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- Materials and Methods
Closed forests are often spatially complex and the light environment can also be spatially and temporally variable. Irradiance generally decreases vertically from the top of the canopy to forest floor. Average irradiance on the floor of a closed forest may be as little as 0.5% of full sunlight whilst leaves in the upper canopy receive full sunlight (Björkman & Ludlow, 1972). The understorey of some evergreen closed forests is amongst the darkest environments habitable by higher plants (Allen & Pearcy, 2000a). In this layer, the light environment is characterised by diffuse irradiance of low intensity interrupted by intense sunflecks that might last from a second or less to 15 min or more. Depending on the ecosystem in question, sunflecks may account for between 20 and 80% of the total irradiance (Watling et al., 1997). Pearcy (1987) estimated that sunflecks resulted in 30–60% of the total daily carbon gain, and noted that the utilisation of sun flecks is of paramount importance for the overall assimilation balance of understorey plants.
For leaves acclimated to shade, the photosynthetic response to a sudden increase in irradiance (via sunflecks) is not instantaneous owing to biochemical and stomatal limitations. Biochemistry is initially limiting because after prolonged shading, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other photosynthetic enzymes are deactivated, and because of the small size of the pools of Calvin cycle intermediates. Appreciable periods of time are required for enzyme re-activation and to build the pools of intermediates. The stomatal limitation arises because stomata typically close at least partially in the shade and, again, it takes some time for the stomata to re-open and thereby reduce the limitation on supply of CO2 for photosynthesis. The efficient use of sunflecks is thus a function of the rapidity with which biochemical and stomatal limitations are removed due to, inter alia, light activation of enzymes and stomatal opening, and/or the maintenance of high enzyme activation and wide-open stomata during low-light periods (Pepin & Livingston, 1997; Allen & Pearcy, 2000a,b; Naumberg & Ellsworth, 2000; Timm et al., 2002).
Previous studies in diverse ecosystems (tropical rainforests, Allen & Pearcy, 2000a; temperate pine plantations, Naumberg & Ellsworth, 2000) have shown considerable variation in the relative contribution of stomatal control to sunfleck utilisation. In a tropical ecosystem, stomatal response times determined the photosynthetic response times to sunflecks when water was limiting, but stomata exhibited little or no control when water was not limiting (Allen & Pearcy, 2000a). In a temperate pine plantation, stomatal responses generally limited sunfleck responses, but to different extents in different species (Naumberg & Ellsworth, 2000).
Sunflecks are also potentially harmful to plants because of the time-lag between the beginning of a sunfleck and the realisation of maximum rates of photosynthesis. This time-lag leads to the quantum of absorbed irradiance being, at least temporarily, in excess of what is used in photochemistry (Logan et al., 1997; Watling et al., 1997; Thiele et al., 1998) and can cause ‘photoinhibition’ (defined as a persistent decrease in quantum use efficiency). Photoprotective mechanisms that function to dissipate excess energy are thus likely to be active in shade-adapted species subject to sunflecks. Foremost among these are: (1) the energy (pH) dependent dissipation that reduces the quantum efficiency of photosystem II; (2) a dissipation mechanism related to the conversion (de-epoxidation) of the xanthophyll cycle pigments violaxanthin via antheraxanthin to zeaxanthin; and (3) the quenching of excited chlorophylls and scavenging of active oxygen species in thylakoids (Niyogi et al., 2001; Demmig-Adams, 2003; Morosinotto et al., 2003).
Only a handful of studies have examined photoinhibition in relation to sunflecks. Investigations on (sub)tropical plants showed that fast responses of photo-protective systems accounted for flexible protection, but that photoinhibition (a persistent decline in quantum efficiency) was not completely avoided (Logan et al., 1997; Watling et al., 1997; Thiele et al., 1998). A reasonable primary hypothesis is that shade-adapted leaves are more susceptible to photoinhibition during sunflecks because they contain fewer protective pigments (xanthophylls) than sun foliage (Logan et al., 1997). Previous studies on sunfleck responses have focused on emerging saplings or understorey shrubs in comparison with gap plants, but did not compare differently light-adapted foliage within the same species (i.e. sun leaves vs shade leaves). Similarly, while photoinhibition and sunflecks have been studied in detail in tropical and subtropical forests (Logan et al., 1997; Watling et al., 1997; Stegemann et al., 1999; Allen & Pearcy, 2000a,b) and in northern hemisphere temperate ecosystems (Pepin & Livingston, 1997; Naumberg & Ellsworth, 2000), the same is not true for cool-temperate rainforests in the southern hemisphere.
