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- Materials and Methods
A lichen is a symbiotic association between a species-specific fungal partner, the mycobiont and its autotroph photobiont (green algae and/or cyanobacteria). The mycobiont depends on the delivery of photosynthates from its photobiont that often grows inside a dense mass of screening fungal tissues. The fungal partner uses the photosynthates as an energy source and for new thallus growth (as reviewed by Palmqvist, 2000), allowing both partners to colonize new ground. At the same time, the mycobiont invests a substantial part of the fixed carbon in a diverse secondary chemistry with > 800 secondary compounds identified so far (Huneck & Yoshimura, 1996; Huneck, 2001). Individual species can contain as much as 30% of dry weight (DW) (Huneck, 1973), but the relationship between lichen growth and investments in secondary compounds is not well known.
A few secondary compounds, such as the orange parietin, the yellow usnic acid, the brown melanins and the colourless atranorin, are located in the upper cortex directly above the photobiont layer. During the last decade, there has been a focus on cortical lichen compounds. There is now solid evidence that some coloured cortical compounds are induced by UV-B (Solhaug & Gauslaa, 1996; Solhaug et al., 2003; McEvoy et al., 2006), boosted by photosynthates (Solhaug & Gauslaa, 2004; McEvoy et al., 2006), correlated with solar radiation at spatial (Gauslaa & Solhaug, 2001; Gauslaa & Ustvedt, 2003) and temporal scales (Bjerke et al., 2005; Gauslaa & McEvoy, 2005), and that they screen (Gauslaa & Solhaug, 2001) and protect underlying photobionts against excessive solar radiation (Gauslaa & Solhaug, 2004; Vráblikováet al., 2006). These functions serve to optimize lichen photosynthesis in a changing light environment and hence expand the organic carbon pool in a lichen thallus. Among the mentioned cortical compounds, melanins are the only compounds that cannot be extracted. They are complex and poorly known polymers of phenolic compounds (Plonka & Grabacka, 2006), and can only be quantified by indirect methods.
The vast majority of secondary lichen compounds are located in the medulla. Most are colourless, but with strong UV-B absorbance bands. Medullary compounds often occur in largest quantities in the photobiont layer, which may still be consistent with a photoprotective role (as hypothesized by, for example, Fahselt & Alstrup, 1997). However, the ecological role of medullary compounds is not clear. Additional hypotheses for functional roles exist, such as antiherbivore, antimicrobial and antifungal agents and mineralization agents (as reviewed by Fahselt, 1994). One hypothesis supported by experimental evidence, infers herbivore deterrent roles of medullary compounds for lichen-feeding snails (Lawrey, 1980; Gauslaa, 2005) and insects (Giez et al., 1994; Pöykkö & Hyvärinen, 2003; Pöykköet al., 2005). The compounds are carbon-based, and most ought to be present in large concentrations in order to deter herbivores efficiently (Gauslaa, 2005). However, the natural variation in medullary compound concentration is still unknown for most species.
This study deals with the tripartite foliose lichen, Lobaria pulmonaria (L.) Hoffm., a spectacular and large old forest species. It contains both green algae (main photobiont) and cyanobacteria in small, internal cephalodia. The upper cortex forms visible cortical melanins that screen UV and PAR radiation from the photobiont (Gauslaa & Solhaug, 2001). In addition to melanins, UV-absorbing depsidones are deposited as numerous tiny crystals outside medullary hyphae. It is not known whether these two groups of secondary metabolites are regulated by common factors. If the depsidones add to the protection against excessive UV-B, the content of both compound types should be highest in sun-exposed habitats. If depsidones serve other functions, one would expect the melanin content to follow a light gradient, but the depsidones may not necessarily concur.
