Water deficit modifies the carbon isotopic composition of lipids, soluble sugars and leaves of Copaifera langsdorffii Desf. (Fabaceae)

ABSTRACT

Water deficit is most frequent in forest physiognomies subjected to climate change. As a consequence, several tree species alter tissue water potential, gas exchange and production of carbon compounds to overcome damage caused by water deficiency. The working hypothesis, that a reduction in gas exchange by plants experiencing water deficit will affect the composition of carbon compounds in soluble sugars, lipids and vegetative structures, was tested on Copaifera langsdorffii. Stomatal conductance, leaf water potential, and CO₂ assimilation rate declined after a period of water deficit. After rehydration, leaf water potential and leaf gas exchange did not recover completely. Water deficit resulted in ¹³C enrichment in leaves, soluble sugars and root lipids. Furthermore, the amount of soluble sugars and root lipids decreased after water deficit. In rehydration, the carbon isotopic composition and amount of root lipids returned to levels similar to the control. Under water deficit, ¹³C-enriched in root lipids assists in the adjustment of cellular membrane turgidity and avoids damage to the process of water absorption by roots. These physiological adjustments permit a better understanding of the responses of Copaifera langsdorffi to water deficit.

Keywords:
carbon assimilation; carbon isotopic composition; lipids; soluble sugars; water deficit

Introduction

Climate change is causing an increase in the intensity and duration of the dry season of Cerrado (Neotropical savannas) and the Atlantic Forest (IPCC 2013). Drought duration is the main determinant of the different levels of resistance and resilience of tree species to water deficit (Keersmaecker et al. 2015), which affect growth (Pasho et al. 2011; Lévesque et al. 2013) and gain in biomass (Ivits et al. 2016). Quantifying the duration, intensity, and spatial extent of water deficit is a difficult task due to the numerous physical variables to be considered (Vicente-Serrano et al. 2013). Evaluating the ability of plants to overcome water deficit and recover after rehydration may be an alternative approach to understanding the adaptation of tree species to climate change (Vaz et al. 2010; Farrell et al. 2013). Furthermore, this approach can provide insight into the distribution of tree populations in forest physiognomies.

Under water deficit, trees induce stomata closure, decrease CO₂ assimilation (Kagotani et al. 2015), suppresses photosynthesis and activate respiration (Reddy et al. 2003; Meng et al. 2014). Furthermore, the breakdown of starch into soluble sugars increases the levels of carbon compounds in leaves (Chaves et al. 2009). The accumulation of soluble sugars, obtained from the breakdown of starch, facilitates osmotic adjustment by plants during water deficit. This, in turn, results in the maintenance of turgor in cells, which allows photosynthesis to remain constant, thereby enabling the redistribution of carbon in plants (Hu et al. 2015). Thus, water deficit inhibits lipid biosynthesis and alters the lipid concentration of cellular membranes (Guerfel et al. 2008). These events remodel cellular membranes, reduce their fluidity and permeability, and prevent the excessive loss of water (Larsson et al. 2006). These physiological responses are observed in Neotropical tree species in regions with strong seasonality and water restriction.

Copaifera langsdorffii is a Neotropical tree that is distributed in environments with strong seasonality and water restriction, such as the Cerrado (Neotropical savannas) and the Atlantic Forest (Cysneiros et al. 2011). Copaifera langsdorffiiproduces oilresin in cavities and resiniferous channels. The oil of this plant is a mixture of volatile and non-volatile terpenes, which are used in popular medicine to treat diseases and pharmaceutical, cosmetic, and biodiesel industries (Rodrigues et al. 2014). The response of C. langsdorffii to water deficit is related to the seasonality of the physiognomies where it occurs and its adaptive strategies (Franco et al. 2016). The species possesses physiological mechanisms and morphological adaptions of its leaves to deal with the wide variation in water availability in the environments in which it grows. During the dry season, foliar senescence reduces the level of gas exchange (Freitas & Oliveira 2002), thereby activating mechanisms to breakdown energy reserves in leaves and transport carbon compounds to the roots via phloem (Ronquim et al. 2003). According to Ronquim et al. (2009), C. langsdorffii possesses the ability to alter carbon acquisition and partitioning between shoots and roots in response to drought. Therefore, we chose to study this species as a model for the analysis of mechanisms of CO₂ assimilation under water deficit.

