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Plant growth and yield are enhanced by exposure to elevated CO2 (Norby et al., 1996;Rogers et al., 1983; Ceulemans & Mousseau, 1994; Idso & Idso, 1994; Saxe et al., 1998). An increase in total leaf area (Ceulemans et al., 1995; Norby et al., 1999) and leaf photosynthetic rate per unit leaf area, coupled with a decrease in shoot respiration rate are thought to be responsible for this increase (Ceulemans et al., 1999). We do not, however, know why plant responses are so species specific.
Elevated CO2 often increases total leaf area, leaf weight and leaf weight-to-area ratio (Ceulemans, 1997; Norby et al., 1999). Leaf area development is an important determinant of total plant productivity and varies with environmental conditions (Taylor et al., 1994). However, much uncertainty remains as to how elevated CO2 affects leaf growth and anatomy. Short-term responses to elevated CO2 have indicated changes in leaf anatomy (Wullschleger et al., 1997), but it remains questionable whether individual leaf size, total leaf number, or both are influenced. Moreover, highly variable responses have been observed between tree species with determinate growth as compared with those with indeterminate growth (Gardner et al., 1995; Jach & Ceulemans, 1999). Anatomical features are plastic because leaf structure, leaf shape and cell distribution can change, within limits, together with leaf functions, which enable an adaptive response to rising CO2 (Long, 1998; Pritchard et al., 1998).
The leaf is the key organ for photosynthesis and transpiration. Therefore, leaf morphology and cell distribution may be important in influencing physiological processes (Parkhurst, 1986). The leaf contains both the assimilating and conducting tissues, and either or both tissues could be affected by CO2 (Assmann, 1999). Although leaf morphological features have been considered for understanding plant responses to rising CO2, the present knowledge based on morphology is still insufficient. It seemed necessary, therefore, to discriminate between responses of mesophyll and vascular tissues to elevated CO2, and also to take into account other microscopic features related to source-sink relations.
There have been studies concerning the responses of leaf anatomy to rising CO2, but most have focused on stomatal acclimation (Beerling, 1997; Jarvis et al., 1999). Few studies have examined changes in internal leaf structure and in leaf surface wax under elevated CO2, which may be important for bridging data collected at the physiological level to whole plant and canopy level processes (Prior et al., 1997; Pritchard et al., 1999). The aim of this study was to investigate the responses of Scots pine (Pinus sylvestris L.) needles after 4 yr of exposure to elevated CO2 to elucidate the response mechanism of different tissue types and to provide data for further interpretation of whole-plant source-sink relationships.
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Stomatal density affects gas exchange, stomatal conductance and instantaneous water-use efficiency (Woodward & Bazzaz, 1988). Thus, stomatal acclimation to the prevailing CO2 environment has been the subject of various studies (Peñuelas & Matamala, 1990; Woodward & Kelly, 1995; Poole et al., 1996; Bettarini et al., 1998). These studies generally reported a reduction in stomatal density when plants, including coniferous trees, were exposed to elevated CO2. For example, Beerling (1997) separated out the different stomatal density responses of the needle surfaces in P. sylvestris and reported a reduction in stomatal density in response to elevated CO2 in yr 1 of treatment. Contradicting observations, however, have been reported for P. banksiana (Stewart & Hoddinott, 1993) and P. pinaster (Guehl et al., 1994). The results presented here indicated that Scots pines, after 4 yr exposure to elevated CO2, reduced their stomatal density. There were no significant differences in the average number of rows of stomata between the two CO2 treatments. This might indicate that the lower stomatal density under elevated CO2 could largely be explained by an increase in epidermal cell expansion that gave rise to the larger needle size in the elevated CO2 treatment (Ceulemans et al., 1995). It was further observed that both adaxial and abaxial surfaces responded differently to the elevated CO2 treatment in terms of their number of rows of stomata and stomatal density, as also noticed in other experiments (Pearson et al., 1995). Such differences in CO2 sensitivity of the adaxial vs the abaxial surface could be attributed to the different light environments at each surface (Ceulemans et al., 1995) or different intrinsic cellular development (Sachs et al., 1993). Although we did not measure stomatal conductance in this experiment, Beerling (1997) and Medlyn et al. (2001) provided a direct evidence of reduction in stomatal conductance for P. sylvestris under elevated CO2. Considering the role of stomata in various physiological processes, the parallel changes in the reduction of stomatal density and stomatal conductance may have important consequences for the response of pine trees to water stress under elevated CO2 (Wang & Kellomaki, 1997). Recently a gene involved in the signal transduction pathway responsible for controlling stomatal numbers at elevated CO2 has been identified in Arabidopsis (Gray et al., 2000).
