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Environmental and Experimental Botany 87 (2013) 49– 57

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h omepa g e: www.elsev ier .com/ locate /envexpbot

The functional divergence of biomass partitioning, carbon gain and water use inCoffea canephora in response to the water supply: Implications for breedingaimed at improving drought tolerance

Paulo E.M. Silva, Paulo C. Cavatte, Leandro E. Morais, Eduardo F. Medina, Fábio M. DaMatta ∗

Departamento de Biologia Vegetal, Universidade Federal de Vic osa, 36570-000 Vic osa, MG, Brazil

a r t i c l e i n f o

Article history:Received 3 June 2012Received in revised form12 September 2012Accepted 13 September 2012

Keywords:BreedingCoffeeDrought tolerancePhotosynthesisWater deficit

a b s t r a c t

Robusta coffee (Coffea canephora) is widely cultivated in regions where water availability is the majorenvironmental constraint affecting crop production. The functional divergence associated with biomasspartitioning, carbon gain and water use in response to water supply was examined in 10 one-year-oldclones of robusta coffee with varying degrees of drought tolerance. The plants were grown outdoors in 24 Lpots and either irrigated or subjected to a four-month water deficit. Under conditions of ample irrigation,clones with superior water use ability (i.e., a higher water potential, transpiration rate, apparent hydraulicconductance and biomass partitioning into roots and a lower wood density) displayed enhanced carbongains. In contrast, under drought conditions, clones that postponed dehydration via more conservativewater use rates showed lower relative decreases in stomatal conductance, photosynthetic rates andbiomass accumulation. Isotopic signatures (�13C) might be useful for identifying clones with improvedperformance under drought conditions. Our results suggest that combining useful morphological andphysiological traits facilitates the successful assessment of coffee clonal performance in response todrought at the seedling stage. This strategy may be valuable when exploring a large number of genotypesin coffee-breeding programs because it reduces the time and resource costs that would otherwise bewasted on potentially undesirable genotypes.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Drought is one of the most important environmental stressesin agriculture, and some efforts have improved crop productivityunder water-limiting conditions (Bray et al., 2000). A variety oftraits affect the performance of plants under water deficit condi-tions, such as alterations in biomass partitioning above and belowthe soil surface, changes in wood density, osmotic adjustment andstomatal control of transpiration (Santiago et al., 2004; Pinheiroet al., 2005; Poorter et al., 2010). However, in many cases, theplant traits associated with performance cannot be optimized

Abbreviations: �13C, carbon isotopic composition ratio; � md, water potentialat midday; � pd, water potential at predawn; � w, water potential; A, net photo-synthetic rate; A/gs, intrinsic water use efficiency; BGR, branch growth rate; Ci/Ca,internal-to-ambient CO2 concentration ratio; Dw, wood density; Ed, daily transpira-tion rate per unit leaf area; FC, field capacity; gs, stomatal conductance; KL, apparentsoil-to-leaf hydraulic conductance; LMR, leaf mass ratio; PC, principal component;PCA, principal component analysis; RMR, root mass ratio; R/SR, root-to-shoot ratio;SLA, specific leaf area; SMR, stem mass ratio; TB, total biomass; VPD, leaf-to-airvapor pressure deficit; WD, water deficit; WUE, water use efficiency.

∗ Corresponding author. Tel.: +55 31 3899 1291; fax: +55 31 3899 2580.E-mail address: fdamatta@ufv.br (F.M. DaMatta).

simultaneously because of underlying biophysical constraints(Mitchell et al., 2008). For example, a reduction in stomatal andhydraulic conductance under drought conditions prevents exces-sive water loss and limits the potential for leaf damage caused bylow leaf water potentials. However, this reduction also restrictsthe uptake of carbon dioxide and hence biomass accumulation andproductivity. In fact, the proper coordination of plant hydraulicproperties and photosynthetic capacity might be crucial for attain-ing greater carbon gains under water deficit conditions (Santiagoet al., 2004; Brodribb, 2009; Franks and Brodribb, 2010; Poorteret al., 2010).

