Leaf traits and gas exchange in saplings of native tree species in the Central Amazon

Global climate models predict changes on the length of the dry season in the Amazon which may affect tree physiology. The aims of this work were to determine the effect of the rainfall regime and fraction of sky visible (FSV) at the forest understory on leaf traits and gas exchange of ten rainforest tree species in the Central Amazon, Brazil. We also examined the relationship between specific leaf area (SLA), leaf thickness (LT), and leaf nitrogen content on photosynthetic parameters. Data were collected in January (rainy season) and August (dry season) of 2008. A diurnal pattern was observed for light saturated photosynthesis (A max ) and stomatal conductance (g s ), and irrespective of species, A max was lower in the dry season. However, no effect of the rainfall regime was observed on g s nor on the photosynthetic capacity (A pot , measured at saturating [CO 2 ]). A pot and leaf thickness increased with FSV, the converse was true for the FSV-SLA relationship. Also, a positive relationship was observed between A pot per unit leaf area and leaf nitrogen content, and between A pot per unit mass and SLA. Although the rainfall regime only slightly affects soil moisture, photosynthetic traits seem to be responsive to rainfall-related environmental factors, which eventually lead to an effect on A max. Finally, we report that little variation in FSV seems to affect leaf physiology (A pot ) and leaf anatomy (leaf thickness).


Introduction
Global climate models predict that in the Amazon the length of the dry season period will be extended as a result of global warming associated to an increase of atmospheric CO 2 concentration (Cox et al., 2004).Indeed, a prolonged dry period may affect plant growth and physiological processes, such as photosynthesis and respiration (Hughes, 2000).Although severe soil moisture depletion during prolonged drought may lead to stomatal closure and a decline in leaf area (Nepstad et al., 1994), there is still controversy on whether draught-in-duced water deficit limits tree growth in the Central Amazon.During the 2005 drought, for example, Saleska et al. (2007) reported an enhanced vegetation index of the forest based on moderate resolution imaging spectroradiometer (MODIS) satellite data.This is contrary to what should be expected, as changes in precipitation can alter growth rates (Lewis et al., 2004).In a rainfall exclusion experiment, a 60% reduction of incoming throughfall led to a drastic increase (38%) in tree mortality (Nepstad et al., 2007), much higher than commonly recorded in the Central Amazon, about 1.1% per year (Williamson et al., 2000).Although sapling photosyn-Sci.Agric.(Piracicaba, Braz.), v.67, n.6, p.624-632, November/December 2010 thetic traits of canopy trees have received some attention in tropical forest (e.g.Marenco and Vieira, 2005;Poorter and Oberbauer, 1993), how seasonality of the rainfall regime affects seedling and sapling photosynthetic traits in the Central Amazon still remains to be elucidated.
In addition to soil moisture, light availability is one of the most important factors limiting seedling and sapling growth in the forest understory (Denslow et al., 1990;Valladares and Niinemets, 2008).Through the canopy profile light varies not only in total quantity, but also in quality, as the red/far red (R/Fr) ratio declines towards the forest floor (Smith, 1982).Low irradiance often leads to a decrease in leaf thickness and light saturated photosynthesis (A max ), whereas specific leaf area (SLA) commonly increases under low light intensity (Oguchi et al., 2005).Plant growth is the result of a complex of interacting factors intrinsically related to carbon gain via photosynthesis and loss due to respiration.However, over a wide range of plants species and growth conditions there seems to be a positive relationship between plant growth and photosynthetic rates (Kruger and Volin, 2006) In this study we hypothesized that variation in soil moisture and subtle changes in light availability in the forest understory affect leaf traits and carbon gain in saplings of canopy trees.Thus, the aims of this work were to determine the effect of the seasonal rainfall regime and understory irradiance on leaf traits and gas exchange in ten rainforest tree species.We also examined the effect of specific leaf area (SLA), leaf thickness (LT), and nitrogen content on the photosynthetic capacity.

