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- Materials and Methods
Poor regeneration of Engelmann spruce (Picea engelmannii Parry ex Engelm.) after forest harvesting has led to questions about the cause of growth limitations (Dobbs, 1972; Vyse, 1981; Butt, 1990). Observations that the establishment of naturally regenerated (Alexander & Shepperd, 1990; Klinka et al., 2000) and planted (Ronco, 1970b) Engelmann spruce seedlings may be improved by the provision of partial shade suggest that light in open sites may be well in excess of that which can be used by spruce seedlings. Studies with other temperate tree species have shown that in the juvenile stage full sun conditions may not be required for maximum biomass production (Shirley, 1945; Marquis, 1966; Brix, 1970; Eis, 1970; Loach, 1970; Drew, 1983; Mitchell & Arnott, 1995), particularly under conditions of water or nutrient stress (Canham et al., 1996). Where growth does not increase with increasing light availability, mechanisms for avoiding damage to the foliage by excess light can become of principal importance in the response of tree species to high irradiance.
Pigments such as chlorophylls and carotenoids may have an important role in regulating the balance between light absorption and light use, and thus for avoiding damage to foliage under high light conditions. For example, a marked reduction in chlorophyll can occur as a mechanism for balancing light absorption and light use (e.g. Khamis et al., 1990; Bungard et al., 1997; Verhoeven et al., 1997, see also Adams et al., 1995). That balance can also be regulated through increases in carotenoids of the xanthophyll cycle functioning to thermally dissipate light energy when it is absorbed in excess of what can be used in photochemistry (for reviews, see Demmig-Adams & Adams, 1992b; Björkman & Demmig-Adams, 1994). The maximum capacity for thermal energy dissipation is considered to be set by the total pool size of xanthophyll cycle pigments (V + A + Z; Thayer & Björkman, 1990), which consists of the three carotenoids, antheraxanthin (A), zeaxanthin (Z), and violaxanthin (V). Antheraxanthin and zeaxanthin are the components essential for thermal energy dissipation and are formed from violaxanthin in response to excess absorbed light (Björkman & Demmig-Adams, 1994; Demmig-Adams & Adams, 1996; Demmig-Adams et al., 1996).
We chose to study the effects of nitrogen (N) limitation on light use by Engelmann spruce because N can be especially important for physiological acclimation to high irradiance (Ferrar & Osmond, 1986; Seeman et al., 1987), and because the interactive effects of light and N-supply on the xanthophyll cycle and other carotenoids have been little explored in higher plants, and contrasting results have been obtained (compare Bungard et al., 1997 with Verhoeven et al., 1997). Although our primary interest was in the physiological acclimation of spruce to high light and N-stress, we took a whole-plant approach to understanding growth responses by examining biomass allocation in addition to physiological acclimation. Although the effects of light on the growth of angiosperm tree species have frequently been studied in relation to both biomass allocation and physiological responses (e.g. Walters et al., 1993a, 1993b; Kitajima, 1994; Walters & Reich, 1996; Poorter, 1999), there are few such studies of conifer species (Grassi & Minotta, 2000).
The objectives of this study were to determine: whether the constraint to light use by Engelmann spruce seedlings can be alleviated by the provision of adequate N; and to what extent changes in chlorophyll and the xanthophyll cycle carotenoids may have a potentially important role in the protection of spruce from damage by high irradiance.
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- Materials and Methods
Whole-plant biomass of Engelmann spruce seedlings grown at high light (HL, 100% full light) did not differ significantly from those grown at low light (LL, 33% full light), regardless of whether seedlings were grown at high or low N-supply. This is in apparent contrast to the situation observed for some other forest tree species in which light requirements for seedling growth increase with increasing N-supply (Canham et al., 1996). Although in the present study some trend was evident for biomass of high light plants to be greater than that of low light plants under conditions of high N-supply, the apparent improvement was slight given the 3-fold difference in irradiance.