The concentration of secondary compounds is in some way related to the carbon metabolism. They may be produced as a sink for excess carbon (Mosbach, 1973) under some environmental stresses during which major parts of the fixed carbon cannot be invested in new thallus growth. If this is the case, a trade-off between lichen growth and synthesis of secondary compounds would be expected. A trade-off may also occur between the synthesis of melanins and depsidones if they are formed from a common resource pool. On the other hand, if the depsidones serve as a vital protection against herbivores, as recently shown by Gauslaa (2005) in grazing experiments on L. pulmonaria, one would not expect depsidone concentration to vary with lichen growth rate and/or solar radiation in its habitat. To test such hypotheses, lichen growth and secondary compounds need to be quantified. So far, we are not aware of lichen studies relating the concentration of secondary compounds to growth. Recently, growth was assessed in 600 L. pulmonaria thalli transplanted in three successional forest stands with contrasting light climates, in which three irrigation treatments were applied in each stand (Gauslaa et al., 2006b). Growth varied substantially among the replicates because of a dependency on a combination of external and internal factors (Gauslaa et al., 2006b). The main objective of our study is to quantify depsidones and melanins in these transplants, and use these new results to analyse relationships between the two categories of compounds, lichen growth and habitat factors.
Another objective is to study the sun-screening function of melanic compounds in a wider perspective, by including experiments quantifying side-effects of melanins, such as their ability to cause excess heating during strong solar radiation (Gauslaa, 1984). Lobaria pulmonaria is susceptible not only to high light intensity, but also to excessive temperatures (Lange, 1953; Gauslaa & Solhaug, 1999). High light screening by melanins may come at a price, as the darkened thalli absorb more solar radiation, causing the temperature to rise beyond tolerable values. In order to evaluate the effect of various acclimation traits on the total tolerance of excessive light, we also aim to estimate the ability of the photosynthetic apparatus to acclimate by dissipating excess light by increased nonphotochemical quenching (NPQ) achieved after a sudden exposure to clear-cut conditions. Previously, NPQ has only been studied in L. pulmonaria during gradual seasonal variations in light in intact forests (MacKenzie et al., 2001, 2002). Finally, we aim to assess the high light susceptibility of the transplants, acclimated under defined forest stand-specific conditions and with known acclimation traits, by exposing them to natural full, direct sunshine.
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Forest stand strongly influenced the L. pulmonaria thalli studied, especially with respect to the solar radiation-induced synthesis of cortical melanins (Figs 3, 4) and increased NPQ (Fig. 6). These acclimation traits presumably played a role in the high growth rates during the 100 d transplantation period, with an average DW gain of 23.1% for the clear-cut area thalli (total range 0–45%; Gauslaa et al., 2006b). Despite the strong light-induced variation in growth responses during the transplantation period (Gauslaa et al., 2006b), the total concentration of medullary depsidones was conserved (Table 1), apparently regardless of external factors such as the indirect site factor reflecting the openness of the canopy. This is strong evidence against the hypothesis inferring a solar radiation-protective role for UV-B-absorbing medullary depsidones, violating the inducibility criterion (Cockell & Knowland, 1999).
The lack of any relationship between thallus growth rate and production of medullary depsidones also suggests that these compounds do not function as waste products, or as a sink for excess carbon during oxidative stress, as was proposed by Mosbach (1973). In plants, various hypotheses exist regarding the effects of environmental factors on secondary metabolite concentrations. The carbon-nutrient balance (CNB) hypothesis (Bryant et al., 1983) states that the concentration of carbon-based secondary compounds (CBSC) will increase whenever excess amounts of carbohydrates accumulate in relation to growth requirements, or, put another way, factors that inhibit photosynthesis (shading) or stimulate growth more than photosynthesis (fertilization) will decrease the carbohydrate pool available for CBSC. A second hypothesis, the growth-differentiation balance (GDB) hypothesis (Loomis, 1932; Lorio, 1986; Herms & Mattsson, 1992; Stamp, 2004), states that growth is limited by water and nutrients, whereas differentiation depends on the available carbohydrates. Therefore, differentiation, and, hence, the production of CBSC, dominate when conditions (excluding carbohydrate supply) are suboptimal for growth. The growth of the L. pulmonaria thalli used in our study has previously been shown to be determined by a combination of external (forest stand, site factors) and internal factors (Chl content and thallus area; Gauslaa et al., 2006b). Application of the plant CNB and GDB hypotheses to our transplants would suggest that the concentration of depsidones varies in accordance with growth rate, higher growth rates implying lower concentrations of secondary compounds. This was not the case for any of the depsidones. A fixed percentage of the weight gain was deposited in medullary depsidones regardless of growth rate, with the exception of the minor methylnorstictic acid that occurred at the highest concentrations in the fastest-growing clear-cut area thalli.