Among agricultural commodities, coffee has a monetary valuethat is surpassed only by oil. The international trade of coffeegenerates over US$ 90 billion each year, and the management ofthe coffee industry involves approximately 500 million peoplefrom cultivation to the final consumable product (DaMatta et al.,2010a). Of the approximately 100 species of the genus Coffea,only the C. arabica L. (arabica coffee) and C. canephora Pierre exA. Froehner (robusta coffee) species are economically importantworldwide, being responsible for approximately 99% of worldproduction. In Brazil, which is a major area of coffee production,coffee is largely cultivated in regions where water availabilityis the major environmental constraint affecting crop production

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(DaMatta et al., 2010a). Short drought periods can substantiallydecrease coffee yields; consequently, irrigation is vital to coffeeproduction. However, the amount of water available for irrigationhas steadily decreased, especially in the drier years of manyBrazilian coffee-growing regions. Thus, water limitations to cof-fee production are expected to become increasingly importantbecause of global climate changes (DaMatta et al., 2010b). In thiscontext, improvements in water use should be a major target forincreasing coffee yields in water-limited environments (Blum,2005).

The first requirement for a successful breeding program ofdrought-tolerant coffee cultivars should be developing cultivarsthat withstand severe drought spells and produce acceptable yieldsunder water-limiting conditions. Plant breeders have empiricallyselected some promising robusta coffee clones that satisfy thisrequirement. Physiological evaluations of a few of these cloneshave suggested that the maintenance of the following traits is ofthe utmost importance: adequate water status using a combina-tion of deep rooting, satisfactory stomatal control of transpirationand leaf area maintenance (DaMatta et al., 2003; Pinheiro et al.,2005); biochemical traits, such as improved tolerance to oxidativestress (Lima et al., 2002; Pinheiro et al., 2004); and assimilate export(Praxedes et al., 2006). More recently, Marraccini et al. (2012)posited that a complex network of responses, probably involvingthe abscisic acid signaling pathway and nitric oxide, should act asmajor molecular determinants to explain the control of transpi-ration in robusta coffee clones under drought conditions. Despitethese evaluations, the development of an efficient breeding methodfor drought tolerance has yet to be achieved; in the screening of elitecoffee genotypes, no single trait has sufficient predictive power toidentify coffee clones with improved yields in water-limited envi-ronments (DaMatta and Ramalho, 2006). Therefore, it is necessaryto gain an understanding of the mechanisms that govern droughttolerance in coffee plants.

In this study, we examined the contribution of morphologicaland physiological variation in hydraulic and photosynthetic pro-cesses to the functional diversity of 10 robusta coffee clones ina long-term, controlled factorial experiment. One group of cof-fee plants was continuously irrigated, while a second group wassubjected to water deprivation to promote a drought response.Physiological measurements based on leaf-level gas exchange andwater-related traits provided direct assessments of the functionsthat are related to short-term carbon gains and water use, whereaslonger-term inferences were made from isotopic signatures andmorphological analyses. Our specific objectives were as follows:(1) to assess the extent and mechanisms of intra-specific varia-tions in carbon gains with respect to hydraulic constraints and(2) to identify drought-tolerant coffee clones by examining sets oftraits and selecting key traits for relevant integrators of droughteffects. Overall, our results facilitate the selection of promisingrobusta coffee clones for future coffee production in drought-proneregions.

2. Materials and methods

2.1. Plant material, growth conditions and experimental design

The experiment was conducted in Vic osa (20◦45′ S, 42◦54′ W,650 m altitude), southeastern Brazil, under full sunlight conditions.Ten clones of C. canephora, classified as drought tolerant (14 and120), moderately drought tolerant (02, 03, 16, 22 and 48), droughtsensitive (109 and 201) and extremely drought sensitive (Apoatã),were evaluated. This classification was based on the clonal abilityto maintain yields under drought conditions, i.e., under droughtconditions, the most and least tolerant clones lose the fewest andmost crop yield, respectively.