If Engelmann spruce seedlings in the high light treatment were using the additional light available in that treatment, then for seedling growth to be similar in high and low light environments, seedlings in the low light treatment would be expected to exhibit morphological and physiological traits associated with enhanced growth efficiency. However, for the same whole-plant biomass achieved in high light and low light environments, allocation to above-ground vs below-ground biomass (S/R), allocation to leaves (LWR) and leaf area (LAR) were similar for seedlings in both light treatments. In addition, although light compensation points differed between high and low light plants, whole-shoot dark respiration rates and apparent quantum yields were only marginally different (nonsignificant) and high and low light plants did not differ in terms of whole-shoot photosynthetic capacity (Amax) or foliar N concentration (%N). Thus, the similar growth responses of seedlings reared at high and low light appeared to be related instead to a lack of capacity of seedlings in the high light treatment to use the additional light available. Given that Amax is in general linearly dependent on foliar N (Field & Mooney, 1986; Evans, 1989), the lack of response to light of whole-shoot photosynthetic capacity (Amax) and growth of spruce appeared related in particular to an inability to increase foliar %N in response to the increase in light availability. Indeed, that photosynthesis and growth of spruce were N-limited at high light is evidenced by the marked and concomitant increases in foliar %N, Amax, and growth observed for this species when grown at high N-supply in comparison to low N-supply at a given level of irradiance.
By contrast to what was observed here for Engelmann spruce, studies of the response of Picea species to ‘release’ from suppression (overstory removal) suggest that acclimation of spruce to high irradiance may involve increases in both foliar N concentration and shoot-level photosynthetic capacity (Lieffers et al., 1993). However, increases in foliar N concentration (%N) and photosynthetic capacity reported in such studies may be dependent on a concomitant increase in N-availability associated with the removal of competing vegetation, and the situation may also be different for plants not suddenly exposed to light levels much higher than the growth irradiance. Some studies have examined the physiological acclimation of spruce grown continuously in sun vs shade environments, but the contrast which has often been made is that between the extremes of the natural light gradient where, as expected, photosynthetic capacity is higher for seedlings or saplings in open sites than in deep understory shade (Carter & Smith, 1985, 1988; Man & Lieffers, 1997). On the other hand, studies comparing the physiological performance of spruce grown at high light and partial shade suggest that photosynthesis may be comparable or higher in partially cut areas than in clear-cut areas (Ronco, 1970a; Man & Lieffers, 1999, but see Carter & Smith, 1988).
Although in this study Engelmann spruce was apparently unable to use the additional light available in the high light treatment for increased photosynthesis and growth, damage to seedlings by excess light appeared to be avoided through adjustments in foliar pigments important to the regulation of the balance between light absorption and light use. In addition to a light-dependent increase in the chlorophyll a/b ratio (Boardman, 1977; Björkman, 1981), at high N-supply Engelmann spruce seedlings exhibited two biochemical adjustments which may reduce the potential for damage by light in excess of that which could be used in photochemistry (Björkman & Demmig-Adams, 1994): a reduction in chlorophyll content (capacity for light absorption); and an increase in the xanthophyll cycle pigment pool size (capacity for thermal dissipation of excess absorbed light energy).
At low N-supply, high irradiance acclimation of Engelmann spruce appeared less complete. Where Chl a + b was already low as a result of growth at low N-supply, the light-dependent reduction in Chl a + b was not observed and although V + A + Z increased, the increase in V + A + Z was proportionately less than that observed at high N-supply (76% vs 92%). Engelmann spruce therefore exemplifies a third pattern of response of Chl a + b and V + A + Z to high light and N-stress (low N-supply) to that which has previously been reported. In Spinacia oleracea (Verhoeven et al., 1997) both a reduction in Chl a + b and an increase in V + A + Z occurred in response to high light under conditions of N-stress, while in Clematis vitalba (Bungard et al., 1997), Chl a + b was reduced without changes in V + A + Z or other carotenoids. By contrast, at low N-supply foliar acclimation of Engelmann spruce to high light (HL-LN) involved only adjustments in V + A + Z, because light-dependent reductions in Chl a + b were inhibited.
