Understanding the Low Photosynthetic Rates of Sun and Shade Coffee Leaves: Bridging the Gap on the Relative Roles of Hydraulic, Diffusive and Biochemical Constraints to Photosynthesis

Samuel C. V. Martins; Jeroni Galmés; Paulo C. Cavatte; Lucas F. Pereira; Marília C. Ventrella; Fábio M. DaMatta

doi:10.1371/journal.pone.0095571

Abstract

It has long been held that the low photosynthetic rates (A) of coffee leaves are largely associated with diffusive constraints to photosynthesis. However, the relative limitations of the stomata and mesophyll to the overall diffusional constraints to photosynthesis, as well as the coordination of leaf hydraulics with photosynthetic limitations, remain to be fully elucidated in coffee. Whether the low actual A under ambient CO₂ concentrations is associated with the kinetic properties of Rubisco and high (photo)respiration rates also remains elusive. Here, we provide a holistic analysis to understand the causes associated with low A by measuring a variety of key anatomical/hydraulic and photosynthetic traits in sun- and shade-grown coffee plants. We demonstrate that leaf hydraulic architecture imposes a major constraint on the maximisation of the photosynthetic gas exchange of coffee leaves. Regardless of the light treatments, A was mainly limited by stomatal factors followed by similar limitations associated with the mesophyll and biochemical constraints. No evidence of an inefficient Rubisco was found; rather, we propose that coffee Rubisco is well tuned for operating at low chloroplastic CO₂ concentrations. Finally, we contend that large diffusive resistance should lead to large CO₂ drawdown from the intercellular airspaces to the sites of carboxylation, thus favouring the occurrence of relatively high photorespiration rates, which ultimately leads to further limitations to A.

Citation: Martins SCV, Galmés J, Cavatte PC, Pereira LF, Ventrella MC, DaMatta FM (2014) Understanding the Low Photosynthetic Rates of Sun and Shade Coffee Leaves: Bridging the Gap on the Relative Roles of Hydraulic, Diffusive and Biochemical Constraints to Photosynthesis. PLoS ONE 9(4): e95571. https://doi.org/10.1371/journal.pone.0095571

Editor: Gerrit T.S. Beemster, University of Antwerp, Belgium

Received: September 7, 2013; Accepted: March 28, 2014; Published: April 17, 2014

Copyright: © 2014 Martins et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by the Foundation for Research Assistance of the Minas Gerais State, Brazil (Fapemig, Grant APQ-01138-12), by the National Council for Scientific and Technological Development (CNPq) (Grants 302605/2010-0 and 475780/2012-4) to FMD, and by Plan Nacional (Spain) (GrantAGL2009-07999) to JG. A PhD scholarship granted by CNPq to SCVM is also gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Because CO₂ influx and water vapour efflux share a common pathway through the stomatal pores on leaf surfaces, a trade-off between transpirational costs and CO₂ assimilation is implicitly unavoidable. The coupling between stomatal conductance (g_s) to CO₂ and water vapour (and the need to maintain a proper leaf water balance) has often been evidenced by the strong positive scaling between g_s and the leaf hydraulic conductance per unit area, K_L [1]–[3]. In turn, the significance of K_L as a potentially limiting component of the vascular system has been further emphasised by the strong hydraulic-photosynthetic coordination observed across a large sample of diverse species [4]. Furthermore, K_L is closely related to the anatomy of the leaf: K_L has been shown to be positively related to both the theoretical axial conductivity of the midrib (determined from xylem conduit numbers and dimensions) and the venation density, D_v [3], [5]. A unified control of hydraulic and photosynthetic traits may also be further highlighted by comparing shade and sun leaves: the former have lower rates of net CO₂ assimilation (A) and g_s, therefore leading to lower demand for water and correspondingly lower K_L and D_v [4], [6].

In addition to stomatal limitations, A is currently known to be constrained by 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) [7]. According to Flexas et al. [8], g_m limitations to photosynthesis are of similar magnitude as stomatal constraints and generally greater than biochemical limitations. Increasing evidence has shown that g_m is often intrinsically co-regulated with g_s [8]. More recently, Ferrio et al. [9] showed a positive scaling between g_m and K_L and proposed that water and CO₂ share an important portion of their respective diffusion pathways through the mesophyll; thus, any downregulation of leaf hydraulics may reduce not only g_s but also g_m, both of which contribute to reducing A. Quantification of g_m has become important in predicting leaf photosynthetic parameters using the Farquhar-von Caemmerer-Berry (FvCB) model of leaf photosynthesis [10] because such a model underestimates the maximum Rubisco carboxylation rate (V_cmax) by considering g_m as being infinite [11], [12]. Furthermore, changes in C_i –C_c because of variations in g_m among species result in different A values in plants with the same biochemical activity and g_s [7].

At lower C_c, A is limited by RuBP carboxylase activity, which in turn depends on the concentration, activation and kinetic properties of Rubisco [13]. For example, Rubisco with a higher specificity factor, S_c/o (which determines the relative rates of carboxylation and oxygenation by Rubisco at given CO₂ and O₂ concentrations), could be regarded as conferring an advantage in minimising photorespiration rates, R_P [14]. Interestingly, Rubisco seems to have evolved towards higher S_c/o under stressful conditions leading to low C_c, such as drought, salinity and high temperature [14]. Additionally, species evolved under stressful conditions with sclerophyll leaves tend to display a higher S_c/o, which may again be related to lower C_c [14], [15]. However, trade-offs between S_c/o (or particularly the affinity for CO₂ (1/K_C)) and the carboxylase turnover rate (k_cat^c) have been described, so that there is no Rubisco in nature with both high affinity for CO₂ and fast activity [16]. At saturating C_c, A becomes limited by the capacity for the regeneration of RuBP (often dominated by the electron transport capacity) but also by S_c/o [17]. In any case, when studying the ecophysiology of plants, Rubisco kinetic parameters should not be considered a constant but rather as an active part determining the biochemical potential of plants to assimilate CO₂ under optimal and suboptimal conditions.

Coffee is an evergreen perennial shrub with hypostomatous leaves that evolved in the African forest understoreys and is thus considered a shade-demanding species. However, in many situations, modern coffee cultivars grow well without shade and even out-yield shaded coffee [18], [19]. At atmospheric CO₂ concentrations and saturating light, coffee displays low A, typically in the range of 4–11 µmol CO₂ m⁻² s⁻¹ [20], [21], which is in the lower range recorded for trees [22]. However, the maximum A values obtained in common A/C_i curves can surpass 20 µmol CO₂ m⁻² s⁻¹ [23], whereas the photosynthetic capacity, determined under true CO₂ saturation (∼50 mmol CO₂ mol⁻¹ air), reaches values exceeding 30 µmol O₂ m⁻² s⁻¹ [21]. Taking the above information together, the low A in coffee might largely result from diffusive constraints to photosynthesis [24]. However, the relative contributions of the stomata and mesophyll to the overall diffusional limitations to photosynthesis, as well as the coordination of leaf hydraulics with photosynthetic limitations, remain to be fully elucidated in coffee. Furthermore, the anticipated low g_s and g_m would compromise the transference of CO₂ through stomata and leaf mesophyll to Rubisco sites, therefore decreasing C_c and ultimately favouring RuBP oxygenation in relation to carboxylation [25]. Under these conditions, Rubisco kinetic properties with a high affinity for CO₂ may be critical to secure optimal carbon balance and yield.

Although the general descriptors of the ecophysiology of sun-shade plants are well-known, an integrative analysis of these descriptors has rarely been performed in conjunction (hydraulics, stomata, mesophyll, Rubisco, etc). Here, we provide a holistic analysis to understand the causes associated with low A in coffee by measuring a variety of key anatomical/hydraulic and photosynthetic traits, including D_v, K_L, the actual and maximum theoretical g_s (g_wmax) and several parameters from A/C_i and A/C_c curves. We have centred our attention on coffee given that, in addition to being a highly important commodity [18], it could also be considered as a model plant for other important crops which evolved as understorey trees, such as cacao, citrus and tea; these crops are traditionally considered to have low A (seldom above 10 µmol CO₂ m⁻² s⁻¹, even in the field under favourable conditions) likely as consequence of large diffusive, rather than biochemical, limitations to photosynthesis [26]. Our main goals were (i) to examine the role of leaf hydraulics as a prime limiting factor for photosynthetic gas exchange, (ii) to calculate g_m to properly parameterise the responses of A to C_c, and (iii) to disentangle the relative contributions of stomatal, mesophyll and biochemical limitations to photosynthesis, and how all of these facts may be affected by the light supply. A further goal was to analyse how the kinetic properties of coffee’s Rubisco [27] may affect the actual A and evaluate if Rubisco in coffee is well tuned for operating at low C_c by comparing it with the highly efficient Rubisco of Limonium gibertii; this shade-intolerant species is particularly adapted to highly stressing environments, whose Rubisco has evolved under extremely low C_c ranges [14]. We demonstrate that leaf hydraulic architecture imposes a major constraint on the maximisation of the photosynthetic gas exchange of coffee leaves and that A was mainly limited by stomatal factors followed by similar limitations associated with mesophyll and biochemical constraints, regardless of the light supply. We also found that, despite the relatively high S_c/o of coffee Rubisco, the large diffusive resistances favour the occurrence of relatively high R_P, which ultimately leads to further limitations to A.

