Sustained enhancement of photosynthesis in coffee trees grown under free-air CO₂ enrichment conditions: disentangling the contributions of stomatal, mesophyll, and biochemical limitations

Abstract

Coffee (Coffea spp.), a globally traded commodity, is a slow-growing tropical tree species that displays an improved photosynthetic performance when grown under elevated atmospheric CO₂ concentrations ([CO₂]). To investigate the mechanisms underlying this response, two commercial coffee cultivars (Catuaí and Obatã) were grown using the first free-air CO₂ enrichment (FACE) facility in Latin America. Measurements were conducted in two contrasting growth seasons, which were characterized by the high (February) and low (August) sink demand. Elevated [CO₂] led to increases in net photosynthetic rates (A) in parallel with decreased photorespiration rates, with no photochemical limitations to A. The stimulation of A by elevated CO₂ supply was more prominent in August (56% on average) than in February (40% on average). Overall, the stomatal and mesophyll conductances, as well as the leaf nitrogen and phosphorus concentrations, were unresponsive to the treatments. Photosynthesis was strongly limited by diffusional constraints, particularly at the stomata level, and this pattern was little, if at all, affected by elevated [CO₂]. Relative to February, starch pools (but not soluble sugars) increased remarkably (>500%) in August, with no detectable alteration in the maximum carboxylation capacity estimated on a chloroplast [CO₂] basis. Upregulation of A by elevated [CO₂] took place with no signs of photosynthetic downregulation, even during the period of low sink demand, when acclimation would be expected to be greatest.

Carbohydrates, Coffea arabica L., FACE, nitrogen, photosynthetic limitations, photosynthetic acclimation, starch.

Introduction

The actual increase in atmospheric CO₂ concentration ([CO₂]) is one of the most well-documented aspects of global climatic change. In 2014, [CO₂] exceeded, for the first time in at least the past 650 000 years, 400 µmol mol^–1 of air, as recorded at the Mauna Loa Observatory in Hawaii. Indeed, over the last decade, atmospheric [CO₂] has increased at a rate of approximately 2 µmol mol^–1 of air year^–1, and projections suggest that atmospheric [CO₂] will exceed 936 µmol mol^–1 of air (Representative Concentration Pathway 8.5) by the end of this century (IPCC, 2013). Given that photosynthesis is limited by the CO₂ supply at the current atmospheric [CO₂] (Long et al., 2004), an increase in [CO₂] is expected to increase the net photosynthetic rate (A) of plants (Ainsworth and Long, 2005). In fact, theoretical analyses suggest that A values might increase by 38% as atmospheric [CO₂] increases from 380 to 550 µmol mol^–1 of air (von Caemmerer and Furbank, 2003). Nevertheless, the results from free-air CO₂ enrichment (FACE) experiments reveal that the photosynthesis of crop plants fails to match the theoretical increase that could be obtained under elevated [CO₂] and that increases in A do not always translate into concordant increases in biomass production and crop yield (Long et al., 2006; Leakey et al., 2009). In any case, increased A under elevated [CO₂] is accompanied by consistent, although not universal, reductions in stomatal conductance (g_s) (Medlyn et al., 2001). Meta-analyses of FACE experiments have reported mean reductions in g_s of 16–19% (Ainsworth and Long, 2005; Ainsworth and Rogers, 2007). The dichotomous responses of A and g_s to elevated atmospheric [CO₂] ultimately lead to improvements in the water-use efficiency of the plant, reduced soil moisture depletion, and ameliorated stress during periods of drought (DaMatta et al., 2010a).

In addition to g_s, an increasing body of evidence suggests that the mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c), may also be a key constraint to photosynthesis (Flexas et al., 2012; Sun et al., 2014). However, the potential responses of g_m to climate changes, including elevated atmospheric [CO₂], have largely been neglected up to now (Flexas et al., 2012, 2014). In contrast to the well-known overall pattern of decreased g_s under long-term [CO₂] exposure, the available information shows no general trend for g_m in plants grown under elevated [CO₂] (i.e. 500–600 µmol mol^–1 of air); indeed, no change, either decreased or increased g_m, has been reported, possibly depending on the species and time (Flexas et al., 2012, and references therein). In any case, not considering g_m in photosynthesis calculations can significantly alter our interpretation of the effects of environmental factors, including CO₂ supply, on photosynthesis (Sun et al., 2014). For example, mis-interpretations of photosynthetic downregulation (see below) based on decreases in the maximum apparent carboxylation capacity (V_cmax) on a C_i basis may occur when a decreased g_m under elevated [CO₂] is not considered.

