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The relationship between stem CO2 efflux (ES), cambial activity and xylem production in Pinus cembra was determined at the timberline (1950 m a.s.l.) of the Central Austrian Alps, throughout one year. ES was measured continuously from June 2006 to August 2007 using an infrared gas-analysis system. Cambial activity and xylem production was determined by repeated microcore sampling of the developing tree ring and radial increment was monitored using automated point dendrometers. Aside of temperature, the number of living tracheids and cambial cells was predominantly responsible for ES: ES normalized to 10°C (ES10) was significantly correlated to number of living cells throughout the year (r2 = 0,574; p < 0,001). However, elevated ES and missing correlation between ES10 and xylem production was detected during cambial reactivation in April and during transition from active phase to rest, which occurred in August and lasted until early September. Results of this study indicate that (i) during seasonal variations in cambial activity non-linearity between ES and xylem production occurs and (ii) elevated metabolic activity during transition stages in the cambial activity-dormancy cycle influence the carbon budget of Pinus cembra. Daily radial stem increment was primarily influenced by the number of enlarging cells and was not correlated to ES.
Stem CO2 efflux (ES) is an important component of the carbon balance of trees and forest stands (Sprugel et al. 1995). ES is commonly estimated by measuring the CO2 efflux from the stem surface into chambers sealed to the tree stem. This approach assumes that CO2 respired from phloem, cambium, and xylem parenchyma tissues diffuses radially from the stem into the chamber (Maier and Clinton 2006, Teskey et al. 2008) and thus reflects actual stem respiration (RS) of living woody tissue cells (Saveyn et al. 2008). On the other hand there is evidence that CO2 produced by respiration, instead of being released directly through the bark is partly solved in xylem sap and carried upward by the transpiration stream, while part of CO2 derived from root or soil microbial respiration is carried upward into the stem and released there (Sprugel et al. 1995, Maier and Clinton 2006, Saveyn et al. 2008, Teskey et al. 2008).
ES is strongly related to woody tissue temperature (cf. Lavigne 1987, Larcher 2003, Atkin and Tjoelker 2003). But even after adjusted to a reference temperature, ES still varies substantially throughout growing season (Lavigne 1996, Lavigne and Ryan 1997, Wieser 2007) as a result of changes in phenology, above all due to growth processes (cf. Sprugel and Benecke 1991, Ryan et al. 1994, Sprugel et al. 1995, Lavigne and Ryan 1997, Wieser et al. in press). Havranek (1981, 1985) and Lavigne et al. (2004) found a linear correlation between xylem production and ES in Pinus cembra and Larix decidua during the year and in Abies balsamea and Acer rubrum during spring, respectively. But still phenological control over ES during the year remains poorly understood. Therefore a monitoring of radial growth and xylogenesis is important to enlighten ES dynamics throughout the year. Radial stem growth is a complex process and involves cell division in the cambial zone, followed by cell enlargement and secondary wall thickening (Deslauriers et al. 2003a, Rossi et al. 2006b). Micro-core sampling and histological analyses (Rossi et al. 2006 a,b) can thereby provide direct and detailed insight on xylem cell production, while dendrometer measurements are used to monitor radial stem increment.
In the temperate zones ES might not only be influenced by radial growth but also by the cambial active-dormancy cycle (Vose and Ryan 2002). Reactivation of the cambium at the end of dormancy is a complex process that involves changes in ultra-structure and cell biochemistry (Riding and Little 1984, Catesson 1994, Little and Pharis 1995) that cause high metabolic activity leading to elevated ES. Lavigne et al. (2004) observed such an increase of ES due to cambial reactivation before the onset of cell division in spring in Abies balsamea, Acer rubrum and Fraxinus americana. Like cambial reactivation, transition to dormancy is characterised by changes in ultra-structure and cell biochemistry. Fragmentation of central vacuole and endoplasmatic reticulum, as well as an increase in RNA, protein and insoluble carbohydrate in cambial cells are required to reach cold hardiness (Riding and Little 1984, Little and Pharis 1995, Catesson 1994, Rensing and Samuels 2004). Based on sporadic respiration measurements Havranek (1981) assumed that apart from cambium reactivation also transition to dormancy might influence ES in Pinus cembra.
Purpose of this study was to determine the relation of intra-annual dynamics of ES to radial stem growth and cambial activity in Pinus cembra throughout the year. Continuous records of ES together with repeated wood microcore analysis allowed a determination of periods, when changes in cambial activity and xylem production affected ES.