The clones, which were grown from rooted stem cuttings, wereobtained from the Institute for Research and Rural Assistance ofthe Espírito Santo State (INCAPER) in Brazil. A total of 120 uni-form seedlings (with four leaf pairs) were planted in February2008 and grown in 24 L pots containing a mixture of soil, sandand composted manure (4:1:1, v/v/v). The plants were irrigatedand fertilized as needed; no restriction of root development wasobserved at the end of the experiment. Eight-month-old plantsof each coffee clone were separated into two groups. One group(i.e., the control plants) received irrigation to ensure that the soilmoisture was the same as the field capacity (FC). The other groupdid not receive water until the soil water content reached approx-imately 66% of the FC; the plants were kept under these conditionsfor 90 days, and then the soil water content was further reducedto 33% of the FC for an additional 30 days (i.e., the water deficit(WD) treatment). After these treatments, the plants were ana-lyzed and harvested. The available water was calculated throughthe values of soil volumetric moisture at both field capacities(−0.010 MPa) and permanent wilting points (−1.5 MPa) follow-ing a soil moisture retention curve. Previously, the weights of allpots were standardized, using the same substrate amounts. Subse-quently, pot weights at FC and WD conditions were gravimetricallyestablished using a balance (0.1 g precision). Moisture levels werecontrolled by monitoring the weights of the pots. Adjustments inpot weights due to increased biomass over time were carried outfortnightly. The plants were irrigated every 2 days. Rainwater wasexcluded by covering the pot surfaces with a plastic film that tightlyadhered to the base of the main plant stem. The youngest, fullyexpanded leaves, which corresponded to the third or fourth leafpair from the apex of the plagiotropic branches, were used forsampling and measurement. In the WD treatments, the sampledleaves expanded after the initiation of water shortage conditions.Overall, the coffee plants were submitted to 20 treatment com-binations in the form of a 10 × 2 factorial experimental design(10 clones and two levels of available water). This design wascompletely randomized; six coffee plants in individual pots pertreatment combination were used as replicates, and the experi-mental plot consisted of one plant per container. The pots wererandomized periodically to minimize any variation between treat-ments. During the period of watering treatment imposition, theaverage air temperature was 21.7 ◦C (maximum and minimumaverage temperatures were 26.9 ◦C and 18.0 ◦C, respectively), andrelative humidity was 80.6%, as measured with sensors installed atthe experimental site (Fig. 1).

2.2. Growth traits and wood density

At the end of the experiment, the plants were harvested andseparated into orthotropic and plagiotropic branches, leaves androots. The total leaf areas were estimated from the maximum leafwidths and lengths using the equations described by Antunes et al.(2008). The roots were washed thoroughly with tap water usinga 0.5 mm screen sieve. The plant tissues were oven-dried at 70 ◦Cfor 72 h, after which the dry weights of the leaves, branches androots were determined. Based on these data, the total biomass(TB), leaf mass ratios (LMR; leaf mass per TB), stem mass ratios(SMR; stem mass per TB), root mass ratios (RMR; root mass per TB)and root-to-shoot ratios (R/S) were obtained. The branch growthrate (BGR) was measured according to Silva et al. (2004). Thespecific leaf area (SLA; leaf area per unit of leaf dry mass) wascomputed using the dry mass of 20 (1.7 cm diameter each) leafdiscs. We further calculated the wood density (Dw) using a stemsegment (2 cm length) that was collected 10 cm below the inser-tion point of the first (older) pair of plagiotropic branches. TheDw was calculated as the dry stem mass per unit of stem vol-ume; the stem volume was calculated according to Markesteijn and

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Fig. 1. The time course of maximum and minimum air temperature (A) and the rel-ative humidity (B) at Vic osa, southeastern Brazil, during the imposition of wateringtreatments, which were initiated on October 15, 2008.

Poorter (2009) with the assumption that the coffee plant stem wasconical.

2.3. Water relations

Leaf water potentials (� w) were measured at predawn(04:30–05:30 h) (� pd) and midday (� md) using a Scholander-typepressure chamber (model 1000, PMS Instruments, Albany, NY,USA). The apparent soil-to-leaf hydraulic conductances (KL) wereexpressed as the ratios of the total transpiration (estimated gravi-metrically) from predawn to midday and the differences in the � w

measured during this interval (Pinheiro et al., 2005). The daily tran-spiration rates per unit leaf area of whole plants (Ed) were estimatedgravimetrically as described in Dias et al. (2007). Transpiration wascorrected for direct evaporative losses by subtracting the averageamount of water lost from pots without plants from each wateringtreatment.

