| Surface structure, wax and methanol-extractable compounds in Scots pine and Norway spruce needles enhanced UV-B | ||
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In the field experiment with mature Scots pines the amount of methanol-extractable UV-absorbing compounds was not affected by supplemental UV-B during the first and second growing seasons (IV). During the third growing season, the amount of these compounds was significantly or slightly elevated in the ambient treatment compared to UV-B (VI). These compounds are synthesised only if necessary and required for protection (Mohr & Drumm-Herrel 1983), and because field-grown plants have higher levels of UV-absorbing compounds compared to controlled environments, supplemental UV-B radiation generally induces only scant additional accumulation under field conditions (Dillenburg et al. 1995). In field experiments with loblolly pine (Sullivan et al. 1996) and two Mediterranean pines (Pinus pinea, Pinus halepensis) (Petropoulou et al. 1995), the amount of UV-absorbing compounds was unaffected after one growing season of UV-B exposure. Still, the even lower amount of UV-absorbing compounds in the present UV-B treatment compared to the ambient treatment was unexpected. The ratio UV-A/UV-B was higher in the ambient treatment compared to the UV-B treatment, and higher levels of UV-A and PAR might stimulate screening compound production at lower or moderate UV-B levels (Day & Demchik 1996), especially in low visible light (Caldwell et al. 1994, Middleton & Teramura 1993). However, it is unlikely that this would be notable in the field conditions (Dillenburg et al. 1995).
The lower amount of UV-absorbing compounds in the UV-B treated needles during the third year of supplemental UV-B might be due to the already inhibited synthesis of UV- protective pigments (Flint et al. 1985, Deckmyn et al. 1994, Sullivan et al. 1996), indicating cumulative effects of UV-B radiation (Sullivan & Teramura 1992). The fact that the inhibited pigment synthesis was observed in all needle age classes suggests that the information of the earlier year´s UV-B exposure is carried to the new, developing needles. Cumulative effects might therefore also be observed in the new needles if the tree itself has been exposed to UV-B for several years. This theory is supported by the glutathione results from the same experiment (Laakso, unpublished data). The author observed that the current-year needles had lower total glutathione concentrations under supplemental UV-B treatment than under ambient treatment throughout the season, suggesting that the antioxidant protection mechanism against enhanced UV-B radiation was disturbed after the third year of experiment (Laakso, unpublished data), whereas no UV-B-induced changes in glutathione status were observed after the first year of experiment (Laakso et al. 1998).
Cumulative effects were expected to be found in the older needles, which developed during the experiment and received supplemental UV-B for several years. In fact, the c+2 needles collected in July had a 56% higher proportion of stress-induced oxidized glutathione (GSSG %) under supplemental UV-B than in the ambient treatment (Laakso, unpublished data), and during the second growing season’s exposure there was a slight trend towards an increasing amount of UV-absorbing compounds in the older needles (11– and 14-month-old needles) (IV). At the end of the third growing season, the c+1 and c+2 needles contained high amounts of UV-absorbing compounds, but no differences were observed between the treatments (VI). However, these previous-year needles were only analysed once at the end of September, when the UV-B-doses were already low.
It has been suggested that young, still developing needles are more sensitive to UV-B because of their lower amount of UV-absorbing compounds and therefore weaker screening ability (DeLucia et al. 1992, Day et al. 1992, 1996, Naidu et al. 1993). However, in Norway spruce the enzyme involved in the formation of kaempferol 3-O-glucoside was already present in the youngest needles at the beginning of May (Strack et al. 1989). In the present study, too, an elevated amount of UV-absorbing compounds was already observed in three-day-old needles (still enclosed their bud scales), where no tubular waxes or chloroform-soluble waxes could yet be detected (VI). These results indicate that secondary metabolites provide protection for young tissues, becoming less abundant and important as the tissue ages and other, non-chemical protective mechanisms may develop, as was concluded by Bennett and Wallsgrove (1994). High amounts of UV-absorbing compounds were also detected in the oldest needles (c+2), where the amount of waxes was already decreased and the wax structure eroded. It may therefore be concluded that Scots pines are well protected against increased UV-B throughout the needle development stages, and only the defence mechanism may vary.