Materials and Methods

Plant Material, Growth Conditions and Experimental Design

The experiment was conducted in Viçosa (20°45′S, 42°54′W, altitude 650 m) in southeastern Brazil. Uniform seedlings of Coffea arabica L. cv ‘Catuaí Vermelho IAC 44′ obtained from seeds were grown in 12 L pots containing a mixture of soil, sand and composted manure (4∶1∶1, v/v/v). Plants were grown either under full sunlight conditions (100% light) or under low light in a shade environment (10% full sunlight). The shade enclosure was constructed using neutral-density black nylon netting, and the plants were kept in these conditions for 12 months before measurements. Throughout the experiment, the plants were grown under naturally fluctuating conditions of temperature and relative air humidity and were fertilised and irrigated as necessary. The pots were randomised periodically to minimise any variation within each light environment. For all samplings and measurements, the youngest fully expanded leaves, corresponding to the third or fourth pair from the apex of the plagiotropic branches, were used. The experiment was arranged in a completely randomised design, with six plants in individual pots per treatment as replicates. The experimental plot included one plant per container.

Morpho-anatomical Features and Related Hydraulic and Diffusive Traits

The specific leaf area (SLA) was computed using the dry mass of 20 leaf discs (1.13 cm diameter each). For anatomical measurements, leaves were collected and fixed in FAA₇₀ for 48 h, followed by storage in 70% (v/v) aqueous ethanol. Samples of the mean region of each leaf blade were embedded in methacrylate (Historesin–Leica Microsystems Nussloch, Heidelberg, Germany) according to the manufacturer. Transverse sections (7 µm thickness), obtained using a rotary microtome (model RM2155, Leica Microsystems Inc., Deerfield, USA), were stained with toluidine blue at pH 4.0 and mounted in synthetic resin (Permount). Anatomical data were quantified using an image analysis program (Image Pro-Plus, version 4.5, Media Cybernetics, Silver Spring, USA). Video images were acquired using a video camera attached to an Olympus microscope (AX70TRF, Olympus Optical, Tokyo, Japan). The following anatomical data were then assessed: (i) total leaf thickness; (ii) palisade and spongy mesophyll thickness; (iii) upper and lower epidermis thickness; (iv) guard cell length (L); (v) vertical distance from the vein to stomatal epidermis, D_v-e; (vi) stomatal density (SD, according to DaMatta et al. [28]); (vii) stomatal index that was estimated as , where S and E are the number of stomata and epidermal cells per unit leaf area, respectively [29]; and (viii) stomatal pore index based on guard cell length (SPI_gcl) that was estimated as . For determining venation traits, samples of leaf lamina were cut from two leaves per plant and cleared as described by Scoffoni et al. [30]. Regions of approximately 6 mm² were imaged at 30X, and D_v was calculated as the sum of the vein lengths divided by the total image area using the image analysis program described above. Leaf size was estimated, using the maximum leaf widths and lengths, with the equations described by Antunes et al. [31]. Stomatal size and epidermal cell size, as well as the level of coordination between anatomical traits and leaf size (quantified as the deviation from the expected proportional relationship to each other), were all determined following Carins Murphy et al. [6].

Midrib xylem conduits were measured to determine the theoretical midrib axial hydraulic conductance (K_t), where the conduits were treated as ellipses to calculate K_t aswhere a and b are the long and short internal vessel diameters, and η is the viscosity of water at 25°C [32], further normalising by leaf length and area.

The maximum stomatal conductance to water vapour (g_wmax) was calculated according to Franks et al. [33] aswhere SD is the stomatal density, d_W is the diffusivity of water vapour in air, a is the maximum area of the open stomatal pore, v is the molar volume of air, and l is the stomatal pore depth for fully open stomata. Values for standard constants d_W and v were those for 25°C (24.9×10⁻⁶ m² s⁻¹ and 24.4×10⁻³ m³ mol⁻¹, respectively). a was calculated as , where p is the stomatal pore length, which was approximated as L/2 according to Franks and Farquhar [34]. l for fully open stomata was taken as L/4, assuming guard cells inflate to a circular cross section [33].

Leaf Hydraulic Conductance

The leaf hydraulic conductance (K_L) was estimated according to Brodribb and Holbrook [35] by following the kinetics of water potential (Ψ_l) relaxation in rehydrated leave as:where C is leaf capacitance, estimated using pressure-volume curves [36], Ψ_o is Ψ_l before rehydration, and Ψ_f is Ψ_l after rehydration for t seconds. Leaf Ψ_l was measured using a Scholander-type pressure chamber (model 1000, PMS Instruments, Albany, NY, USA).

Gas Exchange and Fluorescence Measurements

Leaf gas exchange and chlorophyll a fluorescence were measured simultaneously using an open-flow infrared gas-exchange analyser system equipped with a leaf chamber fluorometer (LI-6400XT, Li-Cor, Lincoln, NE, USA). Environmental conditions in the leaf chamber consisted of a leaf-to-air vapour pressure deficit between 1.2 and 2.0 kPa and a leaf temperature of 25°C.

In light-adapted leaves, the actual quantum yield of PSII (Φ_PSII) was determined by measuring steady-state fluorescence (F_s) and maximum fluorescence during a light-saturating pulse of c. 8,000 µmol m⁻² s⁻¹ (F_m′), following the procedures of Genty et al. [37]:

The electron transport rate (J_F) was then calculated aswhere PPFD is the photosynthetically active photon flux density, α is the leaf absorptance and β is the PSII optical cross section. The product α β was herein determined from the relationship between Φ_PSII and Φ_CO2, obtained by varying the CO₂ concentration under non-photorespiratory conditions in an atmosphere containing less than 1% O₂, as described by Valentini et al. [38].

The light respiration rate (R_L) was determined according to the ‘Laisk-method’ [39], using the y axis intersection of A/C_i (internal CO₂ concentration) curves performed at three different PPFD intensities (750, 250 and 75 µmol m⁻² s⁻¹). The rate of mitochondrial respiration at darkness (R_D) was measured early in the morning in dark-adapted leaves.

The photorespiratory rate of Rubisco (R_P) was obtained according to Valentini et al. [38] using the following equation:

Six A/C_i curves were obtained from different plants per treatment. In light-adapted leaves, A/C_i curves were initiated at an ambient CO₂ concentration (C_a) of 400 µmol mol^–1 under a saturating PPFD of 1000 µmol m^–2 s^–1. Once steady state was reached, C_a was decreased stepwise to 50 µmol mol^–1 air. Upon completion of the measurements at low C_a, C_a was returned to 400 µmol mol^–1 air to restore the original A. Next, C_a was increased stepwise to 2,000 µmol mol^–1 air. A/C_i curves consisted of 13 different C_a values.

C_c was calculated after Harley et al. [40] aswhere Г^* was determined from the in vitro Rubisco specificity factor (S_c/o) as

A was taken from gas-exchange measurements, and the J_F values were obtained from chlorophyll a fluorescence yield. After estimating C_c, g_m was calculated following Harley et al. [40]:

From the A/C_i and A/C_c curves, the maximum carboxylation capacity (V_cmax) and maximum capacity for electron transport rate (J_max) were calculated on a C_i and C_c basis using the kinetic parameters for coffee described in Martins et al. [27]. The FvCB model was fitted to the data by applying iterative curve fitting (minimum least square difference) using the Microsoft Excel Solver tool (Microsoft Corporation, Redmond, WA, USA). Additionally, g_m, V_cmax and J_max were estimated using the Ethier and Livingston [11] method, which is based on fitting A/C_i curves with a non-rectangular hyperbola version of the FvCB model, relying on the hypothesis that g_m reduces the curvature of the Rubisco-limited portion of an A/C_i response curve. Again, the kinetic parameters of Rubisco measured on coffee were used. Corrections for the leakage of CO₂ and water vapour into and out of the leaf chamber of the Li-6400-40 have been applied to all gas-exchange data, as described by Rodeghiero et al. [41].

