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In the present study the seasonal patterns of time lags between diurnal xylem and whole stem diameter variations at the top and at the base of two Scots pine trees (Pinus sylvestris L.) were compared. The diameter variations were measured during the summers of 2001 and 2002. Time lags were determined using the cross-correlation method. The lags were found to vary in time according to the different stages of growth. At the top the xylem lagged behind the whole stem between the beginning of stem growth and the end of shoot growth in both years. In 2001 the time lags at the base showed a similar behaviour during stem growth. That kind of seasonal pattern of the time lags would result from the changes in the sink strength due to changing growth rate at different parts of the tree and the differences in the annual rhythm of growth and water availability in the soil (based on precipitation measurements) between the years 2001 and 2002 were reflected in the patterns. The time lags of shrinking and swelling periods during high and low photosynthetic activity (measured using a shoot chamber) were also compared. It was found, for example, that in 2001 in the middle of the growing season at the top of the tree the whole stem lagged on average 15 min more behind the xylem on the days of high photosynthetic activity than on the days of low or moderate. These results show for the first time that the transportation of carbohydrates and variable sink activity could be detected during the growing season in field conditions using stem and xylem diameter variation measurements. Furthermore, these results provide evidence of the pressure gradient-driven flow also in the phloem of gymnosperms.
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Diurnal tree stem diameter changes have been detected since the early 1900s (MacDougal 1924). The changes have been explained by the variation in the water content of the stem (Kramer & Kozlowski 1979). A close connection to transpiration and the ascent of sap has lately been verified by separating the xylem and whole stem contributions to the variations (Irvine & Grace 1997; Perämäki et al. 2001). Transport of photosynthetic products from leaves to the growing parts or stores occurs via the phloem. In 1930 Edward Münch published a theory about the mechanism of phloem transport. He suggested that the flow is driven by an osmotically created pressure gradient between the sources and sinks of carbohydrates, and the flow is strongest towards the strongest sink. However, this theory, although widely accepted, has not yet been unambiguously verified, especially in larger trees and in field conditions (Taiz & Zeiger 1998).
According to the Münch hypothesis osmotic forces, controlled by active loading and unloading of sugars to and from the phloem, drive the phloem flow. The active loading of sugars from source cells to the phloem results in an osmotic drag of water from the xylem to the phloem. The incoming water increases the turgor of the phloem cells near the sources and causes the sap to flow. Near the sinks active unloading of sugars from the phloem lowers the solute concentration and water flows to the xylem. This deepens the pressure gradient between the sources and sinks and increases the flow rate in the phloem (Taiz & Zeiger 1998).
The elastic properties of the xylem and phloem connect the diurnal stem diameter variations to the sap pressure (Irvine & Grace 1997). In particular, xylem diameter variations follow the patterns of transpiration closely (Perämäki et al. 2001). According to the cohesion theory, suction, due to transpiration, is the driving force of the ascent of sap (e.g. Zimmermann 1983). In terms of the time lags of diameter variations this means that the xylem diameter in the lower parts of the transporting pathway lags a little behind the upper parts (Sevanto et al. 2002).
Sevanto et al. (2002) also compared the time lags between xylem and whole stem diameter variations at different heights. They found that the whole stem lagged behind the xylem, which is logical since the xylem is more directly connected to the transpiration stream than the phloem. However, they found that the time lag was considerably larger at the top of the tree than at the base, which seems contradictory to the explanation provided by the ascent of sap. This led them to suggest that the Münch hypothesis of phloem transport could explain the results. However, their measurements were performed during a 23 d period in the middle of the growing season and only one tree was observed.
It could be argued that the pattern of time lags observed by Sevanto et al. (2002) could follow from variable flow resistances between the xylem and phloem along the stem. In the upper part of the crown a larger number of cells differentiate during growth than at the basal part of the stem and during their development when still alive and expanding osmotically they could form a fairly impermeable layer for water flow (Milburn 1996).
