. After removal of the under-canopy roofs, the difference between both treatments decreased, but treated and control trees only attained similar final values of stem radius variation in BER.

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Table des matières

Summary 
Résumé 
Remerciements 
List of figures 
List of tables 
INTRODUCTION
Problem statement
Current knowledge
Objective and Hypotheses
Study sites and methodological approach
Structure of the thesis
References
CHAPTER 1. XYLOGENESIS IN BLACK SPRUCE SUBJECTED TO RAIN EXCLUSION IN THE FIELD
Abstract
Résumé
Introduction
Material and methods
Study area and experimental design
Tree physiology
Xylem dynamics
Stem radius variations
Results
Soil water content and tree physiology
Xylem dynamics
Xylem characteristics
Stem radius variations
Discussion
Acknowledgments
References
Tables and figures
CHAPTER 2. : HIGH RESOLUTION ANALYSIS OF STEM RADIUS VARIATIONS IN BLACK SPRUCE (PICEA MARIANA (MILL.) BSP) SUBJECTED TO RAIN EXCLUSION FOR THREE SUMMERS
Abstract
Introduction
Methodology
Study sites
Experimental design
Data collection
Data analysis
Results
Weather and site characteristics
Stem radius variations
Discussion
Conclusion
Acknowledgments
References
Table and figures
CHAPTER 3. FOLIAR ABSORPTION IN BLACK SPRUCE [PICEA MARIANA (MILL.) BSP] SAPLINGS: DOES IT EXIST? 
Abstract
Introduction
Methodology
Experimental setup
Measurements
Statistics
Results
Discussion
Absorption
Duration of canopy wetting event and timing of measurements
Drought treatment
Age of saplings
Acknowledgements
References
Tables and Figures
CHAPTER 4. WOOD ANATOMY OF BLACK SPRUCE SUBJECTED TO REPEATED RAIN EXCLUSION
Abstract
Introduction
Materials and methods .
Study sites and experimental design
Sampling
Data treatment
Vulnerability curve
Results
Discussion
Conclusion
Acknowledgments
References
Tables and figures
GENERAL CONCLUSION 
Limitations of the study
Opportunities for further research and alternative explanations
Implications of the results
References
Appendix 1: Isotopes 
Isotopes 
Hypothesis 
Methodology 
Results 
Figure 
References 

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