The chloroplastic CO₂ concentration of transition (C_{c_trans}), where C_c denotes the transition between the Rubisco- and RuBP regeneration-limited states, was estimated as described by Gu et al. [42]:where K_m is the effective Michaelis–Menten constant for CO₂ that considers the competitive inhibition by O₂ which was taken from Martins et al. [27].

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 proposed by Grassi and Magnani [43]:where g_{s_CO2} is the stomatal conductance to CO₂ (), g_m is the mesophyll diffusion conductance according to Harley et al. [40] and g_tot is the total conductance to CO₂ from ambient air to chloroplasts (). ∂A/∂C_c was calculated as:

Statistical Analyses

Data are expressed as the means ± standard error. Student’s t-tests were used to compare the parameters between treatments. Additionally, one sample t-tests were performed to compare the means for shade plants with the expected values if they were proportional to the sun plants. All statistical analyses were carried out using Microsoft Excel.

Results

Sun- and shade-grown individuals differed reasonably in venation architecture and mesophyll structure (Table 1). The sun leaves, compared with shade leaves, displayed a higher leaf thickness (15%) primarily resulting from a higher thickness of palisade (43%) and spongy (14%) mesophyll, which led to a higher palisade-to-spongy parenchyma ratio (25%) and lower SLA (39%). However, the upper and lower epidermis thickness and the maximum distance from the vein to the epidermis (D_v-e) did not change in response to the light treatments. Sun-grown plants had 31% more midrib xylem conduits but with slightly lower mean conduit diameters (7%) than shade-grown plants. The sun plants displayed K_t and K_L values that were 160% and 58% higher, respectively, than in the shade individuals. Adjustments to the light treatments associated with the morphological characteristics of stomata were also observed, as denoted by higher SD (62%), stomatal index (27%) and SPI_gcl (62%) in sun leaves compared with the shade leaves; however, both the guard cell length and stomatal size did not change in response to the light treatments. Vein density was proportional to 1/√ leaf area but slightly higher (17%) than would be expected if it was directly proportional to SD (Figure 1). In line with the differences in the stomatal index, SD and epidermal cell size deviated significantly from the expected proportional relationship to leaf area: SD was 25% higher and epidermal cell size was 31% lower in shade plants than would be expected if these variables were directly proportional (Figure 1). The maximum g_s (g_wmax), determined by the stomatal size and density, was larger (61%) in sun leaves than in the shade leaves.

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Figure 1. The relationships between vein and stomatal density, vein density and 1/√ leaf area, stomatal density and 1/leaf area, and epidermal cell area and leaf area.

Data (± standard error) are shown for coffee plants grown under shade (black circles) or full sun (white circles). Asterisks denote differences (*, P<0.05; **, P<0.01) between the observed values for the shade plants and the expected values (broken line) if these were proportional to the averages for the sun plants.

https://doi.org/10.1371/journal.pone.0095571.g001

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Table 1. Anatomical and hydraulic traits of coffee plants grown under shade or full sunlight conditions.

https://doi.org/10.1371/journal.pone.0095571.t001

Under saturating PPFD (1,000 µmol photons m⁻² s⁻¹), both A and g_s values were higher (c. 55%) in sun leaves than in the shade leaves (Table 2). The values for several other photosynthetic/respiration parameters were also higher in sun leaves than in the shade leaves (Table 2): 60% for g_m (average of two independent methods), 42% for V_cmax, 45% for J_max (for V_cmax and J_max, the values express averages obtained on C_i and C_c bases), 140% for R_D, 200% for R_L, 41% for R_p and 51% for J_F. In contrast, C_i, C_c, C_{c_trans} and the ratios A/g_s, g_m/g_s and J_max/V_cmax on both C_i and C_c bases did not differ significantly in response to the light treatments. V_cmax and J_max calculated on a C_c basis were, on average, 101% and 37% higher than V_cmax and J_max, respectively, calculated on a C_i basis, whereas the J_max/V_cmax ratio was on average 32% lower when calculated on a C_c basis than on a C_i basis.

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Table 2. Mean values for the photosynthetic and respiration parameters of coffee plants grown under shade or full sunlight conditions.

https://doi.org/10.1371/journal.pone.0095571.t002

The overall photosynthetic limitations were essentially similar when comparing sun- and shade-grown plants (Table 2). We found that A was mainly constrained by stomatal limitations (c. 40%), whereas mesophyll and biochemical limitations accounted similarly for the remaining limitations (c. 30% each).

The analysis of Rubisco kinetic properties are summarised in Table 3, showing that coffee presents a Rubisco with a relatively high affinity for CO₂ (low K_c) and fast activity (k_cat^c), resulting in relatively high values for S_c/o. From the combination of in vivo V_cmax and in vitro data (k_cat^c), the concentration of active Rubisco sites was estimated to be 16 and 20 µmol m⁻² s⁻¹ for shade and sun plants, respectively.

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Table 3. Rubisco kinetic constants measured for coffee (taken from Martins et al. [27]).

https://doi.org/10.1371/journal.pone.0095571.t003

Discussion

This study provided a holistic examination of the key steps that could limit the photosynthetic capacity to fix CO₂ and demonstrated that leaf hydraulic architecture imposes a major constraint on the maximisation of the photosynthetic gas exchange of coffee leaves by limiting g_s. It is tempting to suggest that these constraints might to some extent explain the photosynthetic behaviour of other important (sub)tropical crops such as cacao, citrus and tea, which have a photosynthetic performance and a slow-growth behaviour similar to that of coffee [26]. Our results suggest that improvements of the photosynthetic performance of these crops by means of selecting hydraulic traits (e.g., D_v and K_L) that might support higher g_s values (thereby decreasing stomatal limitations of photosynthesis) could be a useful strategy to facilitate the selection of promising genotypes with enhanced crop growth and production.

We have shown that adjustments in leaf hydraulics through increases in D_v and K_L in sun-grown individuals were coordinated with a suite of traits related to water flux and gas exchange per leaf area, such as mesophyll structure (e.g., higher mesophyll thickness and palisade-to-spongy parenchyma ratio) and stomatal attributes (increased SD and stomatal pore index). Importantly, we showed that increases in K_t occurred at the expense of an increased number of midrib conduits with lower lumen in sun plants, suggesting that improvements in the hydraulic safety would not compromise the hydraulic efficiency [44]. In addition, the larger SD coupled with larger stomatal index implies a proportionally greater increase in the number of guard cells than in normal epidermal cells [45]; hence, light availability should directly and positively influence stomatal fate in coffee regardless of passive changes linked by light-induced differences in leaf blade expansion. However, differential leaf expansion was responsible for the adjustment of D_v to SD, which remained nearly proportional to each other. Thus, coffee plants can balance water supply with transpirational demand through a coordination of increased initiation of stomata cells with differential expansion of epidermal cells. Such coordination reflects an optimisation of the trade-off between transpirational costs and CO₂ assimilation, resulting in the higher intrinsic water use efficiency observed in coffee (c. 85 µmol CO₂ mol⁻¹ H₂O on average), which is markedly high compared with other tropical woody species [46], [47].

Our K_L values, determined using the method of relaxation kinetics of Ψ_l, were remarkably lower than those found by Brodribb et al. [1] for other tropical trees using the same method (between 17 and 36 mmol m⁻² s⁻¹ MPa⁻¹). Considerably low values of K_L had already been reported for C. arabica (c. 4.2 mmol m⁻² s⁻¹ MPa⁻¹) by Gascó et al. [48] using the high-pressure method. Consistent with the close relationship between K_L and D_v [4], [5], [49], our maximum D_v values were also within the low range recorded for angiosperms [50], suggesting that the hydraulic architecture of coffee leaves imposes strong resistance for water flow which, in turn, should limit the CO₂ diffusion into the leaf [4]. In addition, our K_L and D_v values (Table 1) fit very well in the general relationship of both K_L and D_v with the maximum A proposed by Brodribb et al. [5], [51], that is, our estimated K_L and D_v values exactly predict our maximum measured A values. Therefore we contend that the leaf hydraulic architecture ultimately should act as a prime factor limiting photosynthetic gas exchange in coffee leaves.