The examination of carbon isotopic composition (δ ¹³C) is a useful way to evaluate gas exchange (Leavitt & Wright 2002), the photosynthetic cycle (Nate et al. 2010), and CO₂ assimilation in plants subjected to water deficit (Adiredjo et al. 2014). Evaluation of δ ¹³C of vegetative structures and carbon compounds allows the detection of changes in the content of stable ¹³C/¹²C carbon isotopes in response to a water shortage. Water deficit changes the isotopic ratio of ¹²C/¹³C due to low photosynthetic rate and stomatal closure (Ebdon & Kopp 2004). Therefore, this study aimed to evaluate the effect of changes in the assimilation of CO₂ during water deficit on the δ¹³C of soluble sugars, lipids and vegetative structures in C. langsdorffii. We hypothesized that the reduction in levels of gas exchange in plants under water deficit will have an affect on the composition of carbon compounds in soluble sugars, lipids and vegetative structures.

Materials and methods

Cultivation and experimental conditions

The experiment was performed on young (24 months of age and approximately 50 cm high) plants of Copaifera langsdorffi Desf., in 4-L plastic pots (32 cm high; 14 cm in diameter). During the 24 months of growth, the young plants were kept at field capacity (soil water potential = -0.005 MPa; soil water percentage = 16 %). The soil used in the experiment was extracted from an area of Cerrado where adult individuals of C. langsdorffii were growing.

The experiment was conducted in the municipality of Botucatu, state of Sao Paulo, Brazil (22°53′12.17″S 48°29′52.45″W). The plants were maintained in a greenhouse under a relative humidity of approximately 50 % and an average temperature of 25 °C. The photosynthetic photon flux density (PPFD) inside the greenhouse reached 800 µmol m⁻². s⁻¹ during the day without clouds.

Experimental design

The experiment was randomized among three treatments (control, water deficit and rehydration) with 13 plants per treatment. The experiment took place over the course of 37 days. The control treatment included plants at 100 % of field capacity, which was monitored by weighting the pots (Thameur et al. 2012) and evaluating soil water potential. In water deficit and rehydration treatments, irrigation was suspended until the soil water percentage reached approximately 5 %. Water deficit lasted 18 days, after which the rehydration treatment, was restarted, when irrigation was performed until the soil water potential approached that of the control treatment, at which it was maintained for 19 days.

Leaf water potential (Ψ _H2Oleaf ) and soil water potential (Ψ _H2Osoil )

Leaf water potential (Ψ_H2Oleaf, MPa) and soil water potential (Ψ_H2Osoil, MPa) were measured in four individuals (n=4) from each treatment at intervals of three days. We used one leaf per individual to obtain Ψ_H2Oleaf, and a soil portion with a depth of 15 cm to obtain Ψ_H2Osoil. We evaluated only four of the 13 plants of each treatment to avoid an influence from the removal of leaves and soil during the evaluation of Ψ_H2Oleaf and Ψ_H2Osoil on the values of other experimental variables. The Ψ_H2Oleaf was evaluated at 5:30 a.m. (predawn - Pd) and at 12:00 p.m. (midday - Md), to document recovery or the accumulation of the effects of water deficit during the day. The Ψ_H2Osoil was evaluated just at Md. The Ψ_H2Oleaf and Ψ_H2Osoil were measured using a potentiometer with a temperature controller (WP4-T, Decagon Devices, USA).

