PHOTOSYNTHETIC TRAITS OF CANOPY LEAVES OF DINIZIA EXCELSA (FABACEAE)

CARACTERÍSTICAS FOTOSSINTÉTICAS DE FOLHAS DE DOSSEL DE DINIZIA EXCELSA (FABACEAE)

ABSTRACT

The response of leaf traits to irradiance and [CO₂] in canopy leaves of several tall trees remains to be determined under natural conditions. Thus, the objective of this work was to determine gas-exchange parameters in sun and shade leaves of Dinizia excelsa Ducke in 35-45 m tall trees of Central Amazonia. We assessed light saturated photosynthesis (A_max), stomatal conductance (g_s), mesophyll conductance (g_m), transpiration rates (E), water use efficiency (WUE), intrinsic water use efficiency (WUEi), maximum electron transport rate (J_max), the maximum carboxylation rate of Rubisco (V_cmax), intercellular CO₂ concentration (C_i)specific leaf area (SLA) and fresh leaf thickness. We also estimated the CO₂ concentration at the chloroplast level (C_c) and determined the light and CO₂ saturated photosynthesis (A_pot). A_max was obtained at light saturation (1200 µmol m^-2 s^-1), whereas A_pot, V_cmax, J_max and g_m were obtained after constructing A/C_i response curves. There was a significant difference between sun and shade leaves in C_i and C_c, but for other parameters no differences were observed. A_max was positively correlated with g_s, g_m and E, and there was also a significant correlation between g_s and gm (p ≤ 0.05), as well as between J_max and V_cmax. Thicker leaves had higher values of A_max, g_s, C_i, C_c and E. A_pot was limited by the electron transport rate and by low g_m. The canopy of the tree caused a decrease in irradiance (30-40%), but this reduction was not enough to reduce important photosynthetic parameters. Thus, all resources allocated to leaf production led to maximum use of the solar energy received by the leaves, which allowed this species to grow at fairly rapid rates.

Keywords:
Mesophyll conductance; Maximum carboxylation rates of Rubisco; Central Amazonia

RESUMO

A resposta das características foliares à irradiância e concentração de CO₂ em folhas de dossel de várias espécies arbóreas de alto porte ainda permanece para ser determinada em condições naturais. Assim, o objetivo deste trabalho foi determinar parâmetros de troca gasosa em folhas de sol e sombra de Dinizia excelsa Ducke, em árvores de 35-45 m de altura, na Amazônia Central. Foram avaliados fotossíntese saturada de luz (A_max), condutância estomática (g_s), condutância do mesofilo (g_m), taxa de transpiração (E), eficiência de uso de água (WUE), eficiência intrínseca de uso de água (WUEi), taxa máxima de transporte de elétrons (J_max), taxa máxima de carboxilação da Rubisco (V_cmax), concentração de CO₂ intercelular (C_i), área foliar específica (SLA) e a espessura da folha fresca. Também foi estimada a concentração de CO₂ no nível de cloroplasto (C_c) e determinada à fotossíntese saturada de luz e CO₂ (A_pot). O A_max foi obtido com saturação de luz (1200 µmol m^-2 s^-1), enquanto que A_pot, V_cmax, J_max e g_m foram obtidos após a construção de uma curva de resposta A/C_i. Houve diferença significativa entre as folhas de sol e sombra em C_i e C_c, mas nenhuma diferença foi observada para outros parâmetros avaliados. A_max foi positivamente correlacionado com g_s, g_m e E, e também houve uma correlação significativa entre g_s e g_m (p ≤ 0,05), bem como entre J_max e V_cmax. As folhas mais espessas apresentaram maiores valores de A_max, g_s, C_i, C_c e E. Apot foi limitado pela taxa de transporte de elétrons e pelo baixo g_m. A folhagem da copa da árvore causou diminuição da luminosidade (30-40%), mas essa redução mostrou-se insuficiente para reduzir parâmetros importantes da fotossíntese. Dessa forma, todo recurso alocado a produção de folhas levou a um máximo aproveitamento da energia solar recebida pelas folhas, possibilitando que esta espécie tenha um crescimento moderadamente rápido.