In this article we analyse the time lags between xylem and phloem diameter variations during the whole growing season and compare the temporal and spatial patterns. We hypothesize that if the variations in time lags are caused by permeability changes between the phloem and xylem along the stem, the lags should remain fairly constant throughout the season. If the changes are connected to phloem transport and source–sink dynamics, we should see considerable variation in the pattern during the growing season. The relative sink activity between parts of a tree has a clear temporal pattern in boreal regions (Leikola 1969) that should be reflected also on the time lags, if the latter hypothesis is true. Similarly, high productivity during the day should increase the driving concentration gradient in phloem. This should, in turn, increase time lags if they are connected to phloem transport.
MATERIALS AND METHODS
We measured the diameter variations on two Scots pine (Pinus sylvesrtis L.) trees during two consecutive years at SMEAR II station in Hyytiälä in Southern Finland (61°51′ N, 24°17′ E) (Vesala et al. 1998). The measurements were made at different heights on the xylem surface and above the bark, referred to as the whole stem (including the xylem, phloem and a thin layer of smoothed bark) measurement henceforth. The trees were growing in a 45-year-old Scots pine stand with a density of about 2000 trees per hectare. The average height of the stand is 15 m and average diameter at breast height (1.3 m) is 15 cm.
The measurements on the first tree were performed during the summer 2001 (1 May −30 September 2001) and on the second tree during the summer 2002 (1 May – 30 September 2002). The measurement heights in the summer 2001 were 2.50 and 11.60 m and in the summer 2002 they were 1.50 and 11.50 m. The total heights of the trees were 14.0 and 15.0 m and the first living branches were at the heights of 8.95 and 9.0 m, respectively. Xylem and whole stem diameter variations were measured in the same internode at each height by linear variable displacement transducers (LVDT; model AX/5.0/S; Solartron Inc., West Sussex, UK). The transducers were attached to rigid metal frames. The frames consisted of four bars: two brass bars opposite to each other and two steel bars making the remaining two sides of a quadrangle around the stem. The sensor was attached to one of the brass bars through a hole and fastened by a small screw. The frames were mounted on the stem using screws through two attachment plates (see Sevanto et al. 2002). In the case of xylem measurements approximately 1 cm2 of bark, phloem and cambium was removed from opposite sides of the stem and the openings were covered by small aluminium plates. One side of the frame was set to rest on one plate and the tip of the sensor on the other so that the sensor measured the variation in the distance between the two plates. In the measurements of 2002 we used small screws instead of aluminium plates for the xylem measurements because of the problems with leaking resin. In the case of whole stem measurements the aluminium plates were attached to the smoothed surface of the bark. In each case the whole stem measurement was 15–30 cm above the xylem measurement and the data was collected at a frequency of one measurement per minute.
The frames and sensors were covered with a conical polyethylene shade to avoid heating by direct radiation. The air temperature at the surface of each frame was measured using copper–constantan thermo-elements and used to correct for the effect of temperature on the expansion of the steel bars of the frame (Sandvik 1802 steel; Sandvik, Sandvixen, Sweden: linear coefficient for thermal expansion 10 × 10−6 m K−1) and wood. The frames (heat capacity approximately 0.08 kJ K−1) were estimated to reach the air temperature in less than 1 min, which makes the use reasonable [see, e.g. Bird, Stewart & Lightfoot (1960) for the estimation of heat transfer coefficients]. The expansion of the steel bars (coefficient of thermal expansion × length of steel bar × temperature change) was added to the output of the sensors. For the thermal expansion of wood we used a coefficient of −3 × 10−6 m K−1 (Salmén 1990). The difference in the temperature between xylem and whole stem measurements at each height was so small that the correction had no effect on the time differences. Furthermore, the difference in the temperature of the stem between the two measurements was assumed to be small. To ensure this we also calculated the results using uncorrected data and the time lags were essentially the same.