2.4. Photosynthetic measurements and isotopic signatures

The net photosynthetic rates (A), stomatal conductances towater vapor (gs) and internal-to-ambient CO2 concentration ratios(Ci/Ca) were determined using a Li-6400 open gas exchange system(Li-Cor, Lincoln, NE, USA) equipped with a blue/red light source(Li-6400-02B). The measurements were made between 09:00 hand 11:00 h of solar time at ambient temperature and CO2 con-ditions under artificial light with 1000 �mol photons m−2 s−1 atthe leaf level, which is an approximation of the ambient irradianceintercepted by the sampled leaves in their natural angles. Duringthe measurements, the average leaf temperature was 30.1 ± 1.3 ◦C,and the average leaf-to-air vapor pressure deficit (VPD) was2.95 ± 0.31 kPa.

The carbon isotope composition ratio (�13C) was measuredin bulk leaf tissues relative to the international Pee Dee Belem-nite standard using an isotope mass spectrometer (ANCA-GSL20–20, Sercon, Crewe, UK) as previously described (Cavatte et al.,2012).

2.5. Statistical analysis

The morphological and physiological variables of clones werecompared using a two-way ANOVA. To avoid heteroscedastic-ity, the variables were transformed when necessary. The meanswere clustered using the Scott–Knott test at the P ≤ 0.05 level.Within each clone, differences due to water treatments weretested using the t-test for each trait. To study the relationshipsbetween variables, Pearson’s linear correlation and principal com-ponent analysis (PCA) were employed. The PCA was performedusing the normalized data because these data preserved the dis-tance between the measured variables. We excluded the KL inthe PCA because this trait (as it was estimated) is necessar-ily auto-correlated with the Ed and � md. The mean trait valuesobtained from the clones under control conditions were usedfor the PCA. A second data set relating to stress performancewas produced for individual traits using the following equation:(control − WD)/control. The principal components (PC) were thenderived from this data set to identify the WD-tolerant coffee clones,i.e., the clones that were least affected by the WD treatment(Ivandic et al., 2000; Luo et al., 2011). The coffee clones with similarbehavior were clustered using a multivariate technique of group-ing analysis according to the method of Tocher, which is basedon average Euclidean distances. This method aims at separatinggenotypes into non-empty and mutually exclusive groups, withhomogeneity within and heterogeneity among groups, based on thecriterion that the mean of the dissimilarity measured within eachgroup must be lower than the mean Euclidean distances betweenany pair of groups (Rencher, 2002). All of the statistical analy-ses were performed using the SAEG software, version 9.1 (SAEG,2007).

3. Results

3.1. Morphology and allometry

Under FC conditions, the TB of coffee clones varied (Table 1)from 88 g (clone 14) to 350 g (clone 03). The WD treatment pro-voked dramatic reductions in the TB. With the exception of clones02 and Apoatã, the WD treatment significantly altered the pat-tern of biomass allocation. Overall, the LMR decreased (e.g., asmuch as 33% in clone 03), whereas the RMR increased (e.g., asmuch as 75% in clone 109) with minor changes (if any) in theSMR (Table 1). As a result, the R/S ratio increased in most cof-fee clones under the WD conditions compared with their controlcounterparts. The SLA ranged from 10.4 (clone 03) to 13.4 (Apoatã)m2 kg−1 under FC and was unresponsive to the WD treatment,except for the Apoatã clone, which had a 19% reduction in the SLA(Table 1).

Under FC conditions, the Dw ranged from 0.28 (Apoatã) to 0.57(clone 14) g cm−3 (Table 1). On average, the Dw increased by 34% inthe WD treatment and ranged from 0.34 (clone 109) to 0.67 (clone14) g cm−3. Notably, the largest relative increase in the Dw (74%)was found in Apoatã, whereas no significant alteration in the Dw

was observed in clone 109 (Table 1).Compared to the control plants, the BGR under WD conditions

was dramatically reduced in all clones (Table 1), and the relativedecreases ranged from 54% (clone 14) to 77% (clone 201).