Palavras-Chave:
Condutância do mesofilo; Taxa de carboxilação da Rubisco; Amazônia Central

1.INTRODUCTION

In the last decades the effect of the increase of atmospheric CO₂ concentration on plant physiology has been intensively investigated due to its presumed consequences on climate changes (Manter and Kerrigan, 2004; Knauer et al., 2019). It is well-known that stomata play an important role on carbon uptake, as they impose the first major diffusional limitation to CO₂ diffusion, from the atmosphere to carboxylation sites (Nascimento and Marenco, 2013; Xiong et al., 2018). The photosynthetic capacity of a leaf depends on diffusive (i.e. stomatal conductance - g_s and mesophyll conductance - g_m) and non-diffusive factors (i.e. the maximum carboxylation rate of Rubisco -V_cmax and maximum electron transport rate, J_max). V_cmax and J_max can be obtained from response curves of photosynthesis to intercellular CO₂ concentration - A/C_i (Farquhar et al., 1980; Stinziano et al., 2019). Both V_cmax and J_max are important parameters in modeling studies that aim to predict the impacts of climate change on plant functioning (Knauer et al., 2019).

Among the factors that affect photosynthesis, the response of stomatal functioning to environmental and endogenous factors has been the subject of much research (Terashima et al., 2011; Mendes and Marenco, 2014; Marenco et al., 2017). Although g_m is also quite relevant to CO₂ diffusion and photosynthesis, it attracted less attention in the past decades, but in recent studies, the importance of g_m to carbon assimilation has been highlighted (Flexas et al., 2016; Knauer et al., 2019). Nevertheless, research is still needed to assess the relevance of g_m in tropical species, particularly the Amazonian species.

The strata of tropical forests, as well as gap opening and closing lead to the formation of a light gradient in the canopy of a tree. This gradient can lead to changes in the strategy of the leaf for light capture and light use efficiency, and thereby to variations in leaf structure and physiology (Givnish, 1984; Clark and Clark, 1992; Marenco et al., 2017). In fact, light is probably the most important environmental factor affecting plant establishment, growth and survival (Niinemets et al., 2015; Gitelson et al., 2017).

The angelim (Dinizia excelsa Ducke, Fabaceae) is an emerging tree that can reach 50-60 m in height and 1.0 to 1.8 m in diameter (Lorenzi, 1992). It has a dense wood -0.91 g cm^-3 (Fearnside, 1997), and trees over 20 cm in diameter have a growth rate of 5.30 mm per year (Schwartz et al., 2016). In the emergent phase, the angelim canopy stands out in the forest landscape; hence it is not shaded by neighboring trees. It has wide distribution in the Brazilian Amazon, can occur in density of one individual per 6 ha, and the trees have good silvicultural performance (Dick, 2001; Ferreira et al., 2004). Angelim is economically important in silvicultural systems and in the timber industry. The wood of angelim is resistant to the attack of fungi and termites, and it is used in the manufacture of decorative plates, construction, shipbuilding, woodworking, and carpentry (Melo and Varela, 2006; Oliveira et al., 2008). In addition, the angelim can also be used in garden and urban afforestation (Lorenzi, 1992).

Gas exchange studies of Amazonian tree species under natural conditions are relevant due to current trends in global warming and atmospheric CO₂ enrichment (Magrin et al., 2014). Thus, measurements of gas exchange parameters (e.g. g_s, A_max, V_cmax and J_max) in Amazonian species is of paramount importance for the construction of models aimed at predicting the performance of Amazonian ecosystems. Thus, the objective of this work was to determine the effect of leaf position in the canopy stratum on leaf traits of angelim trees in the Central Amazon. Leaf trait measurements included light saturated photosynthesis (A_max), g_s, g_m, transpiration (E), water use efficiency (WUE), intrinsic water use efficiency (WUEi), J_max, V_cmax, intercellular CO₂ concentration (C_i), specific leaf area (SLA) and fresh leaf thickness (FLT). It was also estimated the concentration of CO₂ at chloroplast level (C_c) and determined light and CO₂ saturated photosynthesis (A_pot).

2.MATERIALS AND METHODS

The study was conducted at a 10-ha plot of a dense terra-firme forest fragment at the Colosso Reserve (02° 24' 13.2"S, 59°51' 54"W). The region has a humid equatorial climate, with annual precipitation of 2240 mm, distributed over a rainy season from November to May (> 180 mm per month) and a mild dry season from June to September (≤ 100 mm per month). October is a transitional month. The average annual air temperature is 26.7°C, and the average relative humidity is 84%.

In this study, six trees (35-45 m in height and 1.30-2.00 m in diameter) of angelim (Dinizia excelsa Ducke, Fabaceae) were used. In the experiment we used a completely randomized design with two treatments and six replications (trees). The treatments were the leaves from two positions in the canopy: upper part of the tree (hereinafter referred to as sun leaves, which were under direct solar radiation) and lower part of the canopy (shade leaves), which received about 60-70% of total solar radiation. From these canopy strata branches were detached for data collection. They were about 7 to 12 cm in diameter and approximately 12 m in length. All branches were above the canopy of neighboring trees and the lower branches of a tree were shaded only by the upper ones. We used detached branches because of the impossibility of accessing the canopy of the tree for data collection.