Regardless of light regimens, our actual g_s values were relatively similar to the value of 108 mmol H₂O m⁻² s⁻¹ averaged over a range of studies using coffee plants grown under optimal conditions ([10], and references therein). These values are significantly lower than those of the modelled g_wmax, resulting in a g_wmax/g_s ratio above 13, as can be calculated for sun-grown plants. By comparison, that ratio was c. 2.5 in non-water-stressed tomato [52] and 4.5 in Eucalyptus globulus [33]. The high hydraulic resistance of the coffee leaves is most likely the cause of the difference between the theoretical g_wmax and the recorded g_s. Nevertheless, this large difference raises the following questions: why would the plant invest in a large g_wmax if the maximum realisable A is relatively low and constrained by the leaf hydraulic architecture? Furthermore, from an ecological point of view, what would be the advantage of having a large g_wmax if, in the humid, shaded understoreys where coffee evolved, A should be more constrained by light limitation than by CO₂ supply? Despite not having immediate responses to these queries, our results suggest a lack of coordination between the maximum capacity for stomatal aperture and carbon fixation, as also noted in saplings of Bornean rainforest tree species grown in the understorey [53]. A large g_wmax may not be problematic in terms of water loss in the humid understorey, where transpiration rates are expected to be much more dependent on boundary layer resistance, and thus the importance of the stomata in optimising photosynthetic gas exchange should be reduced. In any case, considering that bigger stomata tend to close slower than smaller stomata [33], [54], the relatively large stomata of coffee leaves (combined with low K_L) might result in excessive leaf desiccation if large stomatal apertures are realised. In this sense, the low actual g_s might be a conservative strategy to maintain leaf hydration and minimise the risk of xylem embolism. This observation is in line with recent results obtained for Toona ciliata, where transpirational homeostasis to changes in vapour pressure deficit was achieved through dynamic stomatal control rather than modification of the relationship between veins and stomata [55]. Taken together, these findings lead to the interesting question of why long-term adjustments to the parameters that define g_smax have not been fixed and regulation of g_s comes predominantly from short-term adjustments to environmental conditions but at the cost of inherently low g_s in some species, such as coffee.

Our maximum g_m value was c. two-fold higher than those previously reported for C. arabica seedlings [56]. In any case, our g_m values were similar to those obtained for other evergreen woody species (e.g. [57]–[59]). Greater g_m values for sun leaves, as found here, have been systematically reported and have been often explained by anatomical and morphological differences between shade and sun leaves [60]. Despite the changes in g_m and A between shade- and sun-grown coffee plants, C_i and C_c remained fairly similar. Thus, regardless of the light treatments, when A changed, g_m and g_s scaled accordingly, and, hence, C_c remained constant [61]. This proportional scaling lends support to explain why stomatal, mesophyll and biochemical limitations to photosynthesis were similar between sun and shade leaves. These findings are in agreement with other studies, which show that the approximate scaling of g_s and g_m with A makes the relative limitations to photosynthesis rather conservative between sun and shade leaves, as also noted in Fagus sylvatica [62].

Irrespective of the light environment, the mean drawdown from C_i to C_c (c. 79 µmol mol⁻¹) was lower than that from C_a to C_i (c. 153 µmol mol⁻¹), which is consistent with the fact that the diffusive limitations to CO₂ in mesophyll were lower than those in stomata, thus ultimately reflecting a high g_m/g_s ratio. Indeed, by analysing the dataset reported by Flexas et al. [63], we noted that species operating at low C_i tended clearly to display an increased g_m/g_s ratio (Figure S1) which contributes to an improved performance in photosynthetic water use [63]. We believe that such feature is essential for keeping a positive carbon balance given that stomatal limitations may even be exacerbated in coffee trees grown under field conditions, particularly because g_s peaks in the early morning and decreases progressively throughout the day, reaching values typically ranging from 10 to 50 mmol H₂O m⁻² s⁻¹ from midday onwards [23], [24], [64]–[67] as a consequence of rising vapour pressure deficit.

Under saturating PPFD, A at ambient CO₂ was limited by Rubisco activity regardless of the light treatment given that the estimated C_c was lower than C_{c_trans} (Table 2). However, the realised A and J_F at maximum growth irradiance of shade plants (c. 200 µmol PPFD m⁻² s⁻¹) are c. 60% of their saturating values (Figure S2), indicating that, even though these plants operate during most of their development under light limitation, the balance between RuBP regeneration and Rubisco activity (the J_max/V_cmax ratio) was essentially similar in the shade and sun plants (Table 2). These data indicate that, in contrast to the optimal distribution principle, which suggests that Rubisco and the regeneration of RuBP should co-limit photosynthesis such that no excess capacities remain [68], the coffee plant does not properly optimise its resource allocation. On the one hand, under light saturation, an excess of electron transport capacity is to be expected, given that the actual C_c operates far from C_{c_trans} (cf. Table 2). On the other hand, under light limitation, a great investment in capacity for carbon assimilation and electron transport is retained despite the low realised A and J_F by the shade leaves.

Coffee Rubisco was characterised as displaying (i) a relatively high S_c/o, similar to that of woody evergreen species from xeric habitats [14]; (ii) a higher affinity for CO₂ (low K_c), which ranks coffee Rubisco with the third lowest value of K_c among C₃ and C₄ plants recorded to date [69]; and (iii) a relatively high k_cat^c, superior to the average of species from warm climates [70]. We next modelled the responses of A as a function of C_c by comparing these Rubisco properties with those of several C₃ species (Table S1) including Limonium gibertii, the species with the highest S_c/o and lowest K_c reported to date, thus particularly adapted to low C_c [14]. Importantly, we found that, all else being equal, the A values that would be achieved using the Rubisco kinetic properties of coffee or Limonium would be quite similar and superior to those from all other species analysed (Figure 2) suggesting that coffee presents an “efficient” Rubisco well-tuned for operating at low C_c. Additionally, the higher k_cat^c and lower K_c mean that fewer Rubisco molecules are required to realise a given A. Nevertheless, given that lower K_c leads to a reduction in C_{c_trans}, A of coffee leaves is expected to begin to be limited by RuBP regeneration at relatively low C_c, which could, to some extent, reduce the revenue stream in a scenario of increasing CO₂ atmospheric levels.

Download:

Figure 2. Comparison of the CO₂ assimilation rate as a function of C_c, modelled with the kinetic parameters of Rubisco in coffee and several C₃ species.

The RuBP saturated rates of CO₂ assimilation at 25°C were calculated using the following equation [13]: . C_c, the chloroplastic CO₂ partial pressure; O, the intercellular O₂ partial pressure; Γ*, the CO₂ compensation point in the absence of day respiration; [Rubisco], the catalytic site content of Rubisco. The Rubisco kinetic properties for coffee (Table 3) and the kinetic properties for the other species were retrieved from Savir et al. [69] and are summarized in Table S1. The R_L and [Rubisco] were set as 1.0 µmol CO₂ m⁻² s⁻¹ and 25 µmol sites m⁻², respectively.

https://doi.org/10.1371/journal.pone.0095571.g002

The rates of respiration and photorespiration are other key processes influencing the plant carbon balance. In sun plants, R_D and R_P corresponded to 10% and 38% of A, respectively. However, given that R_L represented only a fraction (25%) of R_D, which is in line with the inhibition of mitochondrial respiration in the presence of light [71], [72], the realised constraint of respiration on A is expected to be relatively low. In turn, R_P is expected to significantly affect A at midday, when the stomatal closure in coffee leaves exacerbates the drawdown from C_a to C_i [24], thus favouring the oxygenase activity of Rubisco.

Conclusion

Regardless of the light treatments, A was mainly limited by stomatal factors followed by similar limitations associated with the mesophyll and biochemical constraints. Our data suggest that an increased g_m/g_s ratio coupled with a Rubisco well-tuned for operating at low C_c might be adaptations to a lower C_i resulting from the low g_s; these adaptations might have helped the establishment of coffee plants when they were moved from the understoreys to the more stressful conditions of open fields, where high vapour pressure deficit and elevated temperatures may constrain gas exchange. Our results also suggest that the coffee plant does not properly optimise its resource allocation: on the one hand, there seems to be an over-investment in capacity for carboxylation and electron transport despite limited light availability under shade; on the other hand, an excess of electron transport capacity is to be expected under full sun. Nevertheless, this excess might be useful in attempts to increase A in coffee through breeding aimed at improving hydraulic traits (D_v and K_L) and expectedly supporting a high g_s. Finally, we contend that the large diffusive resistance should lead to large drawdown from C_a to C_c, thus favouring the occurrence of relatively high R_p despite the relatively high S_c/o of coffee Rubisco, which ultimately leads to further limitations to A.