The study was conducted at Mt. Patscherkofel (2246 m a.s.l.) near Innsbruck, in western Austria (47°12′N, 11°27′E). Mt. Patscherkofel is located in the Central Austrian Alps within an inner-alpine dry zone. During the period 1967-2004 mean annual precipitation at the top of Mt. Patscherkofel was 890 mm with a maximum during summer (June-August: 357 mm) and minimum in winter (December-February: 147 mm). During the same period mean annual temperature at timberline was 2,5 °C and the coldest and warmest months were February (−4,3 °C) and July (10,0 °C), respectively.
The geology of the Mt. Patscherkofel region (Tuxer Alpen as part of the Central Tyrolean Alps) is dominated by gneisses and schist. According to the World Base for Soil Resources (FAO 1998), the soil at the study site is classified as a haplic podzol, a soil type typical for the Central Austrian Alps (Neuwinger 1970).
Cembran pine (Pinus cembra L.) was chosen for this study because it is the dominant conifer in the study area and at the timberline in the central part of the Eastern Alps. Trees were selected on a south-west facing slope at the timberline (1950 m a.s.l.). The trees reached a height of 10-14 m.
Seasonal wood formation dynamics were monitored during the growing seasons 2006 and 2007 by taking small punched cores from 5 trees of the outermost tree rings (micro cores) with a diameter and length of 2,5 mm and c. 2 cm, respectively (Rossi et al. 2006a). Samples were taken on the slope-parallel side of the stem following a spiral trajectory up the stem from c. 15 cm below breast height (1,3 m) to c. 15 cm above. A distance of c. 2 cm in tangential and longitudinal direction was kept to avoid lateral influence of wound reactions on adjacent sampling positions.
Microcores were taken during June through October 2006 in weekly intervals and in 2007 sampling was carried out from late April through October in about ten day intervals. Collected core samples were prepared for light microscopy. Immediately after extraction cores were fixed in a solution of ethanol, propionic acid and 40 % formaldehyde (mixing ratio: 90/5/5), later on embedded in glycolmethacrylate (Technovit 7100) and polymerized after adding an accelerator. Transverse sections of c. 12 μm were cut with a microtome, stained with a water solution of 0,05 % cresyl fast violet and observed under a light microscope with polarised light to differentiate the development of xylem cells, i.e. the discrimination between tracheids in enlarging and cell-wall thickening phase (Antonova and Stasova 1993; Deslauriers et al. 2003a; Rossi et al. 2006b). The number of cambial cells (i.e., fusiform cells lacking radial enlargement), radial enlarging cells, cells undergoing secondary wall thickening and mature xylem cells were counted on all sampled cores in three radial rows. Number of living cells was defined as sum of cambial cells, radial enlarging cells and cells undergoing secondary wall thickening. Total xylem cell number was determined by adding the number of cells in radial enlargement, in cell wall thickening and the number of mature xylem cells (Deslauriers et al. 2003a; Rossi et al. 2006c). Values, i.e. the number of different cells types of five trees per date were averaged.
Because cell number varies within the tree circumference and hence among different samples, standardization is required (Rossi et al. 2003). The total cell number of the previous tree ring was recorded in every sample and used for a cell number correction for each tree. Cell number in each j-sample and by each i-phase (enlarging, wall thickening, mature) was corrected as follows:
where ncij is the corrected cell number, nij is the counted cell number, nm is the mean cell number of previous ring of all j-samples, and ns is the cell number of previous ring for each j-sample
Short-term variation in the measured time series of the number of tracheids were modelled with a Gompertz function using the nonlinear regression procedure included in the Origin software package (OriginLab Corporation, Northampton, MA, USA). The Gompertz equation proved its versatility to describe limiting growth processes and to assess cell dynamics of tree ring growth (Zeide 1993; Camarero et al. 1998; Deslauriers and Morin 2005; Rossi et al. 2003, 2006c).
Xylem cell number increases (including cells in enlarging and wall thickening phase as well as mature cells) were calculated for two-week intervals based on developed Gompertz models.
Air temperature, relative humidity (HMP45C, Vaisala, Helsinki, Finland), solar radiation (SP-Lite, Campbell Scientific, Shepshed; UK), and wind velocity (A100R, Campbell Scientific, Shepshed; UK) were monitored 10 m above ground on top of a scaffolding. While soil temperature (107 Temperature Probe, Campbell Scientific, Shepshed; UK), and soil water potential (EQ3 Equitensiometer, Liu, Dachau, Germany) were monitored in 10 cm soil depth in the rooting zone of the tree used for respiration measurements. Stem temperatures were measured using two 1mm type-T thermocouples inserted into the cambial zone. Measuring intervals for all sensors were 30 min. Daily means were calculated by averaging all daily measurements (48 values/day). Daily precipitation was recorded at a meteorological station on top of Mt. Patscherkofel (2246 m a.s.l.).