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Table 1The total biomass (TB, g), leaf mass ratio (LMR, g g−1), root mass ratio (RMR, g g−1), stem mass ratio (SMR, g g−1), root-to-shoot ratio (R/S, g g−1), branch growth rate (BGR,mm d−1), stem density (Dw, g cm−3) and specific leaf area (SLA, m2 kg−1) for the 10 clones of C. canephora grown under field capacity (FC) or water deficit (WD) conditions. Thevalues followed by the same letter do not differ significantly among the clones; the capital and lowercase letters denote FC and WD conditions, respectively, at the P ≤ 0.05level using a Scott–Knott test. Asterisks indicate significant differences between the irrigation treatments within a clone at the P ≤ 0.05 level using a t-test. n = 6 (SE).

Growth traits

Clone TB LMR RMR SMR R/S BGR Dw SLA

02 FC 300 (30) B 0.40 (0.01) C 0.32 (0.03) C 0.28 (0.01) C 0.49 (0.06) B 0.15 (0.02) D 0.37 (0.01) B 10.9 (0.4) BWD 192 (8) a* 0.38 (0.02) c 0.32 (0.01) b 0.29 (0.02) c 0.48 (0.02) b 0.03 (0.00) b* 0.51 (0.02) c* 11.2 (0.2) b

03 FC 350 (10) A 0.33 (0.01) E 0.37 (0.01) B 0.30 (0.01) C 0.60 (0.02) A 0.21 (0.01) B 0.29 (0.00) D 10.4 (0.2) CWD 195 (13) a* 0.22 (0.01) f* 0.47 (0.02) a* 0.31 (0.01) c 0.90 (0.08) a* 0.03 (0.00) b* 0.45 (0.02) d* 9.67 (0.2) c

14 FC 88 (5) E 0.45 (0.01) B 0.18 (0.00) B 0.37 (0.01) A 0.22 (0.01) D 0.19 (0.00) C 0.56 (0.01) A 12.9 (0.2) AWD 71 (4) c* 0.33 (0.02) d* 0.27 (0.02) c* 0.39 (0.02) a 0.38 (0.04) c* 0.08 (0.00) a* 0.67 (0.02) a* 12.7 (0.5) a

16 FC 269 (24) C 0.36 (0.00) D 0.27 (0.02) A 0.37 (0.01) A 0.38 (0.03) C 0.19 (0.00) C 0.31 (0.00) C 12.3 (0.3) AWD 125 (8) b* 0.33 (0.01) d* 0.32 (0.01) b* 0.35 (0.01) b 0.48 (0.02) b* 0.04 (0.00) b* 0.47 (0.00) c* 12.3 (0.4) a

22 FC 220(3) D 0.40 (0.01) C 0.26 (0.01) B 0.34 (0.01) B 0.35 (0.03) C 0.17 (0.01) C 0.29 (0.01) D 12.5 (0.5) AWD 141 (7) b* 0.40 (0.01) c 0.30 (0.01) b 0.30 (0.01) c* 0.43 (0.02) b* 0.02 (0.00) b* 0.39 (0.01) e* 12.4 (0.3) a

48 FC 265 (8) C 0.49 (0.02) A 0.21 (0.03) B 0.30 (0.01) C 0.28 (0.05) C 0.18 (0.01) C 0.35 (0.00) B 10.1 (0.6) CWD 165 (5) a* 0.44 (0.01) b* 0.22 (0.01) d 0.33 (0.02) b* 0.29 (0.01) c 0.03 (0.00) b* 0.47 (0.00) c* 11.0 (0.7) b

109 FC 130 (10) E 0.51 (0.01) A 0.16 (0.02) B 0.32 (0.02) B 0.20 (0.02) C 0.13 (0.03) D 0.32 (0.01) C 11.0 (0.4) BWD 63 (11) b* 0.37 (0.01) e* 0.28 (0.01) c* 0.35 (0.02) b* 0.39 (0.02) c* 0.03 (0.00) b* 0.34 (0.01) f 10.3 (0.4) c

120 FC 115 (10) E 0.50 (0.01) A 0.17 (0.01) B 0.32 (0.01) B 0.21 (0.01) D 0.17 (0.00) C 0.43 (0.01) B 11.2 (0.3) BWD 92 (2) c* 0.39 (0.01) c* 0.27 (0.01) c* 0.34 (0.01) b 0.37 (0.02) c* 0.06 (0.01) a* 0.56 (0.01) b* 11.3 (0.2) b