Material and Methods
The study was conducted 60 km north of Manaus (02º36'21" S; 60°08'11" W), state of Amazonas, Brazil, in an area of native "terra-firme" forest.The region has characteristics of a humid equatorial climate, with a short mild dry season (July-September, with a rainfall of 50-100 mm per month), and a dry-wet transition month (October).The wet season extends from Novem-ber to May (200-300 mm month -1 ).Annual precipitation is 2240 mm (Inmet, 2008(Inmet, , mean of 1961(Inmet, to 1990)).The area is covered by a dense forest and the predominant soil type is an Oxisol of low fertility, clay texture and pH of 4.2 to 4.5.
We used saplings (1.5 to 2-m tall) of 10 tree species selected taking into account their shade tolerance, relative abundance of saplings in the forest understory (at least three replications per species), and economic importance (Table 1).The gas exchange parameters were measured with an infrared gas analyzer (Li-6400, Li-Cor, NE, USA) using one or two leaves per plant and three saplings per species on each season.Light saturated photosynthesis (A max ) was measured at ambient CO 2 concentration (380 μmol mol -1 ), light saturation (1000 μmol m -2 s -1 , and ambient temperature (28 ± 1ºC).Potential photosynthesis (A pot , hereafter termed photosynthetic capacity) was also measured at light saturation, but at a [CO 2 ] of 2000 μmol mol -1 , rather than ambient [CO 2 ].Gas exchange data were collected after a stabilization period of about 10-15 min (total coefficient of variation < 0.7%).The effect of the time of day on stomatal conductance (g s ) and A max was assessed across species by collecting data between 06h00 and 18h00.Data were collected in January and August 2008 in mature and fully expanded leaves.
Specific leaf area (SLA, the leaf area to leaf mass ratio) was determined in both seasons.As additional information, leaf thickness was determined in the dry season and leaf nitrogen content in the wet season.We measured SLA in a sample of six circles of 240-mm 2 -per leaf obtained from a sample of two to eight leaves per plant, depending on leaf size.We only determined nitrogen (Kjeldahl method) in the wet season (first studied season) in order to preserve the foliage for further studies in the same area.Fresh leaf thickness (FLT) and dry leaf thickness (DLT) were measured with digital calipers in 240-mm 2 -leaf circles (two per leaf) punched from the widest part of the leaf blade, and between the major veins (accuracy of 10 μm).Leaves used for SLA, leaf nitrogen and leaf thickness measurements were the same or similar in appearance (when more than two leaves were re- Sci.Agric.(Piracicaba, Braz.), v.67, n.6, p.624-632, November/December 2010 quired for analysis) to those used for gas exchange determinations.Leaf dry mass was obtained after leaf dehydration at 72°C until constant mass.The fraction of sky visible (FSV) beneath the canopy was measured using a canopy analyzer (LAI-2000, Li-Cor, NE, USA), under overcast sky conditions to improve the accuracy of the instrument, and calculated by integrating the gap fraction to yield the fraction of sky not blocked by foliage.For each sapling, six FSV readings, collected at a distance of about 1.5 m from the stem and forming a circle around the plant (the microsite), were recorded at each microsite and a mean value was obtained.The height of the sensor above the ground corresponded to the height of leaves used for the gas exchange measurements (1 to 2 m above the ground).Finally, we used a second LAI-2000 sensor, operating in the remote mode and installed on the top of a 40-m-tall observation tower (located in a nearby area), to log FSV values above the forest canopy.).We are aware that I und is lower than the actual light availability at sapling height, as it does not take into account the background of diffuse light in the forest understory (about 5-8 μmol m -2 s -1 at midday (Marenco and Vieira, 2005).Temperature and air humidity data were recorded at 30-min-intervals with a sensor (Humitter 50y, Vaisala Oy, Finland) connected to a datalogger (Li-1400, Li-Cor, NE, USA) at a selected site in the understory.In addition, an external quantum sensor (Li-190 SA, Li-Cor, USA) mounted on the Li-6400's irga head was used to log irradiance data at the same time as gas exchange measurements were made.Both in the dry and rainy season, soil moisture was determined gravimetrically: 100(S w -S d )/S w ,where S w and S d represent the mass of wet and dry, undisturbed 110-cm 3soil samples.Soil samples were collected at random in the study area at the depth of 200 mm, both in the wet (26 samples) and dry season (12 samples).All data, but leaf thickness and N content (determined only in one season), were subjected to analysis of variance (ANOVA) to assess the effect of the rainfall seasonality on the parameters.The Lilliefors test was conducted to assess whether experimental errors were normally distributed.As no transformation was needed, all statistical analyses were carried out on untransformed data.When the effect of rainfall seasonality on the variables was not significant (p > 0.05), data were pooled and linear or quadratic regression analyses conducted to examine the effect of FSV and SLA on photosynthetic traits.Tukey post-hoc test was used for mean separation (p ≤ 0.05).