To assess the effect of branch detachment on stomatal conductance (g_s), a previous study was carried out using two gas exchange systems (Li-6400, Li-Cor, Lincoln, USA), collecting data simultaneously from two small branches (about 4 cm in diameter) from the same tree, which was accessible from a 40-m observation tower. After a stabilization period of 15 minutes, one branch was randomly selected and kept intact while the other branch was severed from the tree by a rapid cut, without interrupting gas-exchange measurements. The measurement process on both leaves (i.e. from the undisturbed branch and the severed one) was uninterrupted for 60 min, and during this time stomatal conductance remained similar in both branches. Thus, in the current experiment gas exchange and fluorescence data were measured within that time interval, and for further precaution data were collected on branches thicker than 4 cm in diameter.

Gas exchange and fluorescence measurements were made with a gas exchange system (Li-6400, Li-Cor, Lincoln, NE) with a 2-cm² integrated fluorescence chamber head (Li-6400-40, Li-Cor). Just after detachment, the branch was taken to the gas exchange instrument for data collection. The time lag from branch detachment up to the leaflet insertion in the leaf chamber of the gas exchange system was about 10 minutes. After a stabilization period (5-10 min) at ambient CO₂ (380 µmol mol^-1), photosynthetically active radiation (PAR) of 1200 µmol m^‒2 s^‒1 (light saturation) and ambient temperature (27 °C), photosynthetic rates (A) as a function of the intecellular CO₂ concentration ‒ C_i (A/C_i response curves) were measured. The A/C_i response curves were generated by increasing the reference CO₂ concentration from 50 to 2000 µmol mol^-1 in nine steps, i.e. 380, 250, 100, 50, 380, 550, 1000, 1,500 and 2,000 µmol mol^-1 (Long and Bernacchi, 2003). Light saturated photosynthesis (A_max) was determined at light saturation (a value determined in a previous experiment) and a CO₂ concentration of 380 µmol mol^-1. Whereas the light and CO₂ saturated photosynthesis (hereinafter termed potential photosynthesis ‒A_pot) was measured at light saturation and a reference CO₂ concentration of 2000 µmol mol^-1. The maximum carboxylation rate of Rubisco (V_cmax) and maximum electron transport rate (J_max) were calculated according to Farquhar et al. (1980).

$\begin{array}{c}{\mathit{A}}_{\mathit{c}}=\left[{\mathit{V}}_{c\max }\left({\mathit{C}}_i-\Gamma *\right)\right]/\left[{\mathit{C}}_i+K_c\left(1+\mathit{O}/K_o\right)\right]\hfill \\ {\mathit{A}}_j=\left[{\mathit{J}}_{\max }\left({\mathit{C}}_{\mathit{i}}-\Gamma *\right)\right]/\left.\left(\left[4{\mathit{C}}_{\mathit{i}}+8\Gamma *\right.\right)\right]\hfill \end{array}$

Where A_c and A_j denote the net photosynthetic rates limited by Rubisco activity and electron transport rate (A_j), respectively. G* represents the CO₂ compensation point in the absence of respiration in the light; C_i is the intercellular CO₂ concentration, O represents the oxygen concentration in the intercellular spaces; K_c and K_o represent the Michaelis-Menten constant of Rubisco for carboxylation and oxygenation, respectively. V_cmax values were standardized to 25 ° C (Medlyn et al., 1999).

The quantum yield of the photosystem II system (Φ_PSII) was calculated as previously described (Nascimento and Marenco, 2013). C_c values were determined according to Epron et al. (1995), whereas g_m was obtained as follows: g_m = A/(C_i ‒ C_c). We also calculated, water use efficiency (WUE) the photosynthesis to transpiration ratio (A/E) and the intrinsic water use efficiency (WUEi) the A/g_s ratio. Data were collected from September to November 2010 from two healthy and physiologically mature leaflets per stratum, and from six trees and two canopy strata per tree.

To assess the effect of treatments on leaf morphology, fresh leaflet thickness (FLT) was measured in two leaflets from each canopy stratum on each tree with digital calipers (0.01 mm accuracy). The leaflet thickness was determined in the middle portion of the leaves, avoiding major veins. The leaf area was determined with an area meter (Li-3000, Li-Cor), and the dry matter was determined after oven-drying at 72ºC until constant mass. Specific leaf area (SLA) was determined as the leaf area to leaf mass ratio. For further information we also measured the chlorophyll content of leaf pigment in 80% acetone and measured the SPAD values with a chlorophyll meter (SPAD-502-Minolta, Japan), as previously described (Mendes et al., 2013; Gouvêa and Marenco, 2018).