In mainland Australia these Nothofagus-Eucalyptus forests are confined to relatively small areas and are therefore of high conservational value. One view is that once closed-canopy Nothofagus rainforest is established, it may become self-maintaining and less fire-prone than mixed stands with Eucalyptus spp. (Howard, 1973b). Such a view makes the maintenance and regeneration of these forests an important aim of local forest and park management. Myrtle beech (Nothofagus cunninghamii) – the dominant overstorey species of these ecosystems – can regenerate in the light-limited understorey of a closed canopy rainforest (Howard, 1973b) – a phenomenon supported by previous studies of steady state light compensation points (Howard, 1973b; Ashton & Turner, 1979). In Victoria, coppice shoots are of paramount importance for this regeneration (Howard, 1973b). The response of N. cunninghamii to sunflecks has not yet been investigated, despite the putative importance of sunflecks for carbon gain of coppice leaves and regeneration of this species.
Here we report the responses of gas exchange and photoprotection to simulated sunflecks in N. cunninghamii. The study was carried out in situ in a natural old-growth rainforest gully in Victoria. Measurements were made on more light-adapted leaves from the upper canopy and shade leaves from coppice shoots near the forest floor. We tested the following hypotheses: (1) as a rainforest species that can regenerate under low irradiances –N. cunninghamii displays adaptive responses to sunflecks such as fast induction of photosynthesis or maintenance of open stomata under low irradiances; (2) because coppice leaves receive a larger proportion of irradiance as sunflecks and these are more important for their daily carbon gain their response to sunflecks is faster compared with upper canopy leaves; and (3) because upper canopy leaves experience longer periods of direct sunlight they have stronger photoprotection (measurable as xanthophyll related decrease in quantum use efficiency) at the potential expense of carbon gain compared with coppice leaves.
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- Materials and Methods
The photosynthetic response of N. cunninghamii to sunflecks is amongst some of the faster photosynthetic responses reported and is consistent with our hypothesis that N. cunninghamii responds quickly to sunflecks. There was no evidence that stomata were controlling this photosynthetic response. Instead, stomatal conductance remained high even in deep shade. There was thus a small stomatal response to simulated sunflecks. High stomatal conductance in the shade generally reduces water-use efficiency, but increases the efficiency of sunfleck use. Hence, poor drought tolerance and the ability to survive and regenerate under a shaded canopy may well be functionally related to high stomatal conductance in the shade in this species.
We expected that coppice leaves would have faster sunfleck responses than upper canopy leaves, because of putative differences in the relative importance of sunflecks for daily carbon gain. However the reverse was the case – photosynthesis of upper canopy leaves responded more rapidly to simulated sunflecks than that of coppice leaves. This was related to the higher photosynthetic capacity of upper canopy leaves, and might relate to the lack of difference between upper canopy and coppice leaves in the relative importance of sunflecks for daily carbon gain (Table 4).
Photoprotective mechanisms (xanthophyll cycle) led to a more persistent decline in photochemical efficiency after the simulated sunfleck in upper canopy leaves than in coppice leaves. No persistent photoinhibition (decrease in photochemical efficiency) was observed in coppice leaves after simulated sunflecks, suggesting that fast recovering pH- and xanthophyll-related photoprotection mechanisms prevented photodamage to the photosynthetic apparatus and ensured high efficiency for subsequent shade periods. In upper canopy leaves there was a more persistent decline of maximum quantum efficiency and roughly modelled estimates suggest this could decrease photosynthesis by more than 30% during a shade period after a sunfleck.