Point dendrometers and sap flow sensors for measurements of radial stem increment and sap flow density (F), respectively, were installed at the north-facing side of the tree used for ES measurements and two neighbouring trees.
The dendrometeres consisting of an electronic displacement-sensor (linear motion potentiometer, MM10 Megatron, Putzbrunn Munich, Germany) mounted on a stainless steel rod, were anchored at 1,5m height, whereby the dead outermost layers (periderm) of the bark were slightly removed to reduce the influence of hygroscopic swelling and shrinkage of the bark on dendrometer traces and to ensure close contact with the stem (cf. Zweifel and Häsler 2001).
In 2007 sap flow density (F) was monitored between mid April to mid August 2007, using custom made flow gauges (Granier 1985, 1987) that were installed 10 cm above dendrometers. The sensors were inserted 15 cm vertically apart into the hydroactive xylem (sapwood) and were shielded by a thermally isolating styrofoam cover. The upper probe was supplied with a constant heat of 140 mW, and the temperature difference between the heated upper and the unheated lower reference probe was used for estimating sap flow density according to Granier (1987).
For monitoring ES a stem section of 240 cm2, on the north facing side 1,3 m above ground of an adult tree of about 10 meters height, was enclosed in a clear curved Perspex chamber. The chamber was sealed to the stem with putty (Terostat, Teroson, Ludwigsburg, Germany) and non-hardening insulating foam to ensure a gas-tight seal between the chamber and bark.
During measurements ambient air was continuously sucked through the chamber at a flow rate of 2 l min−1. CO2 concentrations of chamber air stream and reference air sampled 10 m above ground were measured alternately using an infrared gas analyser (Li 6262, Licor, Lincoln, Neb.). The corresponding flow rates were monitored and adjusted with an electronic mass flow controller (LD 20G; Walz Effeltrich, Germany). Each air stream was sampled for 60 s, whereas data were taken during the last 10 seconds of the interval to provide a complete flushing of the system (Wieser and Bahn 2004).
As ES is strongly influenced by woody tissue temperature (cf. Lavigne 1987, Larcher 2003, Atkin and Tjoelker 2003) for some analyses measured ES was adjusted to ES at 10°C (ES10), using an adapted equation introduced by Lavigne et al. 2004 for the temperature normalisation of stem respiration:
The temperature coefficient of respiration, Q10, was calculated using the equation:
The regression slope was taken from linear regression of log10 of ES versus stem temperature (Atkin et al. 2002). Q10 was calculated at two-week intervals, using half hour mean values. Five day mean values of ES10 (2 days before to 2 days after tissue sampling) were used for calculations and in figures to compensate short term variations.
ES measurements and tissue sampling started in early June 2006 and ended mid August and mid October 2007, respectively. After cell development was finished in October 2006 tissue sampling was interrupted until end of March 2007, while respiration measurements were continued.
All the environmental, gas exchange, dendrometer, and sap flow data were transmitted to a AM416 multiplexer (Campbell Scientific, Shepshed, UK) and recorded with a Campbell CR10X data logger programmed to record 30-min means of measurements taken every minute.
Mean average air temperature recorded during the study was 6.4°C and daily means varied between 19.1°C during the growing season (July 20, 2006) and −14.3°C during the winter (January 25, 2007) (Figure 1). Daily mean stem temperature varied between 17.4°C (July 20, 2007) and −11.2°C (January 27, 2007). Lowest daily mean soil temperature recorded was −2.6°C (February 4, 2007) and the highest 13.4°C (July 20, 2006). Due to frequent precipitation in both growth periods (Figure 1), soil water potential always stayed above −0.08 MPa, indicating that the trees did not experience drought stress from the soil side. From beginning of November 2006 to end of March 2007 precipitation primarily fell as snow.
The years 2006 and 2007 showed quite different climate conditions. Climate in 2006 was characterized by a prolonged late frost event occurring between 30 May and 8 June (minimum air temperature at timberline fell to −4,9°C), a warm June and July with mean air temperature exceeding the long-term mean (LTM) in July by 3,8°C, followed by a sharp temperature drop in August (August mean air temperature was 3,2°C below LTM). In 2007 the growing period was extended due to occurrence of exceptionally mild temperatures in spring (March to May, 3,4°C above LTM).
Due to differences in climate conditions dynamics of tracheid development were different during growing seasons 2006 and 2007 (Figure 2) (Gruber et al. 2008). When measurements started in June 2006 cell division had already started. Number of cambial cells reached their maximum at the end of June. Then cambial division declined and terminated end of July. The dormant cambium consisted of 7 to 8 cells. In 2007 cambial division already started end of April 2007 and in early May the number of cambium cells had already increased to about 13 and stayed on a high level until mid June. Cambial division again terminated about end of July.