Lower midday values of Fv/Fm for Engelmann spruce in all treatments under clear-sky conditions than under prolonged overcast-sky conditions suggested that all seedlings exhibited some amount of dynamic (readily reversible) photoinhibition of photosynthesis as a result of exposure to excess light on clear days. Photoinhibition of photosynthesis during the midday period on clear days has been widely reported for plants of diverse taxa and life-forms in open sites and in forest gaps (e.g. Long et al., 1994; Lovelock et al., 1994; Krause & Winter, 1996). Under clear-sky conditions, Fv/Fm was lowest for Engelmann spruce grown at high light and low N-supply (HL-LN), and these same seedlings also had the greatest percentage of the xanthophyll cycle pigment pool in the photoprotectively active state (cf. Bungard et al., 1997). A higher conversion state of the xanthophyll cycle pool has been shown to be associated with a lower percentage use of absorbed light in photochemistry and a higher rate of xanthophyll cycle-dependent thermal energy dissipation in the antennae (Verhoeven et al., 1997, see also Khamis et al., 1990). Thus, these results suggested, in agreement with results for V + A + Z and Fv/Fm, that seedlings grown at high light or low N-supply were making less efficient use of absorbed light than seedlings grown at low light or high N-supply and were dissipating excess light by means of xanthophyll-cycle dependent thermal energy dissipation. The rank-order of treatments in terms of the efficiency of use of absorbed light therefore was: LL-HN > HL-HN > LL-LN > HL-LN. Differences between the two intermediately ranked treatments (HL-HN and LL-LN) were however minor, suggesting that in spruce similar levels of light stress can be achieved by growing seedlings at low light (33% full light) and low N-supply (10 mg N l−1) as by growing seedlings at high light (100% full light) and high N-supply (100 mg N l−1) (see Huner et al., 1996).
That Fv/Fm of Engelmann spruce seedlings grown at low N-supply did not recover following 3-d of overcast-sky conditions (cf. control Fv/Fm value of 0.805 for LL-HN plants) suggested also a level of ‘chronic’ or prolonged, stress-dependent photoinhibition (e.g. Greer & Laing, 1992; Skillman & Osmond, 1998) for spruce seedlings under conditions of N-stress. Thus, in the absence of an increase in photosynthetic capacity in response to growth at high light, changes in foliar pigments (Chl a + b, V + A + Z) appeared sufficient to protect the photosynthetic apparatus of spruce against damage by excess light when N-supply was ample, but the response may possibly have been less than sufficient when N-supply was low.
The light-dependent changes in V + A + Z and other carotenoids observed in Engelmann spruce in this study were in accord with those reported previously (e.g. Thayer & Björkman, 1990; Demmig-Adams & Adams, 1992a; Königer et al., 1995; Logan et al., 1996), but with the exception of neoxanthin. Although neoxanthin does not typically respond to light (Thayer & Björkman, 1990; Demmig-Adams & Adams, 1992a; Königer et al., 1995; Logan et al., 1996), a slight but significant decrease in this pigment at high light was observed in this work. This response, however, appeared largely attributable to that of plants grown at low N-supply (L × N interaction marginally nonsignificant, P = 0.087).
Reported effects of N-supply on the xanthophyll cycle pigments and other individual carotenoids per unit chlorophyll are more variable than the effects of light. In one study V + A + Z, lutein, β-carotene, and neoxanthin (per Chl a + b) all increased under N-stress (Verhoeven et al., 1997, see also Solberg et al., 1998) similar to the present study, but elsewhere N-supply was observed to have no significant effect on these carotenoids (Bungard et al., 1997). Information on α-carotene is less available, but our results for Engelmann spruce are in agreement with the association reported between N-deficiency and low α-carotene concentrations and contents in open-grown individuals of the congeneric species Picea abies (Solberg et al., 1998). Our results show that in Engelmann spruce the decrease in α-carotene at high light relative to low light can be inhibited at low N-supply, similar to what we observed for Chl a + b in this species. This might suggest that the light- or season-dependent changes in α-carotene commonly reported for conifers (Adams & Demmig-Adams, 1994; Siefermann-Harms, 1994; Ottander et al., 1995) and other species in which this taxonomically restricted pigment is known to occur (Thayer & Björkman, 1990; Demmig-Adams & Adams, 1992a; Logan et al., 1996) may not necessarily be observed under conditions of N-stress.
In summary, the lack of growth response to light observed for young Engelmann spruce seedlings in this study appeared related to an inability of this species to increase foliar N concentration and photosynthetic capacity in response to high irradiance. Improved N-availability did not alleviate the constraint to the use of high light. Foliage damage (sunscald, leaf necrosis) from excess light appeared to be avoided through a combination of downward adjustments in chlorophyll and upward adjustments in photoprotective xanthophyll cycle carotenoids. An interaction between light and N-supply was described for α-carotene as well as for Chl a + b in this species.