Data were subjected to analysis of variance (ANOVA) and the t test was used to determine significant differences between sun and shade leaflets. The relationships between quantitative variables (e.g. A_max versus V_cmax, A_pot versus J_max) were examined by regression analysis. Pearson’s correlation coefficient was used to evaluate the relationships between gas exchange variables and morphological traits (SLA and leaflet thickness). Statistical analyzes were performed using Statistica 10.0 software (StatSoft, Tulsa, OK, USA).

3.RESULTS

There was no significant difference between sun and shade leaflets in A_max, A_pot, g_s, g_m, V_max, J_max, VPD, T_leaf, WUE, WUEi SPAD values, and chlorophyll contents (p ≥ 0.05; Table 1). Only C_i and C_c showed differences between canopy strata, i.e. sun versus shade leaflet (p ≤ 0.05; Table 1). On average, the A_max and A_pot values were 8.7 and 24.1 µmol m^-2 s^-1 for the sun leaflets, whereas shade leaflets had A_max and A_pot values of 5.9 and 19.3 µmol m^-2 s^-1, respectively (Table 1). In ambient [CO₂], the g_m values were 46% (sun leaflets) and 29% (shade leaflets) lower than those recorded for g_s (Table 1). The maximum quantum efficiency of PS II (F_v/F_m ratio) did not vary between sun and shade environment. There was a significant effect of the ambient condition on FLT (p ≤ 0.05; Table 1). Mean FLT values were 0.20 and 0.16 mm for the sunny and shade conditions, respectively. However, SLA showed no significant differences between the sun and shade environment (p ≥ 0.05; Table 1). As the ambient condition had no effect on gas exchange parameters (A_max, A_pot, g_s and g_m), data from both environments were pooled for correlation analysis.

Table 1
Light saturated photosynthesis (A_max), light and CO₂ saturated photosynthesis (A_pot), stomatal conductance (g_s), mesophyll conductance (g_m), intercellular CO₂ concentration (C_i), chloroplast CO₂ concentration (C_c), vapor pressure deficit (VPD), leaf temperature (T_leaf), water use efficiency (WUE), intrinsic water use efficiency (WUE_i), the maximum carboxylation rate of Rubisco (V_cmax), electron transport rate (J_max), F_v/F_m ratio, specific leaf area (SLA), chlorophyll content, SPAD values, fresh leaf thickness (FLT), and absolute chlorophyll content (a, b and a + b) in sun and shade leaflets of Dinizia excelsa. Different capital letters denote significant differences (p = 0.05, t-test) between the canopy strata (sun and shade leaflet). Each value represents the mean (± SE) of six trees and two leaflets per tree.
Tabela 1
Fotossíntese saturada por luz (A_max), fotossíntese saturada por luz e CO₂ (A_pot), condutância estomática (g_s), condutância do mesofilo (g_m),concentração intercelular de CO₂ (C_i), concentração de CO₂ de cloroplasto (C_c), déficit de pressão de vapor (VPD), temperatura da folha (T_leaf), eficiência no uso da água (WUE), eficiência intrínseca no uso da água (WUEi), taxa máxima de carboxilação de Rubisco (V_cmax), taxa de transporte de elétrons (J_max),relação F_v/F_m, área foliar específica (SLA), conteúdo de clorofila, valores SPAD, espessura foliar fresca (FLT) e teor absoluto de clorofila (a, b e a + b) em folíolos de sol e sombra de Dinizia excelsa. Letras maiúsculas diferentes indicam diferenças significativas (p = 0,05, teste t) entre os estratos do dossel (folhas do sol e folhas de sombra). Cada valor representa a média (± SE) de seis árvores e dois folhetos (de duas folhas) por árvore.