The dynamics of cell differentiation (enlarging and wall thickening cells) was characterized by delayed bell-shaped curves. In June 2006 cell enlarging had just started as first samples were taken. In 2007 in some individuals radial enlargement commenced in late April. Maximum number of cells in enlarging phase (average about 12 cells) was reached in mid June and some enlarging cells were detected until end of August in 2006 and 2007. In 2006 and 2007 first tracheids were undergoing wall thickening around 16 June and 2 May, respectively. Wall thickening and lignification was completed end of September 2006 (Figure 2E) and mid of October 2007 (data not shown).
Changes of ES generally followed seasonal trends in stem temperature. ES was highest during the growing season and was reduced to the level of maintenance respiration in winter (Figure 2a). Stem temperature accounted for 68% of the variation in ES during the growing season of 2006 and 2007 and 46% during quiescence (end of October to mid April) (Figure 3). The temperature coefficient of respiration (Q10) varied between 2 and 2,25 during growing seasons 2006 and 2007. During quiescence (end of November 2006 to mid April 2007) Q10 decreased to about 1,81.
During the growing season ES at nighttimes (between 22.00h and 05.00h) was significantly higher than during the day (between 09.00h and 16.00h) (p < 0,001) (Figure 4). Average daytime ES was reduced by 18% and 31 % compared to nighttime values, at 10°C and 13°C respectively. Q10 during the growing season was 2,37 and 2,17 at nighttimes and daytimes, respectively.
ES10 was significantly correlated to the number of living cells (cambium, enlarging and wall thickening cells) throughout the whole measuring period and number of living cells accounted for 57% of the variation in ES10 (Figure 5a). Detailed analysis however showed that the influence of the number of living cells on ES10 was higher during spring and early summer 2007 (r2 = 0,902; p < 0,001) (Figure 5c), except for a short period at the end of dormant season. On the other hand from June to end of October number of living cells only accounted for 52% (p < 0,001) of ES10, due to high ES1 value in August and beginning September 2006. (Figure 5b).
Stem increment measured by dendrometers was significantly correlated (r2 = 0,920; p < 0,001) with the number of current year tracheids (number of enlarging, wall thickening and mature cells) in 2007 (Figure 6a). Number of enlarging cells was significantly correlated with daily change in stem increment calculated on basis of the modelled Gompertz functions (r2 = 0,960; p < 0,001) (Figure 6b). No correlation between ES and daily increment could be detected.
Daily mean sapflow density was not significantly correlated to daily means of ES10 (r2 = 0,028, p = 0,068) during the growing season 2007 (Figure 7).
In the recent study stem temperature was the most significant factor influencing ES and accounted for 68% of variations of ES in Pinus cembra (Figure 4). This is in line with Wieser and Bahn (2004) and Maier et al. (1998) who calculated a temperature sensitivity of ES of 71% for Pinus cembra and 61% for Pinus taeda, respectively.
It has been reported that, due to diffusion resistance by cambium and bark, ES can exhibit a lagged response to stem temperature (Ryan et al. 1995, Lavigne et al.1996, Lavigne & Ryan 1997). This effect is usually corrected by using prior sapwood temperatures, at which the appropriate lag time is selected from the best fit to a nonlinear regression. For P. cembra no better fit was detected using sapwood temperatures of up to 5 h before ES measurements, (data not shown), but according to Ryan et al. (1995) the lag between ES and stem temperature can vary between 0 to 5 h among coniferous species.
There is evidence that CO2 produced by respiration, instead of being released directly through the bark into air or soil, may be transported by the transpiration stream. CO2 produced by stem cells or derived from root or soil microbial respiration might be carried upward into foliage, branches and stem and be released there (Sprugel et al. 1995, Teskey and McGuire 2002, Maier and Clinton 2006, McGuire et al. 2007, Saveyn et al. 2008, Teskey et al. 2008). But the relationship between sap flow density (F) and ES substantially differs interspecific (Teskey et al. 2008) and even among trees and during season (Bowman et al. 2005, Maier and Clinton 2006). Martin et al. (1994) found a weak negative correlation in Pinus tadea seedlings and in a study Maier and Clinton (2006) suggest ES in adult Pinus tadea to be reduced by up to 40% at maximum F. Under field conditions it is difficult to separate the effect of CO2 transport by transpiration stream from other effects influencing ES, all the more as both F and ES are highly influenced by temperature. In our study daily mean F and daily means of ES10 were not significantly correlated in 2007. On the other hand, during the same period, ES at a given temperature was significantly higher at nighttimes (between 22.00h and 05.00h) than during the day (between 09.00h and 16.00h) (Figure 4), which is assumed to be a function of F, as sap flow is significantly lower at night (Maier and Clinton 2006, Teskey et al. 2008). This indicates that CO2 transport by transpiration stream might nevertheless influence ES in Pinus cembra. Lavigne et al (1996) report that in A. balsamea night time ES was not higher than day time ES when lagged temperatures were used for correlations. This could not be confirmed for P. cembra (data not shown).