Leaf gas exchange

Measurements of leaf gas exchange were made from 9:00 am to 11:00 am on sunny days on fully expanded and exposed leaves. Photosynthetic photon flux density, used to measure gas exchange, was 1200µmol m⁻²s⁻¹. The gas exchange variables measured were: stomatal conductance (g _s, mol m⁻²s⁻¹), CO₂ assimilation rate (A, μmol CO₂ m⁻²s⁻¹), transpiration rate (E, mol H₂O m⁻²s⁻¹), instantaneous water use efficiency (W _t , μmol CO₂. mmol H₂O⁻¹), and intrinsic water use efficiency (W _g , μmol CO₂. mmol H₂O⁻¹). Instantaneous water use efficiency (W _t ) was calculated as the ratio betweenA andE and intrinsic water use efficiency (W _g ) betweenAandg _s . The measurements were performed using a LI-6400 portable photosynthesis system (LI-COR, USA).

Isotopic analysis

We obtained leaves, the main root, lateral roots and leaf buds from all individuals and, after the separation of plant organs, halted biological processes by freezing all samples in liquid nitrogen. Subsequently, we dried the samples at 65 ºC for 72 hours and ground them in a grinder with liquid nitrogen (Spex - Model 6700). After grinding, we took aliquots of between 50 μg to 70 μg from each sample for analysis. Stable isotope analyses were carried out at the Institute of Biosciences of Botucatu, Universidade Estadual Paulista. The δ ¹³C values were calculated using the equation: /

where R is the isotopic ratio of ¹³C/¹²C, and VPDB is the Vienna Pee Dee Belemnite laboratory standard. The aliquots were placed tin capsules (6 mm high, 4 mm in diameter) and placed in an elemental analyzer EA 1108 (Carlo Erba Instruments, Milan, Italy) coupled to an isotope ratio mass spectrometer (IRMS) (Model Delta S).

Quantification of soluble sugars

We extracted soluble sugars from leaves, roots and leaf buds (100 mg) with 80 % ethanol (1:10, w/v) for 15 min at 80 °C. After cooling, the samples were centrifuged at 12000 × g for 15 min (Garcia et al. 2006). We removed and lyophilized the supernatant. We took aliquots of between 50 μg to 70 μg from each sample of leaf, root and leaf bud soluble sugar extract, placed them in a smooth tin capsule and subjected them to EA/IRMS isotopic analysis. Total soluble sugars were estimated colorimetrically by the phenol-sulfuric method (Dubois et al. 1956).

Quantification of lipids

We extracted lipids from leaves, roots and leaf buds (100 mg) with 1 ml MCW (methanol/chloroform/water; 12:5:3, v/v/v) for 30 min at 70°C. After cooling, the samples were centrifuged at 12000 × g for 5 min. We removed 0.6 ml aliquots of the supernatant, and separated phases by adding 0.2 ml of chloroform and 0.7 ml of water. After centrifugation, the methanol/water phase (upper layer) was removed (Wanek et al. 2001). The chloroform phase (lower layer) was dried in a vacuum concentrator to obtain the lipids. For each treatment, we pipetted aliquots (50 µl) of leaf, root and leaf bud lipid extract into a smooth tin capsule and subjected them to EA/IRMS isotopic analysis. We used the gravimetric method to quantify the lipids of the leaves, roots and leaf buds [milligrams per gram of dry weight (mg g−1 DW)]; (Chen et al. 2011). After lipid extraction, we transferred each aliquot into a pre-weighed vial, dried them under high vacuum and then weighed the dry residues.

Statistical analysis

Means and standard deviations were calculated for all variables. We used four plants (n=4) to evaluate Ψ_H2Oleaf and Ψ_H2Osoil and nine plants (n=9) to evaluate leaf gas exchange, the isotopic variables and the quantification of lipids and soluble sugars (total of 13 plants per treatment). After submitting all variables to Shapiro-Wilk normality test, we used one-way analysis of variance (ANOVA), followed by Tukey’s test, to compare individuals of C. langsdorffi in different treatments (control, water deficit and rehydration). Furthermore, we employed Spearman’s correlation test on δ¹³C_leaf and g _s to observe the relationship between these two variables in all treatments. Statistical analyses were performed using SigmaPlot (version 12.0).