PARAMETER	SUN LEAFLET	SHADE LEAFLET
Amax (µmol m-2 s-1)	8.7 ± 1.3 A	5.9 ± 0.7 A
Apot (µmol m-2 s-1)	24.1 ± 4.0 A	19.3 ± 2.1 A
gs (mol m-2s-1)	0.13 ± 0.03 A	0.07 ± 0.02 A
gm (mol m-2 s-1 bar-1)	0.07 ± 0.009 A	0.05 ± 0.005 A
Ci (µmol mol-1)	232.6 ± 11.4 A	201.8 ± 11.6. B
Cc (µmol mol-1)	107.9 ± 5.3 A	93.8 ± 5.4 B
Vcmax (µmol m-2 s-1)	41.1 ± 4.9 A	38.4 ± 3.3 A
Jmax (µmol m-2 s-1)	101.2 ± 14.6 A	80.6 ± 8.3 A
VPD (kPa)	1.8 ± 0.2 A	1.9 ± 0.2 A
Tleaf (ºC)	33.4 ± 0.8 A	33.6 ± 0.8 A
WUE (mmol mol-1)	4.4 ± 0.6 A	5.2 ± 0.2 A
WUEi (µmol mol-1)	76.4 ± 9.5 A	108.3 ± 13.3 A
Fv/Fm (unitless)	0.78 ± 0.005 A	0.78 ± 0.005 A
SLA (m2 kg-1)	7.8 ± 0.5 A	7.5 ± 0.4 A
FLT (mm)	0.20 ± 0.003 A	0.16 ± B
SPAD	68.1 ± 4.4 A	57.2 ± 3.7 A
Chlorophyll a (µmol m-2)	362.1 ± 42.7 A	317.6 ± 19.4 A
Chlorophyll b (µmol m-2)	93.6 ± 26.0 A	98.1 ± 19.3 A
Chlorophyll a+ b (µmol m-2)	470.8 ± 39.9 A	415.7 ± 27.9 A

A_max was positively correlated with g_s (r = 0.95, Table 2), g_m (r = 0.96), and E (r = 0.92). However, A_max negatively correlated with VPD (r = -0.52), T_leaf (r = -0.32) and WUE (r = -0.28). There was a positive correlation between g_s and g_m (r = 0.83, Table 2), and, it was also found that g_s decreased with increasing in VPD and T_leaf (r = -0.65 and r = -0.46; respectively). However, there was a weak correlation between g_m and VPD (r = -0.37) and T_leaf (r = -0.20), and between WUE and g_s (r = -0.30) and g_m (r = -0.155). WUE and WUEi were positively correlated with the g_m/g_s ratio (r = 0.99; Fig. 1).

Table 2
Pearson's correlation matrix between light saturated photosynthesis (A_max), light and CO₂ saturated photosynthesis (A_pot), stomatal conductance (g_s), mesophyll conductance (g_m), intercellular CO₂ concentration (C_i), chloroplast CO₂ concentration (Cc), transpiration (E), vapor pressure deficit (VPD), leaf temperature (T_leaf), water use efficiency (WUE), intrinsic water use efficiency (WUEi), the maximum carboxylation rate of Rubisco (V_cmax), electron transport rate (J_max), specific leaf area (SLA), fresh leaf thickness (FLT) in Dinizia excelsa. *: significant (p ≤ 0.05); **: significant (p ≤ 0.01), ns: not significant (p > 0.05).
Tabela 2
Matriz de correlação de Pearson entre fotossíntese saturada por luz (A_max), fotossíntese saturada por luz e CO₂ (A_pot), condutância estomática (g_s), condutância do mesofilo (g_m), concentração intercelular de CO² (C_i), concentração de CO₂ de cloroplastos (C_c), transpiração (E), déficit de pressão de vapor (VPD), temperatura da folha (T_leaf), eficiência no uso da água (WUE), eficiência intrisica no uso da água (WUEi), taxa máxima de carboxilação de Rubisco (V_cmax), taxa de transporte de elétrons (J_max), área foliar específica (SLA), espessura da folha fresca (FLT) em Dinizia excelsa. *: significativo (p ≤ 0,05); **: significativo (p ≤ 0,01), ns: não significativo (p > 0,05).