In some FACE studies that were conducted with temperate tree species, the initial stimulation of A was preserved during the long-term exposure to elevated [CO₂] (Liberloo etal., 2007; Bader etal., 2010; Streit et al., 2014). Frequently, however, plants that were exposed to elevated [CO₂] in FACE trials show reductions in photosynthetic capacity (Rogers and Ellsworth, 2002; Hyvönen et al., 2007; Norby etal., 2010), termed photosynthetic downregulation (acclimation), associated with end-product accumulation (Ainsworth and Rogers, 2007; Leakey et al., 2009) that is usually linked to nutrient limitations, particularly nitrogen (N) (Ainsworth and Long, 2005). A reduced or acclimated stimulation of photosynthesis has been mechanistically and quantitatively attributed to decreased V_cmax and investment in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Ainsworth and Long, 2005), but may also be linked to a reduced ribulose-1,5-bisphosphate (RuBP) regeneration, decreasing the in vivo maximum rate of carboxylation that is limited by electron transport (J_max) due to either a lowered electron transport capacity or inorganic phosphate availability in the chloroplast for ATP synthesis (Ainsworth and Rogers, 2007; Kirschbaum, 2011; Zhu et al., 2012). As a rule, limited sink strength predisposes plants to a greater acclimation of photosynthetic capacity and decreases the stimulation of photosynthesis by growth under elevated [CO₂] (Long et al., 2004; Ainsworth and Rogers, 2007). Overall, trees, particularly fast-growing individuals, display a large sink capacity (root–trunk system) compared with that of annuals, which, to a great extent, explains their higher stimulation of photosynthesis when grown under elevated atmospheric [CO₂] compared with shrubs and annual crops (Ainsworth and Long, 2005).

Coffee is one of the most heavily globally traded commodities, with retail sales worldwide estimated at US$90 billion (DaMatta et al., 2010b). The coffee tree is a slow-growing evergreen species that displays relatively low A, with maximum values typically around or below 10 μmol m^–2 s^–1 with current atmospheric [CO₂] and saturating light (DaMatta, 2004). Such low values have largely been associated with diffusive rather than biochemical limitations to photosynthesis (Batista et al., 2012), particularly diffusional constraints at the stomatal level (Martins et al., 2014). Consequently, coffee trees are expected to largely benefit, in terms of photosynthetic performance, by the elevated atmospheric [CO₂], considering that increasing atmospheric [CO₂] increases the gradient that ensures an adequate diffusion of CO₂ from the atmosphere to the chloroplasts. Indeed, the recent efforts of Ramalho et al. (2013), who studied potted coffee plants growing in an enclosure system under different [CO₂] over 1 year, revealed greater A (ranging from 34 to 49%) under elevated (700 μmol CO₂ mol^–1 of air) than under normal (380 µmol CO₂ mol^–1 of air) [CO₂]. This positive effect was later confirmed by Ghini et al. (2015) in coffee plants that were grown using the first FACE facility in Latin America (ClimapestFACE). Interestingly, g_s responds little, if at all, to elevated [CO₂] in coffee; taken together, these results imply that substantial increases in water-use efficiency occur in coffee grown under elevated [CO₂] conditions (Ramalho et al., 2013; Ghini et al., 2015).

In this study, two commercial coffee cultivars with contrasting crop yields were grown under current ambient and elevated [CO₂] at the ClimapestFACE facility (Ghini et al., 2015). We demonstrated previously that, integrated over the course of the day, the A values that were measured in situ were ≥40% higher for the plants that were grown under elevated atmospheric [CO₂], which may in part explain the enhanced crop yields under elevated [CO₂] (Ghini et al., 2015). Given these previous results and those of enclosure studies (Ramalho et al., 2013), our main goal was to examine the mechanisms underlying this response. To achieve this goal, we conducted an in-depth analysis of the photosynthetic performance by calculating g_m to properly parameterize the responses of A to C_c in addition to disentangling the relative contributions of the stomatal, mesophyll, photochemical, and biochemical limitations of photosynthesis. This analysis was conducted in two contrasting growth seasons, which were characterized by strong and weak sink demand, when photosynthetic downregulation, if occurring, would be expected to be minimal and maximal, respectively.

Materials and methods

Site description, CO₂ treatments, plant materials, and samplings

We carried out the experiment using the ClimapestFACE facility located in Jaguariúna (22°43'S, 47°01'W, 570 m above sea level), south-eastern Brazil. The soil at the experimental area is a typical dystroferric red latosol. The climate is humid subtropical, a Cfa type according to the Köppen classification, with hot rainy summers and cold dry winters. Mean monthly air temperature and precipitation were recorded during the experiment (see Supplementary Data at JBX online).

To mimic coffee agrosystems, the FACE system increased the ambient [CO₂] in six 10 m diameter ring plots (elevated CO₂) within a continuous 7 ha coffee field. Six additional 10 m diameter ring plots served as controls, i.e. were left under ambient [CO₂]. Elevated- and ambient-CO₂ plots were at least 70 m apart to minimize cross-plot contamination.

Fumigation with CO₂ began on 25 August 2011. The average atmospheric [CO₂] at the beginning of the experiment was approximately 390 µmol mol^–1. The performance of the FACE system was adjusted so that the [CO₂] as measured at the centre of the ring achieved target levels of 550 µmol mol^–1 of air. The plots were not enriched with CO₂ at night. Further details regarding the experimental site set-up and CO₂ control performance can be found in Ghini et al. (2015).

Two coffee (Coffea arabica L.) cultivars, cv. Catuaí Vermelho IAC 144 and cv. Obatã IAC 1669-20, were assessed. The latter displays significantly greater (>30%) crop yields than the former, as found in test trials under common agronomic practices. Plantlets with three to four pairs of leaves were transplanted into the plots in March 2011. The cultivars were interspersed in rows that were 1.75 m apart, with 0.60 m between plants in the rows. The plots were located in a continuous 7 ha coffee field of cv. Catuaí Vermelho IAC 144, into which plantlets were transplanted on March 2010; the rows were 3.5 m apart, with 0.60 m between plants in the rows. Fertilization per hectare at planting was accomplished with 2300kg of single super phosphate, 2300kg of dolomitic limestone, 285kg of chloride potassium and 570kg of Yoorin Master 2S^® (a granulated fertilizer with fast and slow release of P, Ca, Mg and S). The plants were submitted to routine agricultural practices for commercial coffee bean production, including several applications of herbicides, fungicides, and insecticides. Each tree was fertilized annually with 46g of N, 9g of P and 23g of K plus micronutrients. The fertilizer was applied three times during the growing season, coinciding approximately with periods of supplemental fertilization for most commercial coffee crops in Brazilian Arabica coffee-producing regions. The crop was grown without supplemental irrigation.