Micrometeorological data, namely air temperature (pt-100 type sensors), precipitation (rain gauge AGR-100, Environmental Measurements Ltd, Sunderland UK) and photosynthetic CO2 flux measurements (Hari et al. 1999) (measured using a shoot chamber at the top of the tree) were available for the same period from the SMEAR II station.
We calculated the time lags for each day separately using the cross-correlation method: one data set was moved in time with respect to another until the best correlation was found (Sevanto et al. 2002). During the days when there was a weak temporal pattern in diameter variation (rainy or cloudy days) the method was not applicable and those days were omitted from the analyses. A trend line (calculated using the least-squares method), resulting from the growth, was subtracted from the whole stem data before the analysis.
The time lags between xylem and whole stem diameter variations were found to change during the measurement periods (Fig. 1). To examine the patterns more clearly we calculated 10 d moving averages of the time lags (Fig. 2). In Figs 1 and 2 a positive value means that the diurnal pattern of the whole stem measurement was ahead of xylem and the negative value means that the xylem changed first. Every data set showed a pattern with a peak in the beginning of the growing season followed by a drop and another more gradual rise, especially in the measurements for the top (Fig. 2a). However, the two trees in two different years did not behave in an exactly similar manner. In 2002 the peaks appeared earlier than in 2001 and the whole stem lagged-behind the xylem all the time at the base (2.5 m measurement in 2001 and 1.5 m measurement in 2002) whereas in 2001 the whole stem lagged clearly behind only at the beginning. At the top (11.60 m measurement in 2001 and 11.50 m measurement in 2002) in 2002 the xylem started to lag behind the whole stem in the middle of July whereas at that time in 2001 the whole stem lagged clearly behind.
The growing season was regarded as starting when the daily average temperature exceeded +5 °C for a period of 5 d. In 2001 this happened on 27 April and in 2002 on 25 April. However, the daily average temperature had exceeded +5 °C for the first time a week earlier in 2002 than in 2001. The growing season was taken to end when the daily average temperature dropped below +5 °C for the first time. This resulted in a 34-d-longer season in 2001 than in 2002, since September and October of 2001 were exceptionally warm whereas in 2002 the cold period started abruptly in mid-September.
The differences between the years are large in calendar scale. In relation to phenological events a similar pattern was seen between the years, especially in the top measurements. In 2002 the shoot growth started at the latitude of SMEAR II station on 1 May and ended on the 12 June. In 2001 these dates were 4 May and 21 June, respectively (FFRI 2002). At the top of the tree the period of stem growth was shorter and the growth slower in 2001 than in 2002 (Fig. 2b). The growth started around 15 May in 2002 and around 1 June in 2001 and ended around the 20 August in 2002 and around the 25 August in 2001. At the base the period of stem growth started at the same time as at the top in both years but the growth had already ended after mid-July (Fig. 2d). The time lag values started increasing (whole stem started reacting earlier in relation to xylem) in both years simultaneously as the stem started growing. After the maximum value the following minimum coincided with the end of shoot growth. For the top measurements another positive peak occurred at about the phase when the stem extension stopped.
For the bottom measurements the very noticeable difference is the consistently more negative values in the time lags in 2002. At the top the increase in stem diameter of the two trees was of the same magnitude (approximately 2 mm). However, at the base the increase of stem diameter of the tree of 2002 was only half (approximately 1 mm) that of the tree measured in 2001 (approximately 2 mm). Comparing the cumulative temperature sums of the years, the beginning of growth happened when the sums were of the same order of magnitude. At the beginning of shoot growth the temperature sum was 28 °C in 2001 and 22 °C in 2002 and at the time stem growth started the sums were 96 °C and 115 °C, respectively. These values are in accordance with earlier findings (Leikola 1969).