Results and Discussion
Monthly rainfall was 353 mm in January and 105 mm in August (Table 2), which is in accordance with the historical mean  for the region (Inmet, 2008).In these months, soil moisture ranged between 31% in the dry season to 32% in the wet season, near the soil saturation point of 39% on a wet soil basis (Table 2).Air temperature at the forest floor ranged from 22°C at night to 29°C at noon, and for most of the day the relative humidity was above 90%, with no difference between seasons (Figure 1).Accumulated irradiance at the forest floor was 0.3 and 0.6 mol m -2 day -1 in the wet and dry seasons, respectively.On the other hand, mean maximum understory irradiance was about 10 and 20 μmol m -2 s -1 for the wet and dry seasons, respectively, or about 1.5-2% of the irradiance recorded above the forest canopy (Figure 1).I und values reported in this study are a somewhat higher than those observed by others (Kursar and Coley, 1999;Marenco and Vieira, 2005), perhaps because our I und values were recorded about 1-2 m above the ground rather than at the forest floor.Molion (1987) estimated that the irradiance that reaches the forest floor is 1.2% (approximately 14 μmol m -2 s -1 on a sunny day) of that received above the forest canopy, similar to our I und values observed in the wet season.
Sci. Agric.(Piracicaba, Braz.), v.67, n.6, p.624-632, November/December 2010 Early in the morning and late in the afternoon stomata did not respond to light stimulus, remaining closed even at saturating irradiance (1000 μmol m -2 s -1 ) for photosynthesis in the leaf chamber (Figure 2).Stomata closed and A max declined as the vapor pressure deficit (VPD) increased (Figure 3).However, as the forest understory became brighter, A max and g s tended to linearly increase with irradiance (Figure 4), which indicates that in this environment g s and A max are under the influence of a diurnal cycle, perhaps affected by light and VPD (Figures 3, 4).Our results agree with those reported by Kaiser and Kappen (2000) who observed maximum g s values between 10h00 and 14h00 and a minimum stomatal aperture at sunset.
Light induces stomatal opening (Shimazaki et al., 2007) and thus stomata may open at irradiances above 2-8 μmol m -2 s -1 (Habermann, 1973).However, soon after dawn light was ineffective in triggering stomatal opening.Since irradiance, relative humidity and temperature changed during daytime on the forest floor (Figure 1), it is possible that somehow these environmental factors affected stomatal functioning during the day.Although the light environment had an effect on g s (p < 0.05, Figure 4B), the correlation between g s and irradiance at mea-suring time was tenuous (r 2 = 0.05*).VPD has an important effect on g s , but it only explains 24% of variation (Figure 3B).Thus, we cannot rule out the effect of endogenous factors in modulating stomatal functioning, as reported by others, both in herbaceous plants (Gorton et al., 1993;Holmes and Klein, 1986) and forest trees (Doughty et al., 2006).
Although there was no difference in soil moisture between the dry and rainy season, A max was lower in the dry season (Table 2), which suggests that even a slight decline in soil moisture, or perhaps in leaf water potential associated to a higher irradiance in the dry period, may affect some photosynthetic traits of understory saplings, perhaps mesophyll conductance (g m ).Under progressive drought g m may decline (Flexas et al., 2002).This hypothesis is consistent with the fact that both A pot and g s were unaffected by rainfall seasonality.As g s was not influenced by rainfall seasonality (Table 2), differences in A max between seasons may be ascribed to a limitation of carbon uptake imposed by non-stomatal factors.Had the dry season had any detrimental effect on Rubisco   activity or ATP synthesis (Flexas and Medrano, 2002) A pot should have declined, but it did not.This allows us to conclude that the seasonality may have an effect on g m .In relation to the seasonal effect on A max is important to take into account that climate models predict changes in the total rainfall in the Amazon as a result of global warming (Cox et al. 2004;Oyama and Nobre, 2003).Besides, differences among species were not observed in A max nor in A pot , so data from both rainfall seasons were pooled to obtain a mean value for each species (Table 3).