Results

Soil granulometry and chemical analysis

According to the granulometry analysis performed prior to the experiment, the textural class of the soil was classified as sandy. The remaining physical and chemical soil characteristics are represented in Table 1.

Table 1
Soil granulometry and chemical analysis

Sand	Clay	Silt	F.C.	pH	O.M.	P	H+Al	K	Ca	Mg	V
_______g kg−1_________			MPa	CaCl2	g/dm3	mg/dm3	________mmolc/dm3_______				%
788	110	102	-0.006	5.6	26	9	11	0.7	9	4	57

• Field capacity (F.C.), organic matter (O.M.), phosphorus (P), potential acidity (H + Al), potassium (K), calcium (Ca), magnesium (Mg), base saturation (V).

Leaf and soil water potential

Eighteen days after water deficit, the soil water percentage averaged of 5.62 % and -0.70 MPa of Ψ_H2Osoil, compared to the control. During the same period the Ѱ_H2Oleaf Md was 25.26 % lower than the value of Ѱ_H2Oleaf Pd (Tab. 2), and so we started rehydration on the 19^th day. After rehydration, the water soil percentage averaged 15.23 % and Ψ_H2Osoil increased to -0.22 MPa, statistically equaling the control, and the difference between the values of Ѱ_H2Oleaf Pd and Md was reduced by 25 % (Tab. 2).

Table 2
Leaf water potential (Ψ_H2Oleaf), soil water potential (Ψ_H2Osoil), stomatal conductance (g _s), CO₂ assimilation rate (A), transpiration rate (E), instantaneous water use efficiency (W _t ), and intrinsic water use efficiency (W _g ) of Copaifera langsdorffii Desf. plants in the control, water deficit and rehydration treatments.

	Control	Water deficit	Rehydration
gs (mol m-2s-1)	0.19±24.78a	0.01±8.22c	0.14±22.21b
A (μmol CO2 m-2s-1)	10.34±0.76a	-0.52±0.53c	7.38±1.15b
E (mol H2O m-2 s-1)	2.53±0.50b	0.18±0.06c	8.34±0.94a
Wt (μmol CO2 mmol H2O-1)	4.21±0.87a	-3.78±3.71c	0.89±0.17b
Wg (μmol CO2 mmol H2O-1)	0.05±0.01a	-0.05±0.10b	0.05±0.01a
ΨH2O leaf Pd (MPa)	-1.55±0.16a	-1.89±0.41a	-2.56±0.32b
ΨH2O leaf Md (MPa)	-1.52±0.36a	-7.48±0.32c	-3.60±0.22b
ΨH2O soil (MPa)	-0.005±0.0a	-0.70±0.17b	-0.22±0.20a

• All values are means ± standard deviations (n=4 to Ψ_{H2O leaf} and Ψ_{H2O soil}, n=9 to leaf gas exchange variables). Data were analyzed using one-way ANOVA followed by Tukey’s test (significance at 5 %). Different letters represent statistical difference (P<0.05) between treatments.

Leaf gas exchange

The g _s was 0.01 mol m^-2s^-1 in maximum water deficit (18^th day), reflecting a drastic reduction compared to the control. On the last day of rehydration (37^th day), g _s indicated values (0.14 mol m^-2s^-1) close to the control (0.19 mol m^-2s^-1), and an increase compared to water deficit (0.01 mol m^-2s^-1) (Tab. 2). The water deficit values of A, E, W _t , W _g decreased in relation to control. After rehydration, only Wg values equaled those of the control, while the other variables were higher (E) or lower (A and Wt) (Tab. 2); thus some of the variables exhibited recovery after water deficit.

Isotopic analysis

Leaf gas exchange was evaluated on the first day of the experiment (day 1), the last day of water deficit (day 18) and the last day of rehydration (day 37). However, the carbon isotopic composition was only evaluated on the last day of water deficit and the last day of rehydration (control and rehydration treatments). Significant differences among treatments were observed only for δ¹³C_leaf (Tab. 3). Figure 1 shows the negative correlation (Spearman's coefficient = -0.587) between g _s and δ ¹³C_leaf. The δ¹³C_leaf values increased when g _s decreased, indicating an inverse relationship. The plants subjected to a water deficit had an increased concentration of ¹³C in their leaves, but the plants subjected to water deficit and later rehydrated had higher values of δ¹³C_leaf than plants under full hydration (control treatment) (Tab. 3).