Supporting Information

Figure S1.

The relationship between sub-stomatal CO₂ concentration and mesophyll-to-stomatal conductance ratio in the multi-species dataset from Flexas et al. [63].

https://doi.org/10.1371/journal.pone.0095571.s001

(TIF)

Figure S2.

The response of net photosynthetic rate and electron transport rate to the photosynthetic photon flux density in coffee plants developed under shade or full sun.

https://doi.org/10.1371/journal.pone.0095571.s002

(TIF)

Table S1.

The Rubisco kinetic constants for several C₃ species.

https://doi.org/10.1371/journal.pone.0095571.s003

(DOCX)

Author Contributions

Conceived and designed the experiments: SCVM JG FMD. Performed the experiments: SCVM PCC LFP MCV. Analyzed the data: SCVM JG FMD. Contributed reagents/materials/analysis tools: FMD. Wrote the paper: SCVM JG FMD.

References

1. Brodribb TJ, Holbrook NM, Zwieniecki MA, Palma B (2005) Leaf hydraulic capacity in ferns, conifers and angiosperms: impacts on photosynthetic maxima. New Phytol 165: 839–846.
- View Article
- Google Scholar
2. Sack L, Cowan PD, Jaikumar N, Holbrook NM (2003) The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell Environ 26: 1343–1356.
- View Article
- Google Scholar
3. Sack L, Frole K (2006) Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology 87: 483–491.
- View Article
- Google Scholar
4. Brodribb TJ (2009) Xylem hydraulic physiology: The functional backbone of terrestrial plant productivity. Plant Sci 177: 245–251.
- View Article
- Google Scholar
5. Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiol 144: 1890–1898.
- View Article
- Google Scholar
6. Carins Murphy MR, Jordan GJ, Brodribb TJ (2012) Differential leaf expansion can enable hydraulic acclimation to sun and shade. Plant Cell Environ 35: 1407–1418.
- View Article
- Google Scholar
7. Warren CR (2008) Stand aside stomata, another actor deserves centre stage: the forgotten role of the internal conductance to CO₂ transfer. J Exp Bot 59: 1475–1487.
- View Article
- Google Scholar
8. Flexas J, Barbour MM, Brendel O, Cabrera HM, Carriquí M, et al. (2012) Mesophyll diffusion conductance to CO₂: an unappreciated central player in photosynthesis. Plant Sci 193–194: 70–84.
- View Article
- Google Scholar
9. Ferrio JP, Pou A, Florez-Sarasa I, Gessler A, Kodama N, et al. (2012) The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO₂. Plant Cell Environ 35: 611–625.
- View Article
- Google Scholar
10. Farquhar GD, von Caemmerer S, Berry JA (1980) A biochemical model of photosynthetic CO₂ assimilation in leaves of C3 species. Planta 149: 78–90.
- View Article
- Google Scholar
11. Ethier GJ, Livingston NJ (2004) On the need to incorporate sensitivity to CO₂ transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model. Plant Cell Environ 27: 137–153.
- View Article
- Google Scholar
12. Niinemets Ü, Díaz-Espejo A, Flexas J, Galmés J, Warren CR (2009) Role of mesophyll diffusion conductance in constraining potential photosynthetic productivity in the field. J Exp Bot 60: 2249–2270.
- View Article
- Google Scholar
13. von Caemmerer S (2000) Biochemical models of leaf photosynthesis. CSIRO Publishing: Canberra.
14. Galmés J, Flexas J, Keys AJ, Cifre J, Mitchell RAC, et al. (2005) Rubisco specificity factor tends to be larger in plant species from drier habitats and in species with persistent leaves. Plant Cell Environ 28: 571–579.
- View Article
- Google Scholar
15. Galmés J, Conesa MAN, Ochogavía JM, Perdomo JA, Francis DM, et al. (2011) Physiological and morphological adaptations in relation to water use efficiency in Mediterranean accessions of Solanum lycopersicum. Plant Cell Environ 34: 245–260.
- View Article
- Google Scholar
16. Tcherkez GGB, Farquhar GD, Andrews TJ (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Natl Acad Sci USA 103: 7246–7251.
- View Article
- Google Scholar
17. Farquhar GD, von Caemmerer S (1982) Modeling of photosynthetic responses to environmental conditions. In: Lange OL, Nobel PS, Osmond CB, Ziegler H, editors. Encyclopedia of plant physiology (New Series). Springer-Verlag: Berlin, 549–587.
18. DaMatta FM, Ronchi CP, Maestri M, Barros RS (2010) Coffee: environment and crop physiology. In: DaMatta FM, editor. Ecophysiology of tropical tree crops. Nova Science Publishers: New York, 181–216.
19. DaMatta FM (2004) Ecophysiological constraints on the production of shaded and unshaded coffee: a review. Field Crops Res 86: 99–114.
- View Article
- Google Scholar
20. Franck N, Vaast P, Génard M, Dauzat J (2006) Soluble sugars mediate sink feedback down-regulation of leaf photosynthesis in field-grown Coffea arabica.. Tree Physiol 26: 517–525.
- View Article
- Google Scholar
21. Silva EA, DaMatta FM, Ducatti C, Regazzi AJ, Barros RS (2004) Seasonal changes in vegetative growth and photosynthesis of arabica coffee trees. Field Crops Res 89: 349–357.
- View Article
- Google Scholar
22. Ceulemans R, Saugier B (1993) Photosynthesis. In: Raghavendra AS, editor. Physiology of trees. John Wiley: New York, 21–50.
23. Araújo WL, Dias PC, Moraes GABK, Celin EF, Cunha RL, et al. (2008) Limitations to photosynthesis in coffee leaves from different canopy positions. Plant Physiol Biochem 46: 884–890.
- View Article
- Google Scholar
24. Batista D, Araújo WL, Antunes WC, Cavatte PC, Moraes GABK, et al. (2012) Photosynthetic limitations in coffee plants are chiefly governed by diffusive factors. Trees 26: 459–468.
- View Article
- Google Scholar
25. Galmés J, Medrano H, Flexas J (2007) Photosynthetic limitations in response to water stress and recovery in Mediterranean plants with different growth forms. New Phytol 175: 81–93.
- View Article
- Google Scholar
26. DaMatta FM (2010) Introduction. In: DaMatta FM, editor. Ecophysiology of tropical tree crops. Nova Science Publishers: New York, 1–6.
27. Martins SCV, Galmés JG, Molins A, DaMatta FM (2013) Improving the estimation of mesophyll conductance: on the role of electron transport rate correction and respiration. J Exp Bot 64: 3285–3298.
- View Article
- Google Scholar
28. DaMatta FM, Maestri M, Barros RS (1997) Photosynthetic performance of two coffee species under drought. Photosynthetica 34: 257–264.
- View Article
- Google Scholar
29. Salisbury EJ (1927) On the causes and ecological significance of stomatal frequency with special reference to the woodland flora. Philos Trans R Soc Lond B Biol Sci 216: 1–65.
- View Article
- Google Scholar
30. Scoffoni C, Rawls M, McKown A, Cochard H, Sack L (2011) Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture. Plant Physiol 156: 832–843.
- View Article
- Google Scholar
31. Antunes WC, Pompelli MF, Carretero DM, DaMatta FM (2008) Allometric models for non-destructive leaf area estimation in coffee (Coffea arabica and C. canephora). Ann Appl Biol 153: 33–40.
- View Article
- Google Scholar
32. Lewis AM, Boose ER (1995) Estimating volume flow rates through xylem conduits. Am J Bot 82: 1112–1116.
- View Article
- Google Scholar
33. Franks PJ, Drake PL, Beerling DJ (2009) Plasticity in maximum stomatal conductance constrained by negative correlation between stomatal size and density: an analysis using Eucalyptus globules. Plant Cell Environ 32: 1737–1748.
- View Article
- Google Scholar
34. Franks P, Farquhar G (2001) The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana. Plant Physiol 125: 935–942.
- View Article
- Google Scholar
35. Brodribb TJ, Holbrook MN (2003) Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiol 132: 2166–2173.
- View Article
- Google Scholar
36. Cavatte PC, Oliveira AAG, Morais LE, Martins SCV, Sanglard LMVP, et al. (2012) Could shading reduce the negative impacts of drought on coffee? A morphophysiological analysis. Physiol Plant144: 111–122.