Parameter	A max	A pot	g s	g m	C i	C c	E	VPD	T leaf	WUE	WUEi	V cmax	J max	SLA	FLT
A max	-	0.75**	0.95**	0.96**	0.66**	0.66**	0.92**	-0.52**	-0.32*	-0.28*	-0.70**	0.82**	0.81**	-0.05ns	0.34*
A pot	0.75**	-	0.60**	0.82**	0.30*	0.30*	0.74**	-0.12ns	-0.03ns	-0.21ns	-0.35*	0.73**	0.97**	-0.30*	0.08ns
g s	0.95**	0.60**	-	0.83**	0.78**	0.78**	0.90**	-0.65**	-0.46*	-0.30*	-0.77**	0.65**	0.63**	0.1ns	0.46*
g m	0.96**	0.82**	0.83**	-	0.42*	0.42*	0.85**	-0.37*	-0.20ns	-0.15ns	-0.49*	0.92**	0.89**	-0.18ns	0.20ns
C i	0.66**	0.30*	0.78**	0.42*	-	1.0**	0.74**	-0.60**	-0.38*	-0.59**	-0.96**	0.21ns	0.32*	0.24ns	0.52**
C c	0.66**	0.30*	0.78**	0.42*	1.0**	-	0.74**	-0.60**	-0.38*	-0.59**	-0.96**	0.21ns	0.32*	0.24ns	0.52**
E	0.92**	0.74**	0.90**	0.85**	0.74**	0.74**	-	-0.34*	-0.08ns	-0.57**	-0.79**	0.67**	0.79**	-0.19ns	0.38*
VPD	-0.52**	-0.12ns	-0.65**	-0.37*	-0.60**	-0.60**	-0.34*	-	0.92**	-0.29*	0.42*	-0.2ns	-0.08ns	-0.63**	-0.43*
T leaf	-0.32*	-0.03ns	-0.46*	-0.20ns	-0.38*	-0.38*	-0.08ns	0.92**	-	-0.49*	0.18ns	-0.10 ns	0.05ns	-0.76**	-0.40*
WUE	-0.28*	-0.21ns	-0.30*	-0.15ns	-0.59**	-0.59**	-0.57**	-0.29*	-0.49*	-	0.73**	-0.07ns	-0.29*	-0.39*	-0.19ns
WUEi	-0.7**	-0.35*	-0.77**	-0.49*	-0.96**	-0.96**	-0.79**	0.42*	0.18ns	0.73**	-	-0.31*	-0.41*	-0.04ns	-0.43*
V cmax	0.82**	0.73**	0.65**	0.92**	0.21ns	0.21ns	0.67**	-0.2ns	-0.10 ns	-0.07ns	-0.31*	-	0.81**	-0.33*	0.09ns
J max	0.81**	0.97**	0.63**	0.89**	0.32*	0.32*	0.79**	-0.08ns	0.05ns	-0.29*	-0.41*	0.81**	-	-0.36*	0.09ns
SLA	-0.05ns	-0.30*	0.10ns	-0.18ns	0.24ns	0.24ns	-0.19ns	-0.63**	-0.76**	-0.39*	-0.04ns	-0.33*	-0.36*	-	0.58**
FLT	0.34*	0.08ns	0.46*	0.20ns	0.52**	0.52**	0.38*	-0.43*	-0.40*	-0.19ns	-0.43*	0.09ns	0.09ns	0.58**	-

Figure 1
Water use efficiency (WUE; A) and intrinsic water use efficiency (WUEi; B) as a function of the gm/gs ratio in Dinizia excelsa. Each symbol corresponds to the mean of two leaflets per tree.
Figura 1
Eficiência no uso da água (WUE; A) e eficiência intrínseca no uso da água (WUEi; B) em função da relação g_m/g_s em Dinizia excelsa. Cada símbolo corresponde à média de dois folíolos (de duas folhas) por árvore.

At ambient CO₂ concentration, C_i and C_c were positively correlated with g_s (r = 0.78) and g_m (r = 0.42, Table 2). There was also a positive correlation between J_max and V_cmax (r = 0.81), and between V_cmax (and J_max) and A_max and A_pot (Table 2).

V_cmax and J_max were positively correlated with g_s (r @ 0.6) and g_m (r @ 0.9, Table 2). Gas exchange parameters were correlated with SLA and FLT (Table 2). Thicker leaves had higher values of A_max (r = 0.34), g_s (r = 0.46), C_i (r = 0.52), C_c (r = 0.52) and E (r = 0.38). SLA was negatively correlated with VPD, T_leaf ,V_cmax and J_max (p ≤ 0.05). On the other hand, FLT had no effect on g_m, V_cmax and J_max (p ≥ 0.05). Finally, SLA increased with increasing LFT, which was not expected (r = 0.58, p ≤ 0.001). In Figure 2, irrespective of the environment (sun - shade), the initial portion of the curve showed a linear association between photosynthesis (A) and C_i and C_c indicating a limitation imposed by Rubisco carboxylation rate. The increase of both C_i and C_c led A to increase to the point of photosynthesis limitation by electron transport rate (dashed line in Figure 2). Below the colimitation point (C_i values of 433-544 µmol mol^-1, indicated by the vertical line in Figure 2 A, B), A was limited by Rubisco carboxylation rates. The C_c values at the colimitation point were 201-252 µmol mol^-1 (indicated by the vertical line in Figure 2 C, D). On the other hand, above the colimitation point (high CO₂ concentration) A was limited by electron transport rates (dashed lines in Figure 2).