Table 3
Carbon isotopic composition (δ¹³C) of vegetative structures from C. langsdorffii in the control, water deficit and rehydration treatments.

	Control	Water deficit	Rehydration
Leaf	-28.93±1.20b	-27.57±1.16a	-29.36±0.67b
Leaf buds	-28.15±1.34a	-27.81±0.48a	-28.05±0.71a
Root	-28.18±1.17a	-27.26±0.61a	-28.06±0.90a

• All values are means ± standard deviations (n=9). Data were analyzed using one-way ANOVA followed by Tukey’s test (significance at 5 %). Different letters represent statistical difference (P<0.05) between treatments.

Figure 1
Correlation between carbon isotopic composition (δ¹³C) of leaves and stomatal conductance (g _s) in control, water deficit, and rehydration treatments of Copaifera langsdorffii Desf. The line indicates the relationship between the variables. All values are means ± standard deviations (n=9). Spearman’s test obtained a correlation coefficient of −0.587, R ²=0.3194 and p = 0.00137. p < 0.05 indicates a significant correlation between the two variables.

Isotopic analysis and quantification of soluble sugars

The quantification and δ ¹³C of soluble sugars showed significant differences only in the roots (Fig. 2). In the maximum water deficit (18^th day), the amount of soluble sugars in the roots decreased (0.325 mg g⁻¹ DW) compared to the control (0.443 mg g⁻¹ DW). However, the δ ¹³C for root soluble sugars increased (-27.46 ‰) in relation to the control (-29.00 ‰). On the last day of rehydration (37^th day), the amount of root soluble sugars exhibited an increase (0.832 mg g⁻¹ DW) compared to water deficit (0.325 mg g⁻¹ DW) and control (0.443 mg g⁻¹ DW). In addition, the δ ¹³C root soluble sugars (-28.60 ‰) equaled control values (-29.00 ‰) (Fig. 2).

Figure 2
Quantification and carbon isotopic composition (δ¹³C) of leaf (A), leaf bud (B) and root (C) soluble sugars of Copaifera langsdorffii Desf. subjected to three different treatments (control, water deficit, and rehydration). All values are means ± standard deviations (n=9). Data were analyzed using one-way ANOVA followed by Tukey’s test (significance at 5 %). Different letters represent statistical difference (P<0.05) between treatments.

Isotopic analysis and quantification of lipids

The content and δ¹³C of lipids showed significant differences only in the roots (Fig. 3). Under maximum water deficit (18^th day), the amount of root lipids indicated a drastic reduction (0.033 mg g⁻¹ DW) compared to the control (0.133 mg g⁻¹ DW). Conversely, the δ ¹³C of root lipids was higher (-27.06 ‰) than the control treatment (-28.19 ‰). On the last day of rehydration (37^th day), the content (0.153 mg g⁻¹ DW) and δ¹³C (-28.61 ‰) of root lipids equaled the control (Fig. 3).

Figure 3
Quantification and carbon isotopic composition (δ ¹³C) of leaf (A), leaf bud (B) and root (C) lipids of Copaifera langsdorffii Desf. subjected to three different treatments (control, water deficit, and rehydration). All values are means ± standard deviations (n=9). Data were analyzed using one-way ANOVA followed by Tukey’s test (significance at 5 %). Different letters represent statistical difference (P<0.05) between treatments.