- View Article
- Google Scholar
37. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87–92.
- View Article
- Google Scholar
38. Valentini R, Epron D, Angelis P, Matteucci G, Dreyer E (1995) In situ estimation of net CO₂ assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Q. cerris L.) leaves: diurnal cycles under different levels of water supply. Plant Cell Environ 18: 631–640.
- View Article
- Google Scholar
39. Laisk AK (1977) Kinetics of photosynthesis and photorespiration in C3 plants. Nauka: Moscow.
40. Harley PC, Loreto F, Di Marco G, Sharkey TD (1992) Theoretical considerations when estimating the mesophyll conductance to CO₂ flux by analysis of the response of photosynthesis to CO₂. Plant Physiol 98: 1429–1436.
- View Article
- Google Scholar
41. Rodeghiero M, Niinemets Ü, Cescatti A (2007) Major diffusion leaks of clamp-on leaf cuvettes still unaccounted: how erroneous are the estimates of Farquhar et al. model parameters? Plant Cell Environ 30: 1006–1022.
- View Article
- Google Scholar
42. Gu L, Pallardy SG, Tu K, Law BE, Wullschleger SD (2010) Reliable estimation of biochemical parameters from C₃ leaf photosynthesis-intercellular carbon dioxide response curves. Plant Cell Environ 33: 1852–1874.
- View Article
- Google Scholar
43. Grassi G, Magnani F (2005) Stomatal, mesophyll conductance and biochemical limitations to photosynthesis as affected by drought and leaf ontogeny in ash and oak trees. Plant Cell Environ 28: 834–849.
- View Article
- Google Scholar
44. Sack L, Holbrook MN (2006) Leaf hydraulics. Annu Rev Plant Biol 57: 361–381.
- View Article
- Google Scholar
45. Casson SA, Franklin KA, Gray JE, Grierson CS, Whitelam GC, et al. (2009) Phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr Biol 19: 229–234.
- View Article
- Google Scholar
46. Chaturvedi RK, Raghubanshi AS, Singh JS (2011) Leaf attributes and tree growth in a tropical dry forest. J Veg Sci 22: 917–931.
- View Article
- Google Scholar
47. Nogueira A, Martinez CA, Ferreira LL, Prado CHBA (2004) Photosynthesis and water use efficiency in twenty tropical tree species of differing succession status in a Brazilian reforestation. Photosynthetica 42: 351–356.
- View Article
- Google Scholar
48. Gascó A, Nardini A, Salleo S (2004) Resistance to water flow through leaves of Coffea arabica is dominated by extra-vascular tissues. Funct Plant Biol 31: 1161–1168.
- View Article
- Google Scholar
49. Sack L, Tyree MT, Holbrook NM (2005) Leaf hydraulic architecture correlates with regeneration irradiance in tropical rainforest trees. New Phytol 167: 403–413.
- View Article
- Google Scholar
50. Boyce CK, Lee J-E, Field TS, Brodribb TJ, Zwieniecki MA (2010) Angiosperms helped put the rain in the rainforests: The impact of plant physiological evolution on tropical biodiversity. Ann Missouri Bot Gard 97: 527–540.
- View Article
- Google Scholar
51. Brodribb TJ, Feild TS, Sack L (2010) Viewing leaf structure and evolution from a hydraulic perspective. Funct Plant Biol 37: 488–498.
- View Article
- Google Scholar
52. Galmés J, Ochogavía JM, Gago J, Roldán EJ, Cifre J, et al. (2013) Leaf responses to drought stress in Mediterranean accessions of Solanum lycopersicum: anatomical adaptations in relation to gas exchange parameters. Plant Cell Environ 36: 920–935.
- View Article
- Google Scholar
53. Russo SE, Cannon WL, ElowskyC, Tan S, Davies SJ (2010) Variation in leaf stomatal traits of 28 tree species in relation to gas exchange along an edaphic gradient in a Bornean rain forest. Am J Bot 97: 1109–1120.
- View Article
- Google Scholar
54. Eensalu E, Kupper P, Sellin A, Rahi M, Sõber A, et al. (2010) Do stomata operate at the same relative opening range along a canopy profile of Betula pendula? Funct Plant Biol 35: 103–110.
- View Article
- Google Scholar
55. Carins Murphy MR, Jordan GJ, Brodribb TJ (2013) Acclimation to humidity modifies the link between leaf size and the density of veins and stomata. Plant Cell Environ 37: 124–131.
- View Article
- Google Scholar
56. Hanba Y, Kogami H, Terashima I (2003) The effect of internal CO₂ conductance on leaf carbon isotope ratio. Isotopes Environ Health Studies 39: 5–13.
- View Article
- Google Scholar
57. Manter DK, Kerrigan J (2004) A/C_i curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance. J Exp Bot 55: 2581–2588.
- View Article
- Google Scholar
58. Warren CR, Adams MA (2006) Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use in photosynthesis. Plant Cell Environ 29: 192–201.
- View Article
- Google Scholar
59. Flexas J, Ribas-Carbó M, Díaz-Espejo A, Galmés J, Medrano H (2008) Mesophyll conductance to CO₂: current knowledge and future prospects. Plant Cell Environ 31: 602–621.
- View Article
- Google Scholar
60. Terashima I, Hanba YT, Tholen D, Niinemets Ü (2011) Leaf functional anatomy in relation to photosynthesis. Plant Physiol 155: 108–116.
- View Article
- Google Scholar
61. Monti A, Bezzi G, Venturi G (2009) Internal conductance under different light conditions along the plant profile of Ethiopian mustard (Brassica carinata A. Brown.). J Exp Bot 60: 2341–2350.
- View Article
- Google Scholar
62. Warren CR, Löw M, Matyssek R, Tausz M (2007) Internal conductance to CO₂ transfer of adult Fagus sylvatica: Variation between sun and shade leaves and due to free-air ozone fumigation. Environ Exp Bot 59: 130–138.
- View Article
- Google Scholar
63. Flexas J, Niinemets Ü, Gallé A, Baubour MM, Centritto M, et al. (2013) Diffusional conductances to CO₂ as a target for increasing photosynthesis and photosynthetic water-use efficiency. Photosynth Res 117: 45–59.
- View Article
- Google Scholar
64. Ronquim JC, Prado CHBA, Novaes P, Fahl JI, Ronquim CC (2006) Carbon gain in Coffea arabica during clear and cloudy days in the wet season. Exp Agric 42: 147–164.
- View Article
- Google Scholar
65. Chaves ARM, Ten-Caten A, Pinheiro HA, Ribeiro A, DaMatta FM (2008) Seasonal changes in photoprotective mechanisms of leaves from shaded and unshaded field-grown coffee (Coffea arabica L.). Trees 22: 351–361.
- View Article
- Google Scholar
66. Chaves ARM, Martins SCV, Batista KD, Celin EF, DaMatta FM (2012) Varying leaf-to-fruit ratios affect branch growth and dieback, with little to no effect on photosynthesis, carbohydrate or mineral pools, in different canopy positions of field-grown coffee trees. Environ Exp Bot 77: 207–218.
- View Article
- Google Scholar
67. DaMatta FM, Cunha RL, Antunes WC, Martins SVC, Araújo WL, et al. (2008) In field-grown coffee trees source-sink manipulation alters photosynthetic rates, independently of carbon metabolism, via alterations in stomatal function. New Phytol 178: 348–357.
- View Article
- Google Scholar
68. Niinemets Ü, Kull O, Tenhunen JD (1998) An analysis of light effects on foliar morphology, physiology, and light interception in temperate deciduous woody species of contrasting shade tolerance. Tree Physiol 18: 681–696.
- View Article
- Google Scholar
69. Savir Y, Noor E, Milo R, Tlusty T (2010) Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape. Proc Natl Acad Sci USA 107: 3475–3480.
- View Article
- Google Scholar
70. Sage RF, Cen Y, Li M (2002) The activation state of Rubisco directly limits photosynthesis at low CO₂ and low O₂ partial pressures. Photosynth Res 71: 241–250.
- View Article
- Google Scholar
71. Tcherkez G, Cornic G, Bligny R, Gout E, Ghashghaie J (2005) In vivo respiratory metabolism of illuminated leaves. Plant Physiol 138: 1596–1606.
- View Article
- Google Scholar
72. Tcherkez G, Bligny R, Gout E, Mahé A, Hodges M, et al. (2008) Respiratory metabolism of illuminated leaves depends on CO₂ and O₂ conditions. Proc Natl Acad Sci USA 105: 797–802.
- View Article
- Google Scholar