At ambient CO 2 concentration (380 μmol mol -1 ), A max was closely related with g s (r 2 = 0.60**, Figure 5A), which is consistent with results reported by others (Machado et al., 2002;Marenco et al., 2006;Park and Furukawa, 1999).Nonetheless, the correlation between A pot and g s was very low (r 2 = 0.01 ns ) at saturating CO 2 concentration (Figure 5B), indicating that A pot is little influenced by stomatal opening in the g s range observed for most of the day (08h00 to 16h00).Except very early in the morning, when most stomata were closed, their resistance to CO 2 diffusion into intercellular spaces was offset by an elevated CO 2 concentration in the leaf chamber.However, when g s was very low (less than 0.015 mol m -2 s -1 , denoted by diamonds in Figure 5B) the resistance imposed by stomatal closure was not compensated by a high CO 2 concentration in the leaf chamber, which led to a reduction in photosynthetic capacity.Thus, a g s of 0.015 mol m -2 s -1 most likely reflects a threshold below which leaf conductance is mainly due to cuticular conductance (g c ).Because A pot remained quite constant for most of the day, the effect of FSV, SLA, LT and leaf nitrogen on photosynthetic rates were examined with respect to A pot rather that A max , which was strongly dependent on g s .FSV was positively related to leaf thickness  ), specific leaf area (SLA), fresh leaf thickness (FLT), dry leaf thickness (DLT), and leaf nitrogen content determined in saplings of native tree species of the Central Amazon.Each value represents the mean of two seasons (A max , A pot and SLA) or one season (leaf thickness and leaf nitrogen).
Means followed by the same letters within columns do not differ according to Tukey test 5% probability.
The effect of FSV on A pot , LT and SLA shows that even small changes in intensity of light in the forest floor can alter the performance of the photosynthetic apparatus.This is in agreement with results reported by others (Ellsworth and Reich, 1993;Oguchi et al., 2005;Weston et al., 2000), who observed increases in LT in leaves exposed to brighter environments.The relationship between LT and A pot concurs with previous findings (McMillen and McClendon, 1983;Niinemets, 1999;Reich et al., 1998).Even when A pot and LT and SLA were strongly related (Figures 7, 8), we cannot attribute increases in A pot only to variations in LT or SLA, as pho- ] of 2000 μmol mol -1 and saturating light.Other experimental conditions as described in Figure 2. The diamonds ( ) in Figure 5B show the values of A pot soon after dawn, when g s was very low.Each symbol represents one leaf (one or two leaves per plant).ns: not significant (p > 0.05), **significant at p < 0.01.tosynthetic compounds are less effectively used in thicker leaves, perhaps because of a lower leaf conductance in these leaves (Niinemets, 1999).FSV values increased from 0.014 in January (rainy season) to 0.020 in August (dry season) (Table 2), confirming results obtained previously by Marenco and Vieira (2005).We attributed the difference in FSV values between the evaluated rainfall seasons to differences in leaf area index between the rainy and dry season (5.1 versus 4.7, Table 2) or to the higher solar radiation recorded in the dry season (Table 2).High irradiance in the dry season can result in greater carbon assimilation during this part of the year as suggested by Huete et al. (2006).However, a higher light intensity in the forest canopy in the dry season apparently does not contribute to increase the photosynthetic capacity of saplings at the forest floor, although across species, we found an effect of FSV on A pot irrespective of the seasonal rainfall regime, perhaps due to an effect of the R/Fr ratio on Sci.Agric.