Sampling and measurements were carried out in two contrasting periods of the coffee growth cycle: February and August (2013). In February, the vegetative growth rates are relatively low due to competition with reproductive growth (bean-filling phase), and thus assimilate demand is at its maximum; in August, the plants are fruitless (crop harvests are usually completed in June–July), and vegetative growth is negligible. Thus, assimilate demand by the sinks is at its minimum (Silva et al., 2004).

Gas-exchange and chlorophyll a fluorescence measurements

The net rate of carbon assimilation (A), stomatal conductance [which was subsequently converted into stomatal conductance to CO₂ (g_s)] and internal CO₂ concentration (C_i) were measured simultaneously with chlorophyll a fluorescence parameters in the youngest fully expanded leaves [the third or fourth leaf pair from the apex of the plagiotropic (lateral) branches in the upper third of the plants] using an open system under ambient temperature and a [CO₂] of 390 or 550 µmol mol^–1 of air, depending on the treatment. All of the measurements, which were conducted using three cross-calibrated infrared gas analysers (LI-6400XT; Li-Cor, Lincoln, NE, USA) using an integrated fluorescence chamber head (LI-6400-40; Li-Cor), were made on clear-sky days during four time periods: 08:00–09:00, 10:00–11:00, 13:00–14:00, and 16:00–17:00 (solar time). To improve the uniformity over the course of the day, the measurements were conducted at the leaf level at an artificial photosynthetically active radiation (PAR) level of 1000 µmol of photons m^–2 s^–1. This PAR intensity is sufficiently high to saturate the photosynthetic machinery without causing photoinhibition (Cavatte et al., 2012); in addition, these PAR values approximated the ambient irradiance that was intercepted by the sampled leaves (in their natural angles) in most measurements at each time point. After the leaf tissue was clamped in the leaf chamber, the gas-exchange rates usually stabilized within approximately 3min. In both February and August 2013, the measurements were repeated once using different plants within a given ring of the FACE. Even without supplemental irrigation, the pre-dawn xylem water potential, as measured with a Scholander chamber, was greater than –0.1MPa in both periods, suggesting that our gas-exchange data were not constrained by the water supply.

In light-adapted leaves, the steady-state fluorescence yield (F_s) was measured after registering the gas-exchange parameters. A saturating white light pulse (8000 μmol m^–2 s^–1; 0.8 s) was applied to achieve the light-adapted maximum fluorescence (F_m'). The actinic light was then turned off, and far-red illumination was applied (2 μmol m^–2 s^–1) to measure the light-adapted initial fluorescence (F₀'). Using the values of these parameters, the photochemical quenching coefficient (q_P) and the capture efficiency of excitation energy by open photosystem (PS) II reaction centres (F_v'/F_m') were estimated (Logan et al., 2007). The actual PSII photochemical efficiency (φ_PSII) was determined as φ_PSII=(F_m' –F_s)/F_m' following the procedures of Genty et al. (1989). The electron transport rate (ETR) was then calculated from the equation ETR=φ_PSII×β×α×photosynthetic photon flux density (PPFD), where α is leaf absorptance, and β reflects the partitioning of absorbed quanta between PSII and PSI (Genty et al., 1989). The product of β and α was determined according to Valentini et al. (1995) from the relationship between φ_PSII and φ_CO2 as obtained by varying the light intensity under non-photorespiratory conditions.

The rate of mitochondrial respiration in darkness (R_D) was measured early in the morning in dark-adapted leaves and used to estimate mitochondrial respiration in the light (R_L) according to Lloyd et al. (1995) as R_L=[0.5 –0.05ln(PPFD)]×R_D.

The photorespiratory rate of Rubisco (R_P) was calculated as R_P=1/12[ETR –4(A+R_L)] according to Valentini et al. (1995). The R_P/A_gross ratio was obtained throughout the day by computing the values of R_P, A, and R_L. The values of R_L were corrected for leaf temperature (assessed with temperature sensors coupled to infrared gas analysers over the course of the gas-exchange measurements) using the temperature response for R_L as described by Bernacchi et al. (2001).

Four to six A/C_i curves were obtained in situ (from approximately 07:00 to 10:30 in February, and from approximately 08:30 to 12:00 in August, when g_s values were relatively elevated) from different plants per treatment. These curves were initiated at an ambient [CO₂] (C_a) of 400 μmol mol^–1 under a saturating PPFD of 1000 μmol m^–2 s^–1. Once a steady state was reached, C_a was gradually decreased to 50 μmol mol^–1 of air. Upon the completion of the measurements at low C_a, C_a was returned to 400 μmol mol^–1 of air to restore the original A. Next, C_a was increased stepwise to 1600 μmol mol^–1 of air. The A/C_i curves consisted of 13 different C_a values. This procedure was successfully applied to the measurements that were conducted in February. In August, however, due to the intrinsically low g_s for the season (coupled to the negative g_s response to increases in C_a), we were unable to reliably estimate C_i at C_a values greater than 600 μmol mol^–1. Therefore, A/C_i curves only consisted of seven different C_a values (<600 μmol mol^–1). Regardless of the season, corrections for the leakage of CO₂ into and out of the leaf chamber of the LI-6400 were applied to all gas-exchange data as described by Rodeghiero et al. (2007).