The temperature sum accumulated more rapidly in 2002 (Fig. 3a) and the cumulative CO2 uptake was almost doubled (Fig. 3b). However, in 2002 the growing season included a dry period (from 20 May to 25 June) and in August and September it rained less than in 2001 (Fig. 3c). During the first rainless period the soil was wet enough to make the conditions favourable since the photosynthetic activity (measured by cumulative CO2 flux to the needles over a day [g−2 d−1]) remained high (Fig. 4). However, the drought of August and September was more severe and its effect was observable in the decreasing growth curves at both heights (Fig. 3b & d). It was only at the end of August that the productivity of 2002 started to drop below that of 2001. This may reflect the influence of drought, but also involved preparing for winter. The photosynthetic production in autumn depends on the light and days of extreme cold (Korpilahti 1988), which started earlier in 2002 than in 2001. At the beginning of September 2001 there was a period of heavy rain and as the daily average temperature stayed above 5 °C the temperature-determined growing season continued for an unusually long period.
We compared time lags of the shrinking and swelling periods during the days of high and low or moderate photosynthetic activity. The shrinking period was defined as being from midnight to noon and the swelling period from noon to midnight for each day. Since the CO2 uptake varies along the growing season (Fig. 4) the measurement period was divided into three parts and the different limits between high and low or moderate values was taken for each period according to the average value of the period. In 2001 the first period was from 15 May to 12 June, the second from 13 June to 2 September and the third from 3 September to 30 September and the limits of high and low or moderate CO2 fluxes were 5.5, 6 and 2.5 g−2 d−1, respectively. In 2002 the periods were from 1 May to 1 June, from 2 June to 8 August and from 9 August to 30 September and the limits of high and low or moderate CO2 fluxes 7, 9 and 4 g−2 d−1, respectively. In 2001 the only statistically significant difference (the significance was tested using Student's t-test) was found between shrinking values of days of high and low or moderate CO2 fluxes during the second period at the top of the tree (Table 1). In that case the time lags were on average 15 min longer (P < 0.02) on the days of high CO2 flux than on the days of low or moderate flux. In both cases the whole stem lagged behind but with low or moderate CO2 flux the lag was only 2 min on average. In 2002, at the base of the tree the lags during the shrinking periods were larger than those of the swelling periods for all of the time except in September when the order was reversed. The difference during the first period was 16 min on average (P < 0.01), during the second period 30 min (P < 0.001) and in September it was 10 min (P < 0.01). The photosynthetic activity did not have any effect on these differences. However, during the third period the time lags of the shrinking periods were 9 min larger (P < 0.02) at the base of the tree on days of high photosynthetic activity than on days of low or moderate activity (the xylem lagged behind in both cases). At the top at the same time the phloem lagged behind when the CO2 fluxes were low or moderate (average time lag 9 min) and the xylem lagged behind when CO2 fluxes were high (average time lag 7 min; P < 0.01).
Table 1. The statistically significant differences observed in the time lags of shrinking (from midnight to noon) and swelling periods (from noon to midnight) during the measurements of 2001 and 2002. The time lags are averages of each period and the limits of CO2 fluxes were taken according to the mean value of each period. The statistical significance of the differences is indicated by the P-values (t-test) in brackets
I 15/05–12/06 2001 01/05–01/06 2002
II 13/06–02/09 2001 02/06–08/08 2002
III 03/09–30/09 2001 09/08–30/09 2002
Lags of shrinking periods 15 min longer when CO2 flux > 6 g−2 d−1 (P < 0.02)
Swelling period lags 16 min longer than shrinking period lags (P < 0.001)
Swelling period lags 30 min longer than shrinking period lags (P < 0.01)
The whole stem behind 9 min when CO2 flux < 4 g−2 d−1. The xylem behind 7 min when CO2 flux > 4 g−2 d−1 (P < 0.01)
Shrinking period lags 10 min longer than swelling period lags (P < 0.01)
Monitoring of xylem and whole stem diameter variations at different heights during two consecutive years in two different trees indicate clearly that there are both temporal and positional changes in the time lags during the growing season. We hypothesized (Sevanto et al. 2002) that the time lags could be caused either by different water permeability in the radial direction at different heights or they could reflect the osmotic changes in bark tissue derivable from the Münch hypothesis. The former hypothesis suggests that we should not see very large variation in the time lags or there should be gradual changes connected to the development of secondary xylem. Furthermore, the variation should correlate with the changes in the environmental conditions (i.e. the availability of water).