Figure 2
Photosynthesis as a function of the intercellular [CO2] , C_i (A, B) and cloroplast [CO₂] , C_c (C, D) in sun leaflet (open circle, ○) and shade leaflet (closed circle, ●). The solid line shows the photosynthesis limitation imposed by Rubisco carboxylation rate (A_c), and the dashed line indicates the photosynthesis limitation imposed by electron transport rate (A_j). The vertical solid line shows the value of C_i and C_c at the colimitation point of A_c and A_j. The numerals (in the panels) show the values for C_i and C_c in µmol mol^-1 at the colimitation point. Each symbol represents the mean of six trees and two leaflets per tree.
Figura 2
Fotossíntese em função da concentração intercelular de [CO₂], C_i (A, B) e da [CO₂] no cloroplasto Cc (C, D) no folíolo de sol (círculo aberto, ○) e folíolo de sombra (círculo fechado, ●). A linha sólida mostra a limitação da fotossíntese imposta pela taxa de carboxilação de Rubisco (A_c), e a linha tracejada indica a limitação da fotossíntese imposta pela taxa de transporte de elétrons (A_j). A linha vertical sólida mostra o valor de C_i e C_c no ponto de colimitação de A_c e A_j. Os numerais (nos painéis) mostram os valores para C_i e C_c em µmol mol^-1 no ponto de colimitação. Cada símbolo representa a média de seis árvores e dois folhetos (de duas folhas) por árvore.

The g_m values varied as a function of C_i or C_c (Fig. 3). At low CO₂ concentrations (C_i ≤ 400 µmol mol^-1), g_m increased linearly with increasing C_i or C_c (Fig. 3). On the contrary, g_m decreased at high CO₂ concentrations (Fig. 3). In comparison with g_m values recorded at ambient [CO₂],(C_i of 200-300 m mol mol^-' ), g_m decreased 70% at high CO₂ concentration (Fig. 3).

Figure 3
Mesophyll conductance (g_m) in response to the intercellular CO₂ concentration (C_i - A) and chloroplast [CO₂] (C_c - B) in Dinizia excelsa. Each symbol corresponds to a value recorded on one leaflet.
Figura 3
Condutância do mesofilo (g_m) em resposta à concentração intercelular de CO₂ (C_i - A) e cloroplasto [CO₂] (C_c - B) em Dinizia excelsa. Cada símbolo corresponde a um valor obsservado em um folíolo.

4.DISCUSSION

The mean F_v/F_m value recorded in this study (0.78; Table 1) is within the range of values for non-stressed leaves, i.e. F_v/F_m of about 0.80 (Björkman and Demmig, 1987). Very low F_v/F_m values, e.g. 0.60 or lower (Magalhães et al., 2009) often indicate the occurrence of photoinhibition. Therefore, it is concluded that in this study there was no photoinhibition of photosynthesis in D. excelsa.

The A_max values observed in this study are similar to those found in other studies, such as D. excelsa (Miranda et al., 2005), Minquartia guianensis, Coussapoa orthoneura and Protium opacum (Magalhães et al., 2009; Marenco et al., 2014). The chloroplast CO₂ concentration (C_c) was around 50% lower than the concentration observed in the intercellular spaces (Table 1). Taking C_i as the base line, the decline in C_c is due to the barriers against CO₂ diffusion, from the intercellular space to the carboxylation site in the chloroplast (Niinemets et al., 2015; Tosens and Laanisto, 2018). The greatest resistance to internal diffusion of CO₂ seems to be related to plasma membranes and chloroplast membranes (Warren, 2009; Peguero-Pina et al., 2017).

At the ambient CO₂ concentration (380 µmol mol^-1) there was a close relationship between A_max and g_s and g_m (Table 2). This occurs because both conductances determine the CO₂ flux to the carboxylation site in the chloroplast (Flexas et al., 2013). Stomatal and mesophyll resistance to CO₂ flux account for about 40% of photosynthesis limitation in well-irrigated plants (Yamori et al., 2006; Tosens et al., 2016). Thus, the strong correlation between g_s and g_m indicates a coupling between these parameters. The high correlation between A_max and g_s (and g_m) suggests that diffusive factors were of paramount importance for determining carbon assimilation in D. excelsa. The decline of g_s under high VPD conditions corroborates that some ambient conditions can lead to a reduction in photosynthetic rates. Park and Furukawa (1999) showed that photosynthesis and stomatal conductance measured in tropical trees decreased due to increased VPD. Stomatal response to air humidity, temperature and VPD has been studied for decades, and it is known that stomata respond to changes in leaf tissue water content or to variations in guard cell water potential (Buckley, 2019).