Estimations of the mesophyll conductance to CO₂ (g_m), the maximum rate of carboxylation (V_cmax), the maximum rate of carboxylation limited by electron transport (J_max), and the chloroplastic [CO₂] of transition (C_{c_trans})

C_c was estimated according to Harley et al. (1992) as follows:

$$\text{Cc}=\{\Gamma *[\text{ETR}+\text{8}(\text{A}+\text{RL})]/[\text{ETR}-\text{4}(\text{A}+\text{RL})]\}$$

where ETR and A were obtained from the gas-exchange and chlorophyll a fluorescence measurements as conducted under saturating light; R_L was estimated as described above; and Γ* is the CO₂ compensation point in the absence of mitochondrial respiration (the conservative value of Γ* for coffee was obtained from Martins et al., 2013). We next estimated g_m as the slope of the A versus C_i –C_c relationship as A=g_m(C_i –C_c) so the estimated g_m was an averaged value over the points used in the relationship (C_i <350 μmol mol^–1 of air).

Because all of the available methods for estimating g_m rely on models that include several assumptions as well as technical limitations and sources of error that need to be considered to obtain reliable estimates of this parameter (Pons et al., 2009), g_m was also estimated using an alternative approach: the exhaustive dual optimization (EDO) curve-fitting technique of Gu et al. (2010). For this purpose, the data from the A/C_i curves were uploaded to the Oak Ridge National Laboratory (USA) website (http://leafweb.ornl.gov), which uses EDO to parameterize A/C_i curves based on the Farquhar–von Caemmerer–Berry model (Gu et al., 2010).

The g_m values were used to convert A –C_i into A –C_c curves. From these curves, V_cmax and J_max were calculated by fitting the mechanistic model of CO₂ assimilation as proposed by Farquhar et al. (1980) using the C_c-based temperature dependence of the kinetic parameters of Rubisco (Bernacchi et al., 2002). The curve-fitting procedures have been detailed elsewhere (Martins et al., 2014). Afterwards, the photosynthetic parameters V_cmax, J_max, and g_m were normalized to 25 °C using the temperature response equations from Sharkey et al. (2007).

The chloroplastic [CO₂] of transition (C_{c_trans}), which denotes the transition between the Rubisco- and RuBP regeneration-limited states, was estimated as described by Gu et al. (2010):

$$C_{\text{c}\_\text{trans}}=\left(J_{\text{max}}{K}_{\text{m}}-\text{8}{V}_{\text{cmax}}{\Gamma }^{*}\right)/\left(\text{4}{V}_{\text{cmax}}-J_{\text{max}}\right)$$

where K_m, the effective Michaelis–Menten constant for CO₂ that considers the competitive inhibition by O₂, was taken from Martins et al. (2013). C_{c_trans} was calculated using V_cmax, J_max, K_m, and Г* at the ambient temperatures realized during the gas-exchange measurements at 08:00–09:00h in order to allow proper comparisons with the measured C_c at this time.

Quantitative analysis of the limitations of photosynthesis

The overall photosynthetic limitations were partitioned into their functional components [stomatal (l_s), mesophyll (l_m), and biochemical (l_b) limitations] using the values of g_s, g_m, V_cmax, Г*, K_m, and C_c following the approach that was proposed by Grassi and Magnani (2005) as follows:

$$l_{\text{s}}=\left(g_{\text{tot}}/g_{\text{s}}*\partial A/\partial C_{\text{c}}\right)/\left(g_{\text{tot}}+\partial A/\partial C_{\text{c}}\right)$$

$$l_{\text{m}}=\left(g_{\text{tot}}/g_{\text{m}}*\partial A/\partial C_{\text{c}}\right)/\left(g_{\text{tot}}+\partial A/\partial C_{\text{c}}\right)$$

$$l_{\text{b}}=g_{\text{tot}}/\left(g_{\text{tot}}+\partial A/\partial C_{\text{c}}\right)$$

where g_s is the stomatal conductance to CO₂ and g_m is the mesophyll conductance to CO₂ according to Harley et al. (1992), and g_tot is the total conductance to CO₂ from ambient air to chloroplasts (g_tot=1/[(1/g_s)+(1/g_m)]). ∂A/∂C_c was calculated as

$$\partial A/\partial C_{\text{c}}=\left[V_{\text{cmax}}\left({\Gamma }^{*}+K_{\text{m}}\right)\right]/{\left(C_{\text{c}}+K_{\text{m}}\right)}^{\text{2}}$$

Biochemical, N and P, analyses

Leaf discs were collected on clear-sky days prior to flash freezing in liquid nitrogen and subsequent storage at –80 ºC until analysis. For carbohydrate analyses, a 10mg sample of ground tissue was added to pure methanol and the mixture was incubated at 70 °C for 30min. After centrifugation (13 000g, 5min), the hexoses (glucose and fructose) and sucrose in the supernatant were quantified; the concentration of starch was determined from the methanol-insoluble pellet as detailed previously (Praxedes et al., 2006; Ronchi et al., 2006). The levels of malate and fumarate were determined exactly as reported elsewhere (Nunes-Nesi et al., 2007).