The diurnal diameter variations of xylem are caused by the transpiration-induced tension (see, e.g. Irvine & Grace 1997 and Perämäki et al. 2001). When measuring the whole stem, it has to be kept in mind that the tension is also transmitted to the tissues outside the xylem. If we assume equilibrium in water potential between xylem and phloem at initial state: neither loading nor unloading of sugars into phloem, the whole stem should shrink almost in phase with the xylem. In the light of the Münch hypothesis, at the loading and unloading of sugars, differences in the shrinkage pattern of the xylem and phloem should appear. The loading of sugars should make the whole stem lag behind the xylem near the sources, since carbohydrates and water are loaded into the cells and their radius thus increased. On the other hand, unloading should decrease the lags near the sinks since carbohydrates are transported from the phloem to the growing or storage tissues. This would create a positive pressure gradient that would push water to the xylem (Fig. 5).
Thus, the positive peak in the time lags indicates strong sink activity according to the Münch hypothesis. It occurred in the middle of the shoot growth period when noticeable stem extension started. It has been observed that during early development the growing tips are very strong sinks and attract sugars from the leaves below very efficiently (Taiz & Zeiger 1998). As the shoot grows, the newly formed leaves provide a larger proportion of the carbohydrate requirements of the growing shoots and more of the production of the lower leaves is available to other tree parts, thus decreasing the net sink effect. Another peak in the top measurement was observed at around the termination of stem extension growth. The xylem extension growth ends when the secondary cell wall starts forming and lignifying (Taiz & Zeiger 1998). The secondary wall is a major component of the forming tracheids and therefore large quantities of carbohydrates are needed at that stage (Taiz & Zeiger 1998).
In 2002 at the base of the trees the whole stem lagged behind the xylem all the time, which means low sink activity if we interpret the results in the light of the Münch hypothesis. Drought has been reported to have more serious effects on the growth at the base than at the top of the tree on Pinus taeda (see Zahner 1968). At the top growth is suppressed less and the early and late wood are affected equally. At the base, on the other hand, reduced phloem transport is reported to reduce late wood production. The growth patterns in our data supported those observations. The top growth was very similar between the years but during the early summer of 2002 when it was much dryer, we had lower growth at the base. Simultaneously we also observed very negative time lags (see Fig. 2). There is little other data on Pinus sylvestris but the results of, for example, Irvine et al. (1998) at 0.2 m height and Rigling et al. (2002) at 1.3 m height support a response that is similar to Pinus taeda and the extremely low growth rate towards the end of stem growth observed in 2002 (Fig. 3b) could be an indication of that. Furthermore, in 2002 the xylem started to lag behind the whole stem at the top of the tree during the drought in August and September. At that time also the xylem lagged behind the whole stem on the days of high CO2 fluxes (Table 1) and the whole stem was behind on the days of low or moderate CO2 flux. That kind of behaviour could result from water flow from the phloem to the xylem under water stress, which might have been the case. However, the phloem lagging behind towards the end of September in 2001 is in good agreement with the gradual movement of sink activity lower in tree.