The presented study indicates that apart from other factors, number of living xylem cells and cambium cells (living cells) is a paramount factor influencing ES (Havranek 1981, 1985, Ryan et al.1994). Ryan (1990) reported that in Pinus contorta and Pinus engelmannii, living cells in phloem only accounted for 7 % of whole stem living cells and did not account for significant variations in ES during growing season, which also applies for Pinus cembra. A relationship between stem growth and ES was reported before (Ryan 1990, Mair 2001, Vose and Ryan 2002, Lavigne et al. 2004). However we found that aside of growth processes there are periods when changes in cambial activity substantially influence ES.
On 10 April, about 4 days after permanent unfreezing of the stem, ES rapidly increased (Figure 2a), which we attribute to the onset of cambial reactivation. As soil and root zone were frozen until 14 April it is unlikely that the rise in ES was caused by transport of CO2 in xylem sap from the rootzone as has been described by Teskey and McGuire 2007 and Teskey et al. (2008). We assume that the high metabolic activity due to changes in ultra-structure and cell biochemistry during cambial reactivation causes an increase in ES before the onset of cell division (Riding and Little 1984, Catesson 1994, Little and Pharis 1995). Lavigne et al. (2004) proposed this rise of ES, two to three weeks before the onset of cell division, as a non destructive marker to determine beginning of rapid xylem production.
Ignoring elevated ES10 from August to early September correlation between number of living cells and ES10 throughout the growing season 2006 was highly significant (r2 = 0,895, p < 0,001) (Figure 5b). The period of elevated ES10 started just as cambial division ceased at beginning of August. Number of living cells declined rapidly, whereas ES10 values remained at a high level. The cessation of mitosis is known to initiate the transition from active phase to rest in cambial cells (Riding and Little 1984, Catesson 1994, Rensing and Samuels 2004). Therefore, we suggest that elevated ES10 recorded after cessation of cell division can be attributed to elevated metabolic activity due to changes in cell ultra-structure which characterise transition to rest.
The rise of ES at the end of dormancy in spring 2007 also marked the onset of stem diameter increase in dendrometer measurements (Figure 6), triggered by cambial swelling due to rehydration during reactivation (Deslauries et al. 2003b, Lavigne et al. 2004). The identification of cambial reactivation is crucial in dendrometer measurements especially at higher altitude, where frost shrinkage and thaw expansions cause high variations during the dormant period (Zweifel and Häsler 2000, Deslauries et al. 2007). Mäkinen et al. (2003, 2008) found no correlation between cell development and dendrometer measurements, whereas Zweifel et al. (2006) states that both methods are able to detect the course of intra-annual radial growth. In our study radial increment measured by dendrometer and microcoring method correlated significantly (r2 = 0,920; p < 0,001), even though typical fluctuations in stem radius due to changes in tree water status occurred (Zweifel et al. 2000, 2005). Only number of enlarging cells was significantly correlated to daily increment (r2 = 0,960, p < 0,001), which indicates that daily stem increment is mainly caused by cell enlarging whereas wall thickening (Deslauries et al. 2003b) and cambial cell division are of little account for daily increment. The fact that stem increment is not determined by the number of living cells but primarily by number of enlarging cells, explains the missing correlation between ES and daily stem increment.
We conclude that in Pinus cembra aside of temperature, the number of cambial and living xylem cells influence ES. Variations in ES due to changing numbers of living cells and considerably elevated ES during transition stages in the cambial activity-dormancy cycle must be taken into account in process-based models of forest carbon cycles. Hence, our results imply that short time measurements of ES are problematic for the calculation of forest carbon budgets.
Precipitation data were provided by Zentralanstalt für Meteorologie und Geodynamik, Innsbruck, which is greatly acknowledged.
We would like to thank the editor and all reviewers for their careful revision of our manuscript and valuable suggestions on improving it.
FWF Austrian Science Fund (Project No. FWF P18819-B03 “Temperature dependence of Pinus cembra (L.) stem growth and respiration along an altitudinal transect “).