Leaf samples were used to measure the N (using an elemental analyser; Carlo Erba, Milan, Italy) and P (using routine spectrophotometric methods) contents.

Statistical analyses

The experiment was designed as a split-plot design with CO₂ level as the whole-plot factor and cultivar as the subplot factor with six replicates per treatment (ambient and elevated [CO₂]), and the data were subjected to ANOVA. The treatment differences were also tested using ANOVA. Throughout the text, mean differences were considered significant at P≤ 0.05 (see Supplementary Data at JBX online).

Results

Most of the significant treatment effects on the variables analysed were observed for the [CO₂] factor rather than for the cultivar factor; overall, the traits showed no significant responses to the [CO₂]×cultivar interaction (see Supplementary Data).

During the growing season, A was significantly greater under elevated than under ambient [CO₂] at two of the four time points in both cultivars (Fig. 1). g_s did not respond to the CO₂ enrichment in cv. Catuaí; in cv. Obatã, the g_s values at two time points were lower under elevated than under ambient [CO₂] (Fig. 1). The R_P/A_gross ratio was clearly lower in the morning in both cultivars that were treated with CO₂, with a less clear pattern in the afternoon (Fig. 1). Notably, diurnally integrated A was 40% greater and the R_P/A_gross ratio was 20% lower under elevated than under ambient [CO₂], with no significant change in g_s in either cultivar (Fig. 1). During the period of limited growth (winter), both A and g_s values were, as expected, much lower than during the growing season, whereas an opposite pattern was noted for the R_P/A_gross ratio (Fig. 1). In all of the winter measurements, both of the cultivars responded to CO₂ fertilization by increasing A significantly, with little, if any, alteration in g_s. The R_P/A_gross ratio was consistently lower in cv. Obatã under elevated than under ambient [CO₂], whereas in cv. Catuaí, this ratio was unresponsive to the CO₂ supply. The diurnally mean A values in winter averaged 56% higher in the plants that were treated with CO₂ than in their untreated counterparts. In cv. Obatã, g_s values integrated over the course of the day were significantly lower under elevated [CO₂] (Fig. 1).

Fig. 1.

Effect of elevated (550 µmol mol^–1) and ambient (390 µmol mol^–1) [CO₂] on the leaf gas exchange of two coffee cultivars (Catuaí and Obatã) growing in a FACE trial during the growing season (February) and winter (August): net CO₂ assimilation rate (A), stomatal conductance to CO₂ (g_s), and ratio of photorespiration-to-gross photosynthetic rate (R_P/A_gross). In the insets, the mean diurnal values of A, g_s, and R_P/A_gross are shown. The [CO₂] used during the gas-exchange measurements was identical to the growth [CO₂] (390 or 550 µmol mol^–1). The data for A were taken from Ghini et al. (2015). n=6±SE.

Overall, diurnally integrated gas exchange was essentially similar in both cultivars during the growing season (Fig. 1). In winter, A was significantly greater in cv. Obatã (irrespective of CO₂ fertilization), and R_P/A_gross was lower in this cultivar (only at elevated [CO₂]) than in cv. Catuaí (Fig. 1).

From the chlorophyll a fluorescence analysis, we estimated the ETR, q_P, and F_v'/F_m' over the course of the day (Fig. 2). In both the growing season and winter, all of these traits were virtually unaltered by the CO₂ treatments. However, we noted that, irrespective of cultivar, the overall values of ETR and q_P were higher in the growing season than in the phase of restrained growth, whereas F_v'/F_m' varied little, if at all, between seasons.

Fig. 2.

Effect of elevated (550 µmol mol^–1) and ambient (390 µmol mol^–1) [CO₂] on the photochemical parameters of two coffee cultivars (Catuaí and Obatã) growing in a FACE trial during the growing season (February) and winter (August): electron transport rate (ETR), photochemical quenching coefficient (q_P), and capture efficiency of excitation energy by open PSII reaction centres (F_v'/F_m'). In the insets, the mean diurnal values of ETR, q_P, and F_v'/F_m' are shown. The [CO₂] used during the chlorophyll a fluorescence measurements was identical to the growth [CO₂]. n=6±SE.

We analysed g_m using two independent methods. Overall, similar values and trends for g_m were observed regardless of the experimental approach that was used to estimate g_m (Table 1). Therefore, only the g_m values that were obtained using the method of Harley et al. (1992) are discussed below and were used to parameterize the responses of A to C_c. In both the growing season and winter, g_m did not respond to the CO₂ treatments (Table 1). Both V_cmax and J_max, estimated on a C_c and C_i basis, were unresponsive to the CO₂ supply in the growing season, as was V_cmax during the winter (Table 1 and Supplementary Data at JBX online). In fact, the absolute V_cmax values on a C_i basis were essentially similar regardless of the season (57 μmol CO₂ m^–2 s^–1 on average), as can be deduced from Supplementary Data. Notably, had the results been calculated only on a C_i basis, V_cmax would be underestimated by approximately 40%, thus highlighting the importance of estimating g_m to properly describe the responses of A to the CO₂ supply.

Table 1.