In both years and in both trees we see that the time lags reverse from the xylem shrinking first to the whole stem shrinking first for a short period during stem growth and this can not be explained by water flow driven by xylem tension and changes in the radial permeability. Thus, the results suggest, that the diurnal changes in the phloem are not just passive changes in stored water in the bark. Water can be drawn into the phloem even if the hydrostatic pressure drops considerably in the xylem due to an increasing pull of transpiration. In the upper part of the stem, the time lag between xylem and whole stem varies in good agreement with the predictions made using the Münch hypothesis and the results are logical in terms of seasonal growth pattern in Scots pine in Finland. Furthermore, in some cases, the time lags seem to vary according to the photosynthetic activity of the day. The whole stem lags more behind the xylem during high photosynthetic production than during low (Table 1).
We can estimate the loading rate of sugar (sucrose) needed to cause a lag between the xylem and the whole stem. We simply assume that the shrinking of the xylem diameter has to be compensated by the swelling of the phloem in order to keep the whole stem diameter constant and that the swelling is caused by adding material to the phloem. A typical rate of change in xylem diameter at the upper most measurements is 0.05 µm min−1. Taking the dimensions of the stem from that height (stem diameter 4.5 cm and phloem thickness 2 mm) the increase in the cross-sectional area of the phloem should be approximately 0.003 mm2 min−1. Assuming that the conducting sieve cells account for one-third of the total area of the phloem (Taiz & Zeiger 1998) the conducting cells would have an area of 0.9 cm2. So, the increase needed in the area would be small in comparison with the total area of sieve cells, although it is unclear whether the changes are actually happening in the sieve cells or in the cells surrounding them. If we take the sugar concentration of phloem sap to be 1000 mol m−3[Kramer & Kozlowski (1979) give 777 mol m−3 for the sugar concentration of the phloem sap of Red Ash, Sheehy et al. (1995) give a range 300–2000 mol m−3 for source areas and Thompson & Holbrook (2002) use 1250 mol m−3 in their model], which equates to an osmotic pressure of 3.3 MPa (Nobel 1998), we get a loading rate of 0.006 mg min−1. Taiz & Zeiger (1998) give a mass flow rate 0.2–4.8 g h−1 cm−2 for phloem flow. For a gymnosperm the flow rate may be at the lower end of the range. Taking a mass flow rate of 1 g h−1 leads to a sugar flow rate of 0.01 g min−1 in the phloem of our tree(s). Thus the addition in the sugar flux needed to produce the time lags is only a fraction of the total flux. Furthermore, a typical daytime flux of CO2 to the needles at our site is approximately 0.1 mg m−2 s−1. Estimating the sugar production of the needles above the upper most measurement from the CO2 flux gives 0.02 g min−1[the needle area above the measurement height (17 m2) estimated from the measurements of Ilvesniemi & Liu (2001)]. The estimations show that the photosynthetic production is able to maintain the reported phloem flow rates and that the sugar production is high enough to produce time lags. The effects of the tension in the xylem, the difference in the elasticity of the xylem and the phloem and the water permeability of the layer between them are not taken into account in this calculation. The assumed osmotic pressure of the phloem sap is high enough to allow water flow from xylem to phloem and we simply assumed that the sugar concentration is constant.
Taken together, our measurements suggest that phloem transport does occur as proposed by Münch in trees and also in gymnosperms. However, changes in environmental conditions increase the scatter in time lags and make the analysis more difficult. In the cases of severe drought, for example, the maintenance of water balance seems to outdo the effects of sugar transport in time lags. The time lag studies indicated the feasibility of using diurnal diameter changes of phloem and xylem in analysing phloem transport in situ. As it is a non-destructive method it offers a lot of potential in these studies, especially, when we include the amplitude changes into the analyses. When tissue elasticity is known, this information can be used to quantify the driving pressures of the flow (Irvine & Grace 1997) and then we are closer to being able to quantifying the mass-flow rates in the phloem, which has been intractable before. The method that we present in this paper for measuring phloem transport is still very preliminary. However, as it is both non-destructive and inexpensive, it is a very promising, new application of this established ecological measurement technique.
Nuria Altimir is acknowledged for her work in providing CO2 flux data.