(Piracicaba, Braz.), v.67, n.6, p.624-632, November/December 2010 photosynthetic rates.For Acmena ingens, for example, under low irradiance (≤ 20 % of full sunlight) g s and A max where lower in plants grown under a reduced R/Fr ratio (0.2) than in control plants (R/Fr of sunlight, 1.2) (Turnbull, 1991).SLA ranged from 13.5 m 2 kg -1 in S. guilleminiana to 20.8 m 2 kg -1 in R. racemosa, whereas the leaf nitrogen content varied between 0.9 g m -2 for T. unifoliolata and R. racemosa to 1.7 g m -2 for S. guilleminiana (Table 3).SLA values found in this study are within the range (15 and 24 m 2 kg -1 ) observed by Marenco and Vieira (2005) for saplings of canopy tree species.FSV had a positive effect on LT, and a negative one on SLA (p < 0.05).The positive effect of leaf nitrogen on A pot (Figure 9) is consistent with the results reported by Hikosaka (2004;2005).However, although significant, the relationship between A pot and leaf N was weak (r 2 = 0.14, p < 0.05) (Figure 9), indicating that a substantial fraction of the leaf nitrogen is partitioned into non-photosynthetic structures.On the other hand, differences in determination coefficients (r 2 ) between DLT and SLA against FSV (0.11** versus 0.05* for SLA, Figure 6B, D) occur because SLA depends not only on LT, but also on leaf density (Niinemets, 1999), which suggests that FSV has a lower effect on leaf density.Although LT is affected by growth irradiance, we can not explain the wide variations in SLA among species only by differences in microsite brightness (inferred by FSV values) at the forest floor.This suggests that the genetic background of each species plays a major role in determining adaptive strategies to the physical and ecological environment (soil fertility and acidity, herbivory, etc.), which thereby leads to changes in LT and SLA under a given growth conditions (Lee et al., 2000;Peeters, 2002).Increase in LT (decline in SLA) is often related to higher photosynthetic rates per unit leaf area (McMillen and McClendon, 1983;Yun and Taylor, 1986), because of a greater accumulation of photosynthetic proteins.However, it may also involve an increase in the amount of molecules and compounds not directly related to carbon assimilation but with a key role for plant defense against herbivory and for increasing resistance against other physical hazards (Coley, 1988;Wright and Cannon, 2001).
Although the rainfall regime only slightly affected soil moisture, some photosynthetic traits (perhaps g m ) seem to be responsive to rainfall-related environmental attributes, which eventually lead to an effect on A max .In the forest understory, A max and g s of saplings appear to be highly sensitive to diurnal variation, and even when stomatal functioning is affected by environment factors (e.g., light and VPD), somehow endogenous factors also seem to have a role in stomatal movements.However, Sci.Agric.(Piracicaba, Braz.), v.67, n.6, p.624-632, November/December 2010 the dry season of 2008 was not strong enough to unambiguously negate any potential effect of rainfall seasonality on the photosynthetic capacity of saplings.Further studies are needed to elucidate how a prolonged dry season may affect the diurnal pattern of photosynthesis, Rubisco activity, and electron transport rates, which may ultimately affect tree growth in the Central Amazon.Finally, even though irradiance in the forest floor is usually very low, it remarkably affects leaf physiology and leaf anatomy, as photosynthetic capacity, LT and SLA responded to variations in the fraction of sky visible in the forest understory.
Irradiance and rainfall data were recorded above the forest canopy at the top of the 40-m tall observation tower.Irradiance at the observation tower (I open ) was measured using a quantum sensor (Li-190 SA, Li-Cor, NE, USA).Understory irradiance (I und ) was estimated as the product of FSV by I open (i.e.I und = FSV x I open