[ref1] 1. Brodribb TJ, Holbrook NM, Zwieniecki MA, Palma B (2005) Leaf hydraulic capacity in ferns, conifers and angiosperms: impacts on photosynthetic maxima. New Phytol 165: 839–846.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Sack L, Cowan PD, Jaikumar N, Holbrook NM (2003) The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell Environ 26: 1343–1356.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Sack L, Frole K (2006) Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology 87: 483–491.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Brodribb TJ (2009) Xylem hydraulic physiology: The functional backbone of terrestrial plant productivity. Plant Sci 177: 245–251.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiol 144: 1890–1898.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Carins Murphy MR, Jordan GJ, Brodribb TJ (2012) Differential leaf expansion can enable hydraulic acclimation to sun and shade. Plant Cell Environ 35: 1407–1418.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Warren CR (2008) Stand aside stomata, another actor deserves centre stage: the forgotten role of the internal conductance to CO₂ transfer. J Exp Bot 59: 1475–1487.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Flexas J, Barbour MM, Brendel O, Cabrera HM, Carriquí M, et al. (2012) Mesophyll diffusion conductance to CO₂: an unappreciated central player in photosynthesis. Plant Sci 193–194: 70–84.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Ferrio JP, Pou A, Florez-Sarasa I, Gessler A, Kodama N, et al. (2012) The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO₂. Plant Cell Environ 35: 611–625.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Farquhar GD, von Caemmerer S, Berry JA (1980) A biochemical model of photosynthetic CO₂ assimilation in leaves of C3 species. Planta 149: 78–90.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref11] 11. Ethier GJ, Livingston NJ (2004) On the need to incorporate sensitivity to CO₂ transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model. Plant Cell Environ 27: 137–153.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref12] 12. Niinemets Ü, Díaz-Espejo A, Flexas J, Galmés J, Warren CR (2009) Role of mesophyll diffusion conductance in constraining potential photosynthetic productivity in the field. J Exp Bot 60: 2249–2270.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref13] 13. von Caemmerer S (2000) Biochemical models of leaf photosynthesis. CSIRO Publishing: Canberra.

[ref14] 14. Galmés J, Flexas J, Keys AJ, Cifre J, Mitchell RAC, et al. (2005) Rubisco specificity factor tends to be larger in plant species from drier habitats and in species with persistent leaves. Plant Cell Environ 28: 571–579.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref15] 15. Galmés J, Conesa MAN, Ochogavía JM, Perdomo JA, Francis DM, et al. (2011) Physiological and morphological adaptations in relation to water use efficiency in Mediterranean accessions of Solanum lycopersicum. Plant Cell Environ 34: 245–260.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref16] 16. Tcherkez GGB, Farquhar GD, Andrews TJ (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Natl Acad Sci USA 103: 7246–7251.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Farquhar GD, von Caemmerer S (1982) Modeling of photosynthetic responses to environmental conditions. In: Lange OL, Nobel PS, Osmond CB, Ziegler H, editors. Encyclopedia of plant physiology (New Series). Springer-Verlag: Berlin, 549–587.

[ref18] 18. DaMatta FM, Ronchi CP, Maestri M, Barros RS (2010) Coffee: environment and crop physiology. In: DaMatta FM, editor. Ecophysiology of tropical tree crops. Nova Science Publishers: New York, 181–216.

[ref19] 19. DaMatta FM (2004) Ecophysiological constraints on the production of shaded and unshaded coffee: a review. Field Crops Res 86: 99–114.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref20] 20. Franck N, Vaast P, Génard M, Dauzat J (2006) Soluble sugars mediate sink feedback down-regulation of leaf photosynthesis in field-grown Coffea arabica.. Tree Physiol 26: 517–525.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref21] 21. Silva EA, DaMatta FM, Ducatti C, Regazzi AJ, Barros RS (2004) Seasonal changes in vegetative growth and photosynthesis of arabica coffee trees. Field Crops Res 89: 349–357.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref22] 22. Ceulemans R, Saugier B (1993) Photosynthesis. In: Raghavendra AS, editor. Physiology of trees. John Wiley: New York, 21–50.

[ref23] 23. Araújo WL, Dias PC, Moraes GABK, Celin EF, Cunha RL, et al. (2008) Limitations to photosynthesis in coffee leaves from different canopy positions. Plant Physiol Biochem 46: 884–890.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref24] 24. Batista D, Araújo WL, Antunes WC, Cavatte PC, Moraes GABK, et al. (2012) Photosynthetic limitations in coffee plants are chiefly governed by diffusive factors. Trees 26: 459–468.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref25] 25. Galmés J, Medrano H, Flexas J (2007) Photosynthetic limitations in response to water stress and recovery in Mediterranean plants with different growth forms. New Phytol 175: 81–93.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref26] 26. DaMatta FM (2010) Introduction. In: DaMatta FM, editor. Ecophysiology of tropical tree crops. Nova Science Publishers: New York, 1–6.

[ref27] 27. Martins SCV, Galmés JG, Molins A, DaMatta FM (2013) Improving the estimation of mesophyll conductance: on the role of electron transport rate correction and respiration. J Exp Bot 64: 3285–3298.
View Article
Google Scholar

[70] View Article

[71] Google Scholar

[ref28] 28. DaMatta FM, Maestri M, Barros RS (1997) Photosynthetic performance of two coffee species under drought. Photosynthetica 34: 257–264.
View Article
Google Scholar

[73] View Article

[74] Google Scholar

[ref29] 29. Salisbury EJ (1927) On the causes and ecological significance of stomatal frequency with special reference to the woodland flora. Philos Trans R Soc Lond B Biol Sci 216: 1–65.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref30] 30. Scoffoni C, Rawls M, McKown A, Cochard H, Sack L (2011) Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture. Plant Physiol 156: 832–843.
View Article
Google Scholar

[79] View Article

[80] Google Scholar

[ref31] 31. Antunes WC, Pompelli MF, Carretero DM, DaMatta FM (2008) Allometric models for non-destructive leaf area estimation in coffee (Coffea arabica and C. canephora). Ann Appl Biol 153: 33–40.
View Article
Google Scholar

[82] View Article

[83] Google Scholar

[ref32] 32. Lewis AM, Boose ER (1995) Estimating volume flow rates through xylem conduits. Am J Bot 82: 1112–1116.
View Article
Google Scholar

[85] View Article

[86] Google Scholar

[ref33] 33. Franks PJ, Drake PL, Beerling DJ (2009) Plasticity in maximum stomatal conductance constrained by negative correlation between stomatal size and density: an analysis using Eucalyptus globules. Plant Cell Environ 32: 1737–1748.
View Article
Google Scholar

[88] View Article

[89] Google Scholar

[ref34] 34. Franks P, Farquhar G (2001) The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana. Plant Physiol 125: 935–942.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref35] 35. Brodribb TJ, Holbrook MN (2003) Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiol 132: 2166–2173.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref36] 36. Cavatte PC, Oliveira AAG, Morais LE, Martins SCV, Sanglard LMVP, et al. (2012) Could shading reduce the negative impacts of drought on coffee? A morphophysiological analysis. Physiol Plant144: 111–122.
View Article
Google Scholar

[97] View Article

[98] Google Scholar

[ref37] 37. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87–92.
View Article
Google Scholar

[100] View Article

[101] Google Scholar

[ref38] 38. Valentini R, Epron D, Angelis P, Matteucci G, Dreyer E (1995) In situ estimation of net CO₂ assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Q. cerris L.) leaves: diurnal cycles under different levels of water supply. Plant Cell Environ 18: 631–640.
View Article
Google Scholar

[103] View Article

[104] Google Scholar

[ref39] 39. Laisk AK (1977) Kinetics of photosynthesis and photorespiration in C3 plants. Nauka: Moscow.