Our results are, however, consistent with a herbivore defence role of depsidones. Grazing marks from snails are frequent on L. pulmonaria in calcareous broadleaved deciduous forests (Gauslaa et al., 2006a). Nondestructive acetone rinsing, in reducing the concentration of secondary compounds, consistently increases the grazing by snails of this particular lichen (Gauslaa, 2005). In a herbivore-defensive perspective it makes sense for the long-lived L. pulmonaria to maintain a certain concentration of depsidones regardless of growth and light availability.
The cortical melanins in L. pulmonaria, quantified by means of BRI, were highly influenced by habitat-specific indirect site factors during the transplantation period. In the present paper, we also showed that the BRI, compared with plain reflectance values, is a simple and more responsive quantitative estimator of the brown pigments located in the upper parts of the upper cortex of L. pulmonaria. Melanins screen solar radiation (Gauslaa & Solhaug, 2001) and are induced by UV-B (Solhaug et al., 2003). Therefore, the low reflectance of the clear-cut area thalli was expected (Fig. 2). Irrigation increases melanin formation in sites where ambient UV-B, according to Solhaug et al. (2003), is sufficient for a strong induction of melanin synthesis. This is consistent with the view that melanin formation is an active physiological process requiring hydration. Furthermore, the addition of nutrients reduced the formation of melanic pigments in the clear-cut stand. These thalli also had a slightly higher area gain than those sprayed with water alone (Gauslaa et al., 2006b). In contrast to the depsidones, melanic pigment production appears to respond to nutrient addition and/or thallus area growth. A trade-off between melanin synthesis and lichen growth is evident in Fig. 5, showing negative significant correlations between BRI and area gain within each forest stand. All thalli were pale at the start of the experiment, and earlier induction experiments have shown that melanin formation is rapid, provided there are sufficient intensities of inducing UV-B. Therefore, the decreasing BRI with increasing growth is hardly caused by dilution of the browning resulting from hyphal expansion. The trade-off suggests that melanin formation and growth compete to some extent for a common resource pool. Whereas eumelanins and pheomelanins are nitrogen-based, most allomelanins are carbon-based, as reviewed by Plonka & Grabacka (2006). However, as long as the identity of the L. pulmonaria melanins is unknown, the trade-off can be caused by either nitrogen or carbon pools. Low BRI values at both ends of the growth spectrum (clear-cut area thalli) were probably the result of Chl bleaching in some thalli. Browning requires an active metabolism, and some clear-cut area thalli had severe Chl degradation (Gauslaa et al., 2006b), which probably occurred before the protective melanins were synthesized.