Many investigators observed that elevated CO2 stimulated leaf development (Radoglou & Jarvis, 1990; Taylor et al., 1994). The experiments described here indicated that needle cross-sectional area increased by 10.4% in the elevated CO2 treatment, confirming an earlier observation (Jach & Ceulemans, 1999). Nevertheless, the CO2 treatment had different effects on needle width and needle thickness. When all whorl levels were combined, needle thickness significantly increased, by on average 6.4%, in contrast to a non-significant increase in needle width.
Cell size and the number of cells dictate needle size. Changes in the differentiation of tissues within the needle may result in different physiological functions. Therefore, the relative area of mesophyll cells may be more closely related to the rate of photosynthesis than are the epidermis and the vascular bundles (Parkhurst, 1986; Evans, 1999; Roderick et al., 1999). In the present experiment, we found that the relative area of epidermis plus hypodermis, resin canal and central cylinder remained similar or decreased in the elevated CO2 treatment; only the relative area of the mesophyll tissue showed a significant increase. In some cross-sections, the development of a third mesophyll cell layer could be observed, giving rise to a large increase in area. This observation agreed with earlier findings made in P. taeda (Thomas & Harvey, 1983) and in Populus (Radoglou & Jarvis, 1990). In both studies it was concluded that the enlargement of cross-sectional area of needles resulted primarily from an increase in mesophyll tissue. Given the link between the proportion of mesophyll tissue and total chloroplast number per needle, this is an important finding, suggesting that the higher photosynthetic rate was likely to result from a larger area of mesophyll tissue. A higher photosynthetic rate under elevated CO2 was indeed observed (Gunderson & Wullschleger, 1994; Beerling, 1997; Jach & Ceulemans, 1997;2000).
Responses of vascular tissues to elevated CO2 have already been reported for several pine species (Thomas & Harvey, 1983; Conroy et al., 1986; Pritchard et al., 1997). Thomas & Harvey (1983) demonstrated that the area of the vascular tissue increased in P. taeda following exposure to elevated CO2. A similar result was also reported for P. radiata (Conroy et al., 1986) and for P. ponderosa (Pushnik et al., 1995). Pritchard et al. (1997), however, found that the phloem area decreased under elevated CO2 as a result of fewer sieve cells in the needles of P. palustris. The present study revealed a significant increase in the relative area of the phloem although the CO2 treatment did not have a significant effect on the relative area of the vascular bundle. The enhanced formation of phloem under elevated CO2 was in agreement with the findings of Ewers (1982). This author reported that the unifacial cambium in the vascular bundle produced secondary phloem rather than secondary xylem in several coniferous species including P. sylvestris grown in natural conditions. Such changes of the phloem area may imply that the capacity of the vascular bundle to transport fluids is enhanced since there is a link between vein structure and photoassimilate translocation (Körner et al., 1995; Jokela et al., 1997). After exposure to elevated CO2, photosynthetic rate is enhanced with the production of extra photoassimilates in P. eldarica (Garcia et al., 1994), P. taeda (Teskey, 1997) and P. sylvestris (Beerling, 1997; Jach & Ceulemans, 1999). Therefore, the increase in the area of the phloem cells may be interpreted as a positive response of the anatomical structure to the extra photoassimilates produced under elevated CO2 conditions.
The anatomical structure of the conifer needle has been considered an important diagnostic tool in the field and has been used as a bioindicator of environmental pollution (Sutinen & Koivisto, 1995). Likewise, structural characteristics of conifer needles can also be applied to determine the effects of elevated CO2 (Pritchard et al., 1997). In the light of the variability of certain anatomical characteristics, it was important to use strictly comparable material since the variation encountered among needles from different developmental stages or from different crown levels can be larger than the effects of different treatments (Kinnunen et al., 1999). The limitation of small samples together with the use of plant materials from different sources have been overlooked in some publications, and this may have accounted for much of the disagreement in the conclusions. It is strongly suggested that the mid-portion of mature needles should be selected preferably from one single crown level to further confine the variation to the minimum for comparative analysis.
In conclusion, changes in morphological and anatomical characteristics were found in Scots pine needles following a 4-yr exposure to elevated CO2. Leaf development was altered after exposure to elevated CO2. Nevertheless, stomatal density was reduced on both adaxial and abaxial needle surfaces. The significant increase in needle thickness together with a small increase in needle width accounted for a 10% increase in the needle cross-sectional area. The relative area of the epidermis plus hypodermis, resin canal and central cylinder remained similar or decreased in the elevated CO2 treatment, whereas the relative area of mesophyll significantly increased. Furthermore, the formation of sieve cells was enhanced after exposure to elevated CO2. The change of vascular bundles to CO2 enrichment may be linked to the transport of extra photoassimilates produced following the exposure to elevated CO2. In contrast with less competing species, Scots pine needles might structurally benefit from a prolonged exposure to elevated CO2.