Transpiration (E) was strongly correlated with g_s, which is expected as the stomata play a key role in the control of leaf transpiration. Although g_m is expected to have little impact on leaf transpiration (Ouyang et al., 2017), in this study we found a positive correlation between g_s and g_m, which helps to explain the positive correlation between g_m and WUEi, as reported by Jahan et al. (2014). A high g_m/g_s ratio contributes to increased water use efficiency, particularly under water stress. Thus, it has been suggested that plants with potential for acclimation to drought have a high g_m/g_s ratio (Giuliani et al., 2013). The strong relationship observed between the g_m/g_s ratio and WUE and WUEi found in this study supports the hypothesis of coupling between g_s and g_m. These results suggest that in Dinizia excelsa water efficiency can be increased by improving g_m.

Besides g_s, g_m also affects the photosynthetic capacity of the leaf, as shown by the close correlation between V_cmax, J_max, and g_m (Table 2). Indeed, V_cmax and J_max were more influenced by g_m than by g_s. In Figure 2, one can see that Rubisco carboxylation rate was the most limiting factor for photosynthesis (up to the CO₂ concentration indicated by the arrows), which is in agreement with Sage and Kubien (2007) and Mendes et al. (2017). At higher CO₂ concentrations, however, the electron transport rate became the most limiting factor a closer correlation between A_pot and J_max was observed, which is in agreement with Mendes et al., (2017).

It was observed that g_m showed a rapid response (minutes) to changes in CO₂ concentration. However, it is unknown whether this response is a pattern also shared by other Amazonian tree species. At C_i values of 0-400 µmol mol^-1 (C_c < 200 µmol mol^-1), g_m increased linearly with increasing CO₂ until it reached a maximum value (at C_c ≈ 300 µmol mol^-1). On the other hand, at high C_i values g_m decreased with increasing CO₂ concentration, which suggests that at that CO₂ condition photosynthesis is no longer limited by the availability of CO₂ at the intercellular spaces but by mesophyll resistance and electron transport rates.

Leaf thickness is one of the key leaf traits that affect g_m (Terashima et al., 2011). However, the significance of this effect was not detected in D. excelsa probably because there was little variation in FLT (0.16-0.21 mm). However, it is worth noting that FLT positively affected g_s, C_i and C_c. This suggests that in thicker leaves there was an increase in the volume of intercellular space, perhaps as a mechanism to maximize mesophyll CO₂ concentration. A large intercellular space can contribute to reduce the effect CO₂ limitation under partial stomatal closure (Shao et al., 2008). SLA values recorded at the lower part of the canopy (shade leaflets, Table 2) were lower than those reported for understory trees (12 and 22 m² kg^-1) in the central Amazon (Mendes et al., 2013), which indicates that even leaves of the innermost part of the canopy were receiving relatively high levels of solar radiation. An increase in SLA is often related to a decrease in leaf thickness. Thus, the results presented in this study differ from the classical pattern that shows a negative relationship between SLA and leaf thickness (Niinemets, 1999). This discrepancy can be attributed to the fact that in this study leaf thickness was measured in fresh leaves, and variation in leaf water contents may lead to divergence with the most common SLA-leaf thickness relationship.

5.CONCLUSIONS

The canopy leaves of angelim causes a decrease in irradiance (30-40%), which does not appear to be high enough to negatively affect important gas exchange parameters, such as V_cmax and J_max. This is quite important for the carbon economy of the tree, as it allows a maximum use of the solar energy received by the leaves. This ultimately contributes to enhance carbon uptake. As a result this species has a fairly fast growth rate, even when it produces wood of high density. On the other hand, an open canopy can lead to high transpiration rate, which might have a negative impact on photosynthesis if drought periods become longer as predicted by climate models.

In the study it is shown that the mesophyll resistance plays is an important role in CO₂ diffusion. Therefore, it is recommended whenever possible to include g_m values in gas exchange calculations to obtain more accurate values of photosynthetic parameters. At low [CO₂] the photosynthetic rates were limited by Rubisco carboxylation rate, but at high [CO₂] photosynthesis was limited by both g_m and the electron transport rate. Changes in CO₂ concentrations have an effect on g_m, which has its maximum at C_c values of about 300 µmol mol^-1. However, a substantial increase in atmospheric CO₂ concentration may lead to an increase (> 50%) of the mesophyll resistance. The results presented in this study may be useful in the construction of climate models that aim to predict the effects of global climate change on the Amazon ecosystem.

6.ACKNOWLEDGEMENTS

To the Ministério da Ciência, Tecnologia, Inovações e Comunicações (MCTI-INPA). We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for scholarships (KRM and MB) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) for fellowship to RAM.

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Publication Dates

• Publication in this collection
  21 Feb 2020
• Date of issue
  2019

History

• Received
  28 June 2018
• Accepted
  30 Aug 2019