201 FC 220 (16) D 0.42 (0.01) C 0.24 (0.01) B 0.33 (0.00) B 0.32 (0.02) D 0.28 (0.02) A 0.31 (0.00) B 11.7 (0.2) BWD 135 (3) b* 0.30 (0.00) c* 0.32 (0.00) b* 0.38 (0.00) a* 0.47 (0.00) b* 0.06 (0.00) a* 0.39 (0.00) e* 11.2 (0.6) b

Apoatã FC 284 (13) C 0.51 (0.01) A 0.24 (0.02) C 0.25 (0.02) D 0.32 (0.04) C 0.22 (0.00) B 0.28 (0.00) D 13.4 (0.1) AWD 141 (12) b* 0.54 (0.02) a 0.25 (0.02) d 0.22 (0.01) d* 0.33 (0.04) c 0.04 (0.01) a* 0.49 (0.01) c* 10.9 (0.5) b*

3.2. Water relations

For all clones, the � pd was always greater than −0.10 MPaunder FC (Fig. 2A). Under the WD conditions, the � w was signif-icantly lower. The lowest � pd values (approximately −2.5 MPa)were found in clones 03 and Apoatã; in contrast, the highest � pdvalue (−0.48 MPa) was found in clones 14 and 120 (Fig. 2A). Similartrends were observed for the � md, although the values were morenegative (Fig. 2B).

Differences in the Ed among the coffee clones under FC were notas large as under WD conditions (Fig. 2C). In the WD treatment,the Ed was significantly lower in most of the coffee clones (e.g.,as much as 79% in clone 02). The exceptions were three clones inwhich the Ed increased (clone 14) or was unaltered (clones 16 and120) relative to the controls (Fig. 2C).

Under the FC conditions, the highest KL was found in the Apoatãclone, followed by clones 109 and 16; in contrast, clone 201 dis-played the lowest KL (Fig. 2D). Under the WD conditions, dramaticreductions in the KL were found irrespective of the clone measured.Under these same conditions, the highest KL was observed in clones14 and 120, and the lowest KL was found in clones 02, 22 and 48(Fig. 2D).

3.3. Gas exchanges and isotopic signatures

In the control plants, gs ranged from 98 (clones 14 and 22) toapproximately 330 mmol m−2 s−1 (clones 109 and 201), whereasA ranged from 5.9 (clone 14) to approximately 10.5 �mol m−2 s−1

(clones 03 and 201) (Fig. 3A and B). Under the WD conditions, bothgs and A decreased in all clones (Fig. 3A and B).

Regardless of the soil water availability, the Ci/Ca ratio var-ied only slightly among the coffee clones (Fig. 3C). This ratiosignificantly decreased in response to the WD treatment inclones 48, 109 and 120 compared with their control counterparts(Fig. 3C).

Under FC, the �13C ranged from −28.0‰ (clones 02 and 120) to−26.7‰ (clone 22) (Fig. 3D). Under WD, the �13C increased in allclones, except in clones 14, 22 and 120, in which �13C was unre-sponsive to the WD treatment. The highest (i.e., least negative) �13Cvalues were displayed by clones 201 and Apoatã (approximately

−25.0‰), and the lowest �13C values were displayed by clones 14and 120 (approximately −27.5‰) (Fig. 3D).

3.4. Relationships among variables

Both A and gs were strongly correlated with one another regard-less of the watering treatment (Table 2). Under FC, A was positivelycorrelated with Ci/Ca, � md and BGR and negatively correlated withDw and SLA. No significant correlation was found between �13Cwith A, A/gs, Ed and BGR (Table 2, Fig. 4A–C). Under WD, A was neg-atively correlated with Ci/Ca and positively correlated with � pd,� md, Ed, KL and Dw. �13C was positively correlated with Ci/Ca andcorrelated negatively with A, gs, A/gs, � pd, � md, Ed, KL and Dw

(Table 2, Fig. 4). Notably, KL and Dw were negatively correlatedunder FC and positively correlated under WD conditions (Table 2).