Figure 1 -
Figure 1 -Diurnal irradiance above the forest canopy (A, I open ), and relative humidity (RH), temperature (T) and irradiance at the forest understory (B, I und ).Data were collected in January (rainy season, R) and August (dry season, D) of 2008.
Figure 2 -Diurnal variation in light saturated photosynthesis (A, A max ), stomatal conductance (B, g s ) in January (rainy season, open circle, ) and August (dry season, closed circle, ) of 2008 in saplings of 10 forest tree species of the Central Amazon.Each symbol represents one leaf (one or two leaves per plant).Measurements were made at a [CO 2 ] of 380 μmol mol -1 , irradiance of 1000 μmol m -2 s -1 and leaf temperature of 28 ± 1ºC.The continuous line shows the trend observed throughout the day.**significant at p < 0.01.

Figure 3 -
Figure 3 -Relationship between light saturated photosynthesis (A, A max ) and stomatal conductance (B, g s ) and vapor pressure deficit (VPD), in January (rainy season, open circle, ) and August (dry season, closed circle, ) of 2008 in saplings of 10 forest tree species of the Central Amazon.Each symbol represents one leaf (one or two leaves per plant).Experimental conditions as described in Figure 2. **significant at p < 0.01.

Figure
Figure 5 -Relationship between light saturated photosynthesis (A, A max ) and photosynthetic capacity (B, A pot ) and stomatal conductance (g s ) in January (rainy season, open circle, ) and August (dry season, closed circle, ) of 2008 in saplings of ten native forest tree species of the Central Amazon.A pot was measured at a [CO 2

Figure 4 -
Figure 4 -Relationship between light saturated photosynthesis (A, A max ) and stomatal conductance (B, g s ) and instantaneous irradiance recorded at the forest understory during gas exchange measurements, in January (rainy season, open circle, ) and August (dry season, closed circle, ) of 2008 in saplings of 10 forest tree species of the Central Amazon.Each symbol represents one leaf (one or two leaves per plant).Experimental conditions as described in Figure 2. *significant at p < 0.05; **significant at p < 0.01.

Figure 6 -
Figure 6 -Fresh (A, FLT) and dry leaf thickness (B, DLT), photosynthetic capacity (C, A pot ) and specific leaf area (D, SLA) as a function of the fraction of sky visible (FSV) in January (rainy season, open circle, ) and August (dry season, closed circle, ) of 2008 in saplings of ten forest tree species of the Central Amazon.Experimental conditions as described in Figure 5.Each symbol represents one leaf (one or two leaves per plant).*significant at p < 0.05; **significant at p < 0.01

Figure 8 -
Figure 8 -Relationship between photosynthetic capacity (A pot ) and specific leaf area (SLA) in January (wet season, open circle, ) and August (dry season, closed circle,) of 2008 in saplings of forest tree species of the Central Amazon.Experimental conditions as described in Figure5.Each symbol represents one leaf (one or two leaves per plant).**significant at p < 0.01.

Table 1 -
Families and importance of the species.

Table 2 -
Light saturated photosynthesis (A ), leaf area index, photosynthetic photon flux density (PFD) above the forest canopy, rainfall and soil moisture observed in January (rainy season) and August (dry season) of 2008.

Table 3 -
Light saturated photosynthesis (A max), photosynthetic capacity (A pot