Discussion

The gradual decrease of stomatal conductance (g _s) and leaf water potential (Ѱ_H2Oleaf) in C. langsdorffii under water deficit indicated a reduction in water content of foliar tissues. The reduction of the CO₂ assimilation rate (A) and the transpiration rate (E) promoted a decrease in instantaneous water use efficiency (W _t ) (Hommel et al. 2014). Consequently, the low values for the ratio of A to E (W _t ) are associated with a reduced amount of water in the mesophyll and biochemical limitations to photosynthesis due to water loss by transpiration and low CO₂ assimilation. (Prieto et al. 2010; Ghaderi et al. 2011). Medrano et al. (2003) showed that stomatal closure and reduction in A during water deficit increases intrinsic water use efficiency (W _g ). However, our results demonstrated that a reduction in g _s and A caused a decrease in W _g . This response reduces the intercellular partial pressure of CO₂ inside the leaf, consequently decreasing the flux of CO₂ to the carboxylation enzyme Rubisco and suppressing biomass production (Matteo et al. 2014; Hentschel et al. 2015). After rehydration, the recovery of gas exchange and Ѱ_H2Oleaf were incomplete. Generally, plants subjected to water deficit recover 40-60% of maximum photosynthetic rate days after rehydration (Frosi et al. 2017); however, C. langsdorffii required more days of rehydration to completely recover gas exchange and Ѱ_H2Oleaf.

Under water deficit, resistance to diffusion due to g _s decrease limits the supply of CO₂ to Rubisco, thereby decreasing biomass production in the Calvin cycle. The negative correlation between g _s and δ ¹³C_leaf indicated that resistance to CO₂ diffusion in intercellular spaces of carboxylation sites provoked changes in the isotope discrimination in C3 plants (Warren & Adams 2006). In chloroplasts, Rubisco has greater activity at neutral pH and, at that stage, isotope discrimination causes ¹²C enrichment and a decrease in δ ¹³C values (Gilbert et al. 2012; Ghashghaie & Tcherkez 2013). Stomatal closure reduces Rubisco activity (Flexas & Medrano 2002) and pH decreases inside the chloroplast. This acidification causes ¹³C enrichment and an increase in δ ¹³C_leaf.

Therefore, the reduction of Rubisco enzyme activity and acidification of the chloroplast enriches ¹³C in the soluble sugars produced in leaves. The ¹³C are obtained from CO₂ fixation in the Calvin cycle. These ¹³C-enriched soluble sugars are metabolized in photoassimilates, which are redistributed via phloem to the roots and synthesized into other metabolites such as lipids (Hobbie & Werner 2004). Consequently, root lipids of C. langsdorffii are ¹³C-enriched. The enrichment of ¹³C in root lipids under water deficit occurs due to oxidation of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex resulting in the depletion of lipids (Niro & Epstein 1977). Metabolic products derived from acetyl-CoA are isotopically depleted as a result of enzymatic fractionation (Bowling et al. 2008). The evaluation of the transport and conversion of soluble sugars into root lipids through ¹³C-enriched analysis can assist in further studies on the effect of water deficit in forest species.

Lipids are major components of cell membranes; however, the decrease in the amount of root lipids under water deficit is associated with a reduction of lipids in root cellular membranes (Queiroz et al. 2002). The high δ ¹³C and low amount of root lipids maintain membrane fluidity, reducing the water permeability of the cellular membranes and preserving cell turgidity. Thus, the cellular membranes are preserved and water absorption by roots is not impaired. However, after rehydration, the δ ¹³C and amount of root lipids are similar to the control. This response indicates the recovery of cellular membranes in conditions of full hydration.

In summary, water deficit can modify the assimilation of carbon in C. langsdorffii, changing the values of δ ¹³C of leaf soluble sugars and root lipids. The ¹³C-enriched root lipids assist in the adjustment of root cellular membrane turgidity, and avoids damage to water absorption by roots. Examination of physiological responses to water deficit in forest species (such as C. langsdorffii) will assist in understanding the impacts of climate change and the physiological adjustments of plants of forest physiognomies.

Acknowledgements

This work was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank the Stable Environmental Isotope Center for their contribution to isotope analysis.

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Publication Dates

• Publication in this collection
  06 Nov 2017
• Date of issue
  Jan-Mar 2018

History

• Received
  08 May 2017
• Accepted
  17 Sept 2017