Effect of elevated (550 µmol mol^–1) and ambient (390 µmol mol^–1) [CO₂] on some photosynthetic parameters of two coffee cultivars (Catuaí and Obatã) growing in a FACE trial during the growing season (February) and winter (August)

The following values were determined: mesophyll conductance (g_m), estimated using two independent methods: the EDO curve-fitting technique and the method that was proposed by Harley et al. (1992); the maximum apparent carboxylation capacity (V_cmax) and the in vivo maximum rate of carboxylation as limited by electron transport (J_max), both on a chloroplast [CO₂] basis; and the chloroplastic [CO₂] (C_c) and C_c of transition (C_{c_trans}). Data for J_max and C_{c_trans} were not obtained in August. V_cmax, J_max and g_m were normalized to 25 °C using the temperature response equations from Sharkey et al. (2007). n=5–6±SE.

Parameter	Catuaí
	February		August
	390 µmol mol–1	550 µmol mol–1	390 µmol mol–1	550 µmol mol–1
gm_EDO	0.067±<0.012	0.102±<0.015	0.072±<0.012	0.072±<0.015
gm_Harley	0.073±<0.008	0.061±<0.007	0.071±<0.011	0.105±<0.024
Vcmax	61.0±<1.8	54.8±<2.8	57.3±<5.4	58.3±<3.3
Jmax	95.6±<3.9	81.1±<4.6	-	-
Cc	152±<15	205±<15	107±<9	182±<19
Cc_trans	268±<15	231±<13	-	-

	Obatã
	February		August
	390 µmol mol–1	550 µmol mol–1	390 µmol mol–1	550 µmol mol–1
gm_EDO	0.060±<0.015	0.083±<0.017	0.088±<0.021	0.063±<0.010
gm_Harley	0.091±<0.018	0.065±<0.007	0.058±<0.010	0.082±<0.014
Vcmax	55.2±<3.3	54.8±<2.7	59.9±<5.6	52.3±<1.9
Jmax	86.8±<6.2	80.0±<5.3	–	–
Cc	173±<15	253±<31	104±<19	165±<23
Cc_trans	303±<10	231±<9	–	–

We also determined data for C_c and C_{c_trans} (Table 1) as estimated in the early morning (08:00–09:00h) when A is at its maximum. Overall, C_c was significantly higher under elevated than ambient [CO₂], and C_{c_trans} (estimated only in the growing season due to the available J_max data) decreased under elevated [CO₂]. Regardless of cultivars, C_c was lower than C_{c_trans} under ambient [CO₂]. Under elevated [CO₂], the values of C_c did not differ significantly from those of C_{c_trans} irrespective of cultivar (P>0.05).

The functional components (l_s, l_m, and l_b) of the overall photosynthetic limitations did not respond to [CO₂] in cv. Catuaí in the growing season (Fig. 3). In cv. Obatã, l_b was unresponsive to the [CO₂] supply, regardless of the season; however, elevated [CO₂] decreased l_s (37%) significantly in parallel with an increase in l_m (73%) in the growing season. In winter, regardless of cultivar, significant changes were only noticeable in l_m, which decreased by 50% under elevated [CO₂] compared with under ambient [CO₂]. Importantly, our results indicate that diffusional limitation (l_s+l_m) accounted for the most prominent constraints to photosynthesis (≥60% in the growing season and ≥80% in winter), and these responses were not altered by the CO₂ supply in either cultivar (Fig. 3). By comparing the two growth periods, the greater diffusional limitations in winter were chiefly associated with increased l_s (~60% vs ~37% in the growing season), whereas the magnitude of changes in l_m was much narrower when comparing both seasons. Increased diffusional constraints in winter resulted in a lower l_b that was approximately half relative to that obtained in the growing season (Fig. 3). We also assessed intrinsic physiological changes as a function of growth CO₂ by calculating the limitations using different ambient [CO₂] (390 µmol mol^–1 of air for the plants grown at 550 µmol mol^–1 of air and vice versa); the results were nearly identical to those shown previously (Fig. 3) with the exception that l_s was higher in cv. Obatã even in the winter season (see Supplementary Data at JBX online).

Fig. 3.

Effect of elevated (550 µmol mol^–1) and ambient (390 µmol mol^–1) [CO₂] on the overall limitations to photosynthesis of two coffee cultivars (Catuaí and Obatã) growing in a FACE trial during the growing season (February) and winter (August): stomatal (l_s), mesophyll (l_m) and biochemical (l_b) limitations. The required gas-exchange parameters used in the estimations were obtained at an ambient [CO₂] identical to the growth [CO₂] (390 or 550 µmol mol^–1). n=5–6±SE.

In the growing season, the pools of carbohydrates (and malate/fumarate) were measured at the beginning, middle, and end of the photoperiod. Regardless of the cultivar, the concentrations of glucose, fructose, and sucrose were similar or slightly higher in the plants that were treated with CO₂ compared with their untreated counterparts, but only in a few measurements did these differences reach statistical significance (Fig. 4). The concentrations of starch were not significantly affected by the treatments in cv. Catuaí, whereas in Obatã, the starch levels were consistently higher (64% on average) under elevated than under ambient [CO₂]. In winter, measurements were conducted only at midday for both cultivars. The glucose and sucrose pools were unaffected by the CO₂ supply independently of cultivar, whereas the starch pools averaged 38% higher under elevated than under ambient [CO₂]. Fructose pools increased significantly in response to the CO₂ supply but only in cv. Catuaí. It should be emphasized that there was an almost invariant total soluble sugar concentration [represented by the sum of glucose, fructose, and sucrose (on average, the total soluble sugars totalled 6.6 and 6.8% of leaf dry weight in the growing season and winter, respectively; data not shown)], whereas the levels of starch were remarkably higher (522% on average) in winter than in the growing season.