Acclimation of NPQ also occurred during the transplantation period as a strong response to stand-specific light intensities. According to Demmig-Adams & Adams (2006), both sustained and flexible forms of thermal energy dissipation may occur. The long low light hydration period before NPQ measurements (see Materials and Methods section) suggests that the clear-cut area thalli have achieved a long-lasting increased NPQ value resembling the state in evergreen plants during winter (Demmig-Adams & Adams, 2006). This sustained high NPQ may explain the lower FV/FM values in the clear-cut area thalli recorded by Gauslaa et al. (2006b). In addition to the NPQ reported in Fig. 4, flexible energy dissipation dependent on rapid function of the xanthophyll cycle presumably occurs during daily fluctuations in solar radiation. Such a flexible xanthophyll-based NPQ could hardly have been detected after the prolonged low light hydration period before harvesting. The thalli may have reached higher NPQ values if measured directly after natural high light exposure. Acclimation of L. pulmonaria thalli to seasonal fluctuations in ambient light and temperature has been shown to be temperature-dependent in deciduous forests, but the ability to quench is dependent on seasonal light intensities (MacKenzie et al., 2001, 2002). A higher Chl a : b ratio in clear-cut area thalli at the end of the transplantation period represents another trait of high-light acclimation (Gauslaa et al., 2006b). Quantum yield of PSII, on the other hand, was not forest stand-specific (Fig. 4). However, it is noticeable that the quantum yield of PSII did not become depressed in clear-cut area thalli, because laboratory (Gauslaa & Solhaug, 1996, 1999) and field studies (Gauslaa & Solhaug, 2000) show that L. pulmonaria is susceptible to sudden exposures of high light. One reason for the successful acclimation in the clear-cut area was probably the small clear-cut area size, meaning that lichens were shaded from direct sun by neighbouring canopies 2 h every morning after sunrise (Gauslaa et al., 2006b), allowing repair of photoinhibitory damage and acclimation at low light when hydrated by morning dew. The forest stand-specific acclimation acquired during the 100 d field transplantation greatly influenced their success in the subsequent field exposure to extreme sun (Fig. 6). Higher cortical melanic pigment content, affording better solar protection for the photobiont (Gauslaa et al., 2001; Solhaug et al., 2003), is the likely reason for the increased tolerance exhibited by the air-dry clear-cut area thalli.
Cockell & Knowland (1999) proposed the following set of criteria for determining whether a compound has a screening role: (i) the compound should absorb the relevant radiation; (ii) biosynthesis of the pigment must be inducible by the radiation; (iii) screening activity should be demonstrated in vivo; and (iv) enhanced survival under elevated radiation should be shown to be the result of the compound when other physiological processes are not functioning. Criteria (i)–(iii) have been fulfilled for the melanic compounds in L. pulmonaria (Gauslaa & Solhaug, 2001; Solhaug et al., 2003). Melanic pigmentation reduced photoinhibition in L. pulmonaria under high light exposure in the desiccated state, according to our regression analysis, although a high content of Chl before exposure was highly beneficial. DNA repair mechanisms do not function when thalli are desiccated (Buffoni-Hall et al., 2003), as is probably the case for other physiological repair processes. Thus, the enhanced survival of the darker brown thalli provides the evidence to fulfil the fourth requirement of Cockell & Knowland (1999), assuming that NPQ does not function in desiccated thalli.
Lobaria pulmonaria is a heat-sensitive lichen when desiccated (Lange, 1953; Gauslaa & Solhaug, 1999). Melanin synthesis should ideally reach a concentration that sufficiently shields the photobiont from high irradiance without jeopardizing photochemical processes with excess heat. The excess temperature in melanic thalli relative to pale thalli increased with increasing irradiance (Fig. 5). Air-dry L. pulmonaria thalli experience a linear decrease in maximal PSII efficiency, from below normal to almost zero with increasing temperatures from 40 to 54°C during a 24 h period (Gauslaa & Solhaug, 1999). Together, these results suggest that melanic thalli are more susceptible to heat damage than their paler counterparts at temperatures > 35°C. Thallus temperatures during the outdoor exposure experiment were not recorded, but with irradiance values approaching or exceeding 2000 µmol m−2 s−1 for 8 out of 12 d, it can be assumed that temperatures > 40°C were frequently achieved under the UV/PAR-transmitting screens. Thus, the duration of the exposure to elevated thallus temperatures can be a critical factor for melanic thalli, with adverse effects narrowly tuned against the beneficial photoprotection provided by the melanins. In a long-term perspective, the beneficial light-shielding effect of melanins is more crucial than the detrimental heat-absorbing one, at least in cool climate zones and/or in natural forest habitats.
In conclusion, our study has shown that melanic compounds protect transplanted thalli against high irradiance stress, whereas the depsidones maintain a fixed concentration, regardless of sun exposure and lichen growth rate. The nonresponsiveness of depsidones to climatic factors during transplantation is consistent with a herbivore defence function rather than with the irradiance screening hypothesis.