Recently, Fischbach et al. (1999) observed the UV screening pigments in Norway spruce needles in UV-B field cabinet experiments during two years, and part of the differences in responses were possibly due to the climatic differences between these two years, which was probably also the case in our three growing seasons´ experiment. For example, the higher amount of UV-absorbing compounds in the needles collected in August 1997 could be explained by the warmer and sunnier weather compared to August 1998 (Table 2, Fig. 3). Stephanou and Manetas (1997a,b) also observed a higher amount of UV-absorbing compounds in the sunny and warm summertime compared to the rainy autumn, and even changes in the weather conditions during one day may cause alterations in the amount of UV-absorbing compounds (Veit et al. 1996). Strid and Porra (1992) found more abundant accumulation of UV-absorbing compounds in plants that have had the opportunity to recover than in plants repeatedly exposed to UV-B. This observation might be important, since plants in natural conditions are seldom exposed to continuously high UV-B doses. It is interesting to speculate what this observation could mean for the northern plants, which have the autumn time to recover from the UV-B stress, but are exposed to almost continuous light and UV-B in the summer time, although the UV-B doses are only high in the daytime. The repair mechanisms work ineffectively at low temperatures (Björn et al. 1997), and if the synthesis of UV-absorbing compounds is also ineffective in a cold climate, it may be crucial for plants in the northern early spring conditions.
The maximum absorption peak of methanol extraction was observed at 280 nm, whereafter the absorbances steeply decreased, which is typical of woody species (Day et al. 1994). Methanol extraction does not differentiate between individual compounds and may also include several other phenolics besides flavonoids (Lindroth & Pajutee 1987, Sauvesty et al. 1992), and possibly also some other compounds. The absorption at 310 and 320 nm could indicate some flavone and the maximum absorption at 280 nm could be due to kaempferol (Sharma et al. 1998), which, together with prodelphinidin, procyanidin, quercetin, and isorhamnetin, is the most common flavonoid in Scots pine needles (Lauranson-Broyer & Lebreton 1993). Schnitzler et al. (1996, 1997) found that the main UV-B-induced compounds in Scots pine cotyledons and primary needles were 3”,6”-di-para-coumaroyl-isoquercitrin and 3”,6”-di-para-coumaroyl-astragalin, but no information is available from mature Scots pines. Because the responses of individual flavonoids to UV-B radiation may be different (Lavola et al. 1997, Fischbach et al. 1999), it is possible that the amounts of certain flavonoids also increased significantly in the present study, although the methanol extraction revealed no UV-B-induced changes.
There are also other compounds, such as ferulic acid and other bound phenylpropanoids, which absorb strongly at 260 to 280 nm and are co-polymerized with cutin and lignin in the cell wall, and may play a dominant role in the UV screening of conifer species (DeLucia et al., 1992 and references therein). Because these compounds are released from the cell walls by alkaline hydrolysis and are not methanol-extractable (Strack et al. 1988, van de Staaij et al. 1995), their role in the protection against UV-B remained unclear in the present study. Investment in these cell-wall-bound compounds, thicker epidermis or other protective mechanisms instead of methanol-extractable UV-absorbing compounds could partly explain the lower amount of UV-absorbing compounds in supplemental UV-B compared to ambient treatment (VI). In Scots pine, UV-B radiation did not induce the biosynthesis of wall-bound metabolites in cotyledons (Schnitzler et al. 1996), but there is no knowledge of their role in the protection in mature trees (Laakso & Huttunen 1998).
It is still unknown which protective mechanism is selected in each situation, although it might be concluded that flavonoids are produced when the epidermis itself is not a sufficiently efficient UV-B filter, due to the undeveloped or already eroded wax structure. For example, if the epicuticular waxes of the current year needles have been degraded by air pollutants, the wax structure may be an ineffective barrier against UV-B in the future, because no new waxes are produced after the first year. Furthermore, unlike the UV-absorbing compounds, the hairs or epicuticular wax crystals may also reduce PAR transmission (Cen & Bornman 1990, Bornman & Vogelmann 1991, Krauss et al. 1997). According to Lowry et al. (1980), the cuticular defences are costly in terms of protection per unit of synthesis and only effective in intense visible radiation. Flavonoids are suggested to be the primary means of attenuating UV-B also because they are synthesized rapidly upon exposure to UV-B (Caldwell et al. 1983, Jordan 1996). For example, Scots pine seedlings reacted within 72 hours of UV-B irradiation by accumulation of flavonoids (Schnitzler et al. 1993). The production of secondary metabolites is also a significant cost (Bennett & Wallsgrove 1994), but in contrast to terpenoids, for example, the production of phenolic compounds is not especially costly (Gershenzon 1993). Furthermore, the secondary substances might be recycled by the plant and therefore used for its primary metabolism (see Fagerström et al. 1987).
It should be kept in mind that the possible changes in the amount or distribution of waxes or UV-absorbing compounds are connected to the other functions of the plant. The altered wax composition may change the reflectance properties of the leaves, but may also reduce transpiration, alter the uptake of aqueous chemicals and affect the responses of herbivores to the plants (Jordan 1996). Changes in secondary chemistry may have important implications for plant herbivores, and the decomposition rates and products of plant litter, change the physiology of plants and possibly influence the ecosystem function (Rozema et al. 1997b).