Fig. 4.

Effect of elevated (550 µmol mol^–1) and ambient (390 µmol mol^–1) [CO₂] on the leaf concentrations of carbohydrates and malate of two coffee cultivars (Catuaí and Obatã) growing in a FACE trial during the growing season (February) and winter (August). n=6±SE. DW, dry weight.

We next evaluated the malate and fumarate pools (Fig. 4), given that the concentration of these organic acids often changes when the carbon balance is altered. In the growing season, the levels of malate were not significantly affected by the treatments in cv. Catuaí; in cv. Obatã, malate pools were consistently lower (24% on average) over the course of the day under elevated compared with under ambient [CO₂]. In winter, malate pools were significantly lower (33%) under elevated than under ambient [CO₂] in cv. Catuaí, whereas these pools were not affected by the CO₂ treatment in cv. Obatã. Regardless of the season, fumarate was not detected in this study.

The leaf N and P concentrations were approximately 3.5 and 1.5%, respectively, on a dry-weight basis and remained invariant regardless of treatment (data not shown). These concentrations are within an optimal range for coffee.

Discussion

In this study, we examined in detail the underlying mechanism associated with the photosynthetic enhancement that we demonstrated previously in coffee trees when grown with supplemental CO₂ (Ghini et al., 2015). First (and as expected), a higher A under elevated [CO₂] was associated not only with improved carboxylation rates coupled with a higher availability of CO₂ as substrate (higher C_c) but also with a relatively higher carboxylation over oxygenation activity of Rubisco, resulting in decreased R_P, here noted through the lower R_P/A_gross ratio under elevated [CO₂] (Fig. 1).

Given that the stimulation of A by CO₂ fumigation would increase the ATP demand that is required for RuBP regeneration and that the control of photosynthesis shifts from Rubisco-limited to RuBP regeneration-limited (Ainsworth and Rogers, 2007), we next analysed whether any impairment in leaf photochemistry could constrain the maximization of A under elevated [CO₂] (Fig. 2). We found that the CO₂ stimulation of A was not accompanied by concordant alterations in the efficiency of the excitation energy as captured by the open PSII reaction centres (estimated as F_v'/F_m') and in the fraction of absorbed light that is dissipated photochemically (estimated as q_P). Importantly, the ETR values by far exceeded the photochemical needs that were required to support the observed A values (Martins et al., 2013), irrespective of treatments. Therefore, our data indicated that photochemical events are unlikely to have limited CO₂ fixation in this study. In any case, seasonal photochemical adjustments (decreased q_P and ETR in winter) occurred independently of the CO₂ supply, but these adjustments should represent a consequence, rather than a cause, of the decreases in A in winter. In other words, adjustments probably occurred because the light-capture and light-utilization processes are imbalanced in coffee in winter, and thus adjustments in leaf photochemistry may be a proper way of avoiding the occurrence of photoinhibition in coffee (Chaves et al., 2008; Pompelli et al., 2010), as could also be deduced here by the unchanging variable-to-maximum chlorophyll fluorescence ratio (data not shown).

We subsequently quantified the distribution of the overall photosynthetic limitations between diffusional and biochemical processes. To reach this goal, we first estimated g_m and found no g_m acclimation to elevated CO₂ (Table 1). Regardless of the CO₂ supply, we demonstrated that the major limitations to photosynthesis in coffee are linked to diffusional constraints (Fig. 3), with greater values of l_s than those of l_m, as also reported previously for potted coffee seedlings grown under ambient [CO₂] (Martins et al., 2014). In addition, we found, relative to the growing season, an exacerbation of diffusional constraints to coffee's photosynthesis in winter that was almost entirely traceable by further increases in l_s. Such an increase may be a consequence of the stomatal sensitivity to low nocturnal temperatures in C. arabica (Barros et al., 1997; Silva et al., 2004). In contrast to other studies suggesting that mesophyll constraints to photosynthesis are of a similar magnitude as stomatal limitations (Warren, 2008; Flexas et al., 2012), we found that l_s was exacerbated in coffee trees in the afternoon, particularly because g_s peaked in the early morning and decreased sharply in the afternoon, reaching values that were typically below 40 mmol H₂O m^–2 s^–1 (Chaves et al., 2008; DaMatta et al., 2008) as a consequence of increasing vapour pressure deficit. Therefore, as coffee trees display an inherently low g_s and g_m (see also Martins et al., 2014), it is expected that this species might benefit remarkably from increasing [CO₂] relatively more than other plant species with lower diffusional limitations to photosynthesis (Flexas et al., 2014).

Notably, the absence of CO₂-induced alterations in l_b is consistent with unaltered parameters related to biochemistry (J_max and V_cmax on a C_c basis) and N and P concentrations, suggesting that the amount of resources (e.g. N) that were allocated to electron carriers and Calvin cycle enzymes remained unchanged. Additionally, unchanged V_cmax has been associated with unaltered Rubisco amounts and/or activation state. Overall, these results, which are consistent with a lack of photosynthetic acclimation, contradict what has been noted in many studies in which V_cmax, in particular, has been shown to be downregulated in response to elevated [CO₂] (Ainsworth and Long, 2005, and references therein). Our results, however, agree with the report of Ramalho et al. (2013), who suggested a lack of photosynthetic downregulation in coffee seedlings grown in large pots in enclosure systems. Other evidence demonstrates that photosynthetic downregulation was unlikely to have occurred in this study: (i) the photosynthetic enhancement due to elevated [CO₂] was independent of the CO₂ supply because the A values did not differ significantly between coffee trees that were grown under ambient or elevated [CO₂] when measurements were conducted at 390 or 550 µmol CO₂ mol^–1 of air (Ghini et al., 2015); and (ii) such an enhancement occurred regardless of the higher carbohydrate accumulation under elevated [CO₂].