[ref40] 40. Harley PC, Loreto F, Di Marco G, Sharkey TD (1992) Theoretical considerations when estimating the mesophyll conductance to CO₂ flux by analysis of the response of photosynthesis to CO₂. Plant Physiol 98: 1429–1436.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref41] 41. Rodeghiero M, Niinemets Ü, Cescatti A (2007) Major diffusion leaks of clamp-on leaf cuvettes still unaccounted: how erroneous are the estimates of Farquhar et al. model parameters? Plant Cell Environ 30: 1006–1022.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref42] 42. Gu L, Pallardy SG, Tu K, Law BE, Wullschleger SD (2010) Reliable estimation of biochemical parameters from C₃ leaf photosynthesis-intercellular carbon dioxide response curves. Plant Cell Environ 33: 1852–1874.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref43] 43. Grassi G, Magnani F (2005) Stomatal, mesophyll conductance and biochemical limitations to photosynthesis as affected by drought and leaf ontogeny in ash and oak trees. Plant Cell Environ 28: 834–849.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref44] 44. Sack L, Holbrook MN (2006) Leaf hydraulics. Annu Rev Plant Biol 57: 361–381.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref45] 45. Casson SA, Franklin KA, Gray JE, Grierson CS, Whitelam GC, et al. (2009) Phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr Biol 19: 229–234.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref46] 46. Chaturvedi RK, Raghubanshi AS, Singh JS (2011) Leaf attributes and tree growth in a tropical dry forest. J Veg Sci 22: 917–931.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref47] 47. Nogueira A, Martinez CA, Ferreira LL, Prado CHBA (2004) Photosynthesis and water use efficiency in twenty tropical tree species of differing succession status in a Brazilian reforestation. Photosynthetica 42: 351–356.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref48] 48. Gascó A, Nardini A, Salleo S (2004) Resistance to water flow through leaves of Coffea arabica is dominated by extra-vascular tissues. Funct Plant Biol 31: 1161–1168.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref49] 49. Sack L, Tyree MT, Holbrook NM (2005) Leaf hydraulic architecture correlates with regeneration irradiance in tropical rainforest trees. New Phytol 167: 403–413.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref50] 50. Boyce CK, Lee J-E, Field TS, Brodribb TJ, Zwieniecki MA (2010) Angiosperms helped put the rain in the rainforests: The impact of plant physiological evolution on tropical biodiversity. Ann Missouri Bot Gard 97: 527–540.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref51] 51. Brodribb TJ, Feild TS, Sack L (2010) Viewing leaf structure and evolution from a hydraulic perspective. Funct Plant Biol 37: 488–498.
View Article
Google Scholar

[140] View Article

[141] Google Scholar

[ref52] 52. Galmés J, Ochogavía JM, Gago J, Roldán EJ, Cifre J, et al. (2013) Leaf responses to drought stress in Mediterranean accessions of Solanum lycopersicum: anatomical adaptations in relation to gas exchange parameters. Plant Cell Environ 36: 920–935.
View Article
Google Scholar

[143] View Article

[144] Google Scholar

[ref53] 53. Russo SE, Cannon WL, ElowskyC, Tan S, Davies SJ (2010) Variation in leaf stomatal traits of 28 tree species in relation to gas exchange along an edaphic gradient in a Bornean rain forest. Am J Bot 97: 1109–1120.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref54] 54. Eensalu E, Kupper P, Sellin A, Rahi M, Sõber A, et al. (2010) Do stomata operate at the same relative opening range along a canopy profile of Betula pendula? Funct Plant Biol 35: 103–110.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref55] 55. Carins Murphy MR, Jordan GJ, Brodribb TJ (2013) Acclimation to humidity modifies the link between leaf size and the density of veins and stomata. Plant Cell Environ 37: 124–131.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref56] 56. Hanba Y, Kogami H, Terashima I (2003) The effect of internal CO₂ conductance on leaf carbon isotope ratio. Isotopes Environ Health Studies 39: 5–13.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref57] 57. Manter DK, Kerrigan J (2004) A/C_i curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance. J Exp Bot 55: 2581–2588.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref58] 58. Warren CR, Adams MA (2006) Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use in photosynthesis. Plant Cell Environ 29: 192–201.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref59] 59. Flexas J, Ribas-Carbó M, Díaz-Espejo A, Galmés J, Medrano H (2008) Mesophyll conductance to CO₂: current knowledge and future prospects. Plant Cell Environ 31: 602–621.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref60] 60. Terashima I, Hanba YT, Tholen D, Niinemets Ü (2011) Leaf functional anatomy in relation to photosynthesis. Plant Physiol 155: 108–116.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref61] 61. Monti A, Bezzi G, Venturi G (2009) Internal conductance under different light conditions along the plant profile of Ethiopian mustard (Brassica carinata A. Brown.). J Exp Bot 60: 2341–2350.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref62] 62. Warren CR, Löw M, Matyssek R, Tausz M (2007) Internal conductance to CO₂ transfer of adult Fagus sylvatica: Variation between sun and shade leaves and due to free-air ozone fumigation. Environ Exp Bot 59: 130–138.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref63] 63. Flexas J, Niinemets Ü, Gallé A, Baubour MM, Centritto M, et al. (2013) Diffusional conductances to CO₂ as a target for increasing photosynthesis and photosynthetic water-use efficiency. Photosynth Res 117: 45–59.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref64] 64. Ronquim JC, Prado CHBA, Novaes P, Fahl JI, Ronquim CC (2006) Carbon gain in Coffea arabica during clear and cloudy days in the wet season. Exp Agric 42: 147–164.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

[ref65] 65. Chaves ARM, Ten-Caten A, Pinheiro HA, Ribeiro A, DaMatta FM (2008) Seasonal changes in photoprotective mechanisms of leaves from shaded and unshaded field-grown coffee (Coffea arabica L.). Trees 22: 351–361.
View Article
Google Scholar

[182] View Article

[183] Google Scholar

[ref66] 66. Chaves ARM, Martins SCV, Batista KD, Celin EF, DaMatta FM (2012) Varying leaf-to-fruit ratios affect branch growth and dieback, with little to no effect on photosynthesis, carbohydrate or mineral pools, in different canopy positions of field-grown coffee trees. Environ Exp Bot 77: 207–218.
View Article
Google Scholar

[185] View Article

[186] Google Scholar

[ref67] 67. DaMatta FM, Cunha RL, Antunes WC, Martins SVC, Araújo WL, et al. (2008) In field-grown coffee trees source-sink manipulation alters photosynthetic rates, independently of carbon metabolism, via alterations in stomatal function. New Phytol 178: 348–357.
View Article
Google Scholar

[188] View Article

[189] Google Scholar

[ref68] 68. Niinemets Ü, Kull O, Tenhunen JD (1998) An analysis of light effects on foliar morphology, physiology, and light interception in temperate deciduous woody species of contrasting shade tolerance. Tree Physiol 18: 681–696.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

[ref69] 69. Savir Y, Noor E, Milo R, Tlusty T (2010) Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape. Proc Natl Acad Sci USA 107: 3475–3480.
View Article
Google Scholar

[194] View Article

[195] Google Scholar

[ref70] 70. Sage RF, Cen Y, Li M (2002) The activation state of Rubisco directly limits photosynthesis at low CO₂ and low O₂ partial pressures. Photosynth Res 71: 241–250.
View Article
Google Scholar

[197] View Article

[198] Google Scholar

[ref71] 71. Tcherkez G, Cornic G, Bligny R, Gout E, Ghashghaie J (2005) In vivo respiratory metabolism of illuminated leaves. Plant Physiol 138: 1596–1606.
View Article
Google Scholar

[200] View Article

[201] Google Scholar

[ref72] 72. Tcherkez G, Bligny R, Gout E, Mahé A, Hodges M, et al. (2008) Respiratory metabolism of illuminated leaves depends on CO₂ and O₂ conditions. Proc Natl Acad Sci USA 105: 797–802.
View Article
Google Scholar

[203] View Article

[204] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Plant Material, Growth Conditions and Experimental Design

Morpho-anatomical Features and Related Hydraulic and Diffusive Traits

Leaf Hydraulic Conductance

Gas Exchange and Fluorescence Measurements

Statistical Analyses

Results

Discussion

Conclusion

Supporting Information

Figure S1.

Figure S2.

Table S1.

Author Contributions

References