In the early morning, when the A and g_s values are at their maxima, the A in the plants under elevated [CO₂] was at the region co-limited by Rubisco and RuBP regeneration, given that C_c was statistically equal to C_{c_trans} regardless of cultivar, as observed in the growing season (Table 1). On the one hand, this response means no investment excess in Rubisco or ETR; on the other hand, it would also imply diminishing returns in A with further increments in [CO₂] and no adjustments in g_s and g_m (Martins et al., 2014). Such a fast transition from the Rubisco-limited to the electron transport-limited phase is due to a higher affinity for CO₂ displayed by coffee's Rubisco (Martins et al., 2013, 2014), which, despite being highly advantageous under low CO₂ conditions, diminishes the benefit from elevated [CO₂] (Martins et al., 2014). In contrast, in winter, the remarkably higher diffusional limitations to A coupled with unaltered V_cmax (assuming a constant J_max/V_cmax ratio) would lead the C_c values to be in the Rubisco-limited phase, where the response of A to C_c is more prominent. This pattern, regardless of season, is also expected from midday onwards due to the low g_s values (Fig. 1) and highlights the importance of elevated [CO₂] for overcoming the high constraints to CO₂ diffusion for maximizing A in coffee.

The remarkable increases in starch levels with no detectable alteration in V_cmax in winter suggests, overall, that coffee displays a relatively high ability to accumulate starch in its leaf tissues without compromising its photosynthetic performance. Although there are a number of studies supporting direct couplings between starch accumulation and photosynthetic acclimation in response to elevated CO₂ (e.g. Eguchi et al., 2008; Dawes et al., 2013), our results agree with other studies in which uncoupling between high starch pools and photosynthetic downregulation has not been observed, as reported for fast-growing poplar clones (grown in FACE under elevated [CO₂]) displaying a combination of a high capacity for starch synthesis and a high sink demand; Davey et al., 2006; Liberloo et al., 2009). Given that starch concentrations changed paralleling an almost invariant total soluble sugar concentrations (Fig. 4), it is tempting to suggest that increased starch levels, especially under elevated [CO₂], instead of feeding back to decrease photosynthetic performance, allowed the coffee trees to avoid photosynthetic acclimation by preventing the cycling and/or accumulation of soluble sugars. When it occurs, soluble sugar accumulation could in turn more directly repress photosynthetic gene expression (Paul and Foyer, 2001; Paul and Pellny, 2003) and ultimately provoke downregulation. Nevertheless, in sharp contrast to fast-growing poplar clones, we demonstrated here a lack of photosynthetic downregulation in a tropical, slow-growing species, and this fact was observed not only during the growing season but also during the period of the lowest sink demand when acclimation would be expected. This information implies a sustained ability of coffee trees that are rooted freely in the soil to benefit from elevated [CO₂]; it additionally highlights the fact that the remarkable decreases in A in winter (Fig. 1) could not be associated with a starch build-up and concomitant photosynthetic acclimation but rather are chiefly associated with diffusional constraints regardless of the CO₂ supply.

In the growing season, there were no cultivar differences in A, but cv. Obatã displayed increased starch concentrations, which might support an improved bean-filling capacity; in winter, on the other hand, cv. Obatã displayed significantly higher A values than cv. Catuaí with similar leaf carbohydrate pools, which might be translated into a higher photoassimilate production that could be stored in the root–trunk system, guaranteeing improved vegetative growth when the environmental conditions become more conducive for growth. Overall, these facts may help to explain why cv. Obatã is better able to sustain higher vegetative growth rates and crop yields than cv. Catuaí under elevated [CO₂] (Ghini et al., 2015).

In conclusion, we demonstrated under plantation conditions that coffee photosynthesis is strongly limited by diffusional constraints, particularly at the stomata level, and that this pattern is little, if at all, affected by elevated [CO₂]. Stimulation of A by elevated [CO₂] occurred without any photosynthetic downregulation, even during the period of lowest sink demand by the crop. Given these facts, we therefore suggest that coffee will greatly benefit from the rise in atmospheric [CO₂], as will other species where photosynthesis is largely limited by CO₂ diffusion.

Acknowledgements

The authors are grateful to Embrapa (project 01.07.06.002.00: Climapest—Impacts of global climate changes on plant diseases, pests and weeds; and project 02.12.01.018.00: Impact of increased atmospheric carbon dioxide concentration and water availability on the coffee agroecosystem under the FACE facility) for financial support. This research was further supported by the Foundation for Research Assistance of Minas Gerais State, Brazil (FAPEMIG, project APQ 04784/10), granted to FMD and by the National Council for Scientific and Technological Development, Brazil (CNPq), granted to FMD (project 562157/2010-7) and RG (project 304189/2013-8). We thank the scholarships that were granted by the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES), FAPEMIG, and CNPq.

Author notes

Editor: Elizabeth Ainsworth