Under FC, five PCs explained 85% of the observed variation andthe first two PCs explained 50% (PC1 = 30%; PC2 = 20%) of the vari-ation. The first PC was characterized by high positive scores forcarbon gain traits (i.e., TB and A) and water uptake ability (i.e.,RMR). The first PC had a strong negative correlation with Dw andLMR (Fig. 5). Considering all clones, this finding suggested thatan increase in biomass was negatively correlated with the LMR,a result supporting the univariate correlations of the TB and LMR.The second PC was negatively associated with A, gs, Ed, LMR and� md and positively associated with Dw and RMR (Fig. 5). SLA, BGRand �13C had minor effects on the first two PCs analyzed. Basedon the PCA, four groups of coffee clones with different strategiesassociated with carbon gain and water use were identified (Fig. 5).(I) The data from clones 02, 03 and 16 clustered together, local-ized mostly to the PC1 and characterized by elevated carbon gainsthat were associated with an enhanced ability for water uptake andtransport. (II) The data from clones 14 and 120 were also mostlylocalized to the negative quadrant of PC1, therefore in the oppo-site direction of the first group, suggesting that diminished carbongains reflected a more conservative use of water. (III) The data fromclones 109 and Apoatã clustered together and localized mostly tothe PC2; these data were characterized by elevated Ed and relativelylow Dw coupled with high LMR and low RMR. (IV) Finally, the datafrom clones 22, 48 and 201 clustered together and had a behaviorthat was intermediary between those of groups I and III.

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Table 2The pairwise Pearson correlations of the net carbon assimilation rate (A), stomatal conductance (gs), internal-to-ambient CO2 concentration ratio (Ci/Ca), leaf-to-air vaporpressure deficit (VPD), predawn water potential (� pd), midday water potential (� md), diurnal transpiration rate (Ed), soil-to-leaf apparent hydraulic conductance (KL), stemdensity (Dw), carbon isotopic composition ratio (�13C), leaf mass ratio (LMR), root mass ratio (RMR), stem mass ratio (SMR), root-to-shoot ratio (R/S), specific leaf area (SLA),branch growth rate (BGR) and total biomass (TB) for the 10 C. canephora clones that were grown under ample irrigation (above the diagonal) or WD (below the diagonal)conditions. For each watering regime, n = 60. The bold correlation coefficients have a P-value < 0.05, and the bold, underlined coefficients have a P-value < 0.01.

A gs Ci/Ca VPD � pd � md Ed KL Dw �13C LMR RMR SMR R/S SLA BGR TB

A 0.87 0.62 0.26 0.01 0.29 0.02 0.04 −0.51 −0.19 −0.18 0.28 −0.18 −0.16 −0.40 0.24 0.33gs 0.94 0.86 −0.10 −0.02 0.17 −0.06 0.01 −0.33 −0.04 0.05 −0.28 −0.04 0.01 −0.46 0.11 0.01Ci/Ca −0.30 −0.03 −0.22 0.00 −0.10 −0.22 −0.11 −0.06 −0.54 0.10 −0.14 0.06 −0.11 −0.43 0.10 −0.15VPD −0.41 −0.46 −0.17 0.08 0.19 0.08 0.15 −0.18 0.14 −0.20 0.27 −0.14 0.17 0.06 0.22 0.06� pd 0.34 0.31 0.04 −0.22 −0.23 −0.12 −0.48 0.19 0.07 −0.15 0.22 −0.13 0.25 −0.35 0.16 0.13� md 0.38 0.32 −0.09 −0.24 0.86 0.41 0.63 −0.71 0.11 0.22 0.05 −0.44 0.04 −0.01 −0.06 0.37Ed 0.35 0.33 0.02 −0.41 0.62 0.55 0.29 −0.29 0.18 0.11 −0.05 −0.08 −0.06 0.27 0.06 0.08KL 0.33 0.28 0.03 −0.37 0.43 0.65 0.75 −0.34 −0.03 0.25 −0.08 −0.26 −0.08 0.20 −0.18 0.11Dw 0.42 0.34 −0.23 −0.47 0.36 0.31 0.56 0.38 0.06 0.15 −0.36 0.36 −0.34 0.09 −0.14 −0.58�13C −0.44 −0.32 0.33 0.12 −0.50 −0.45 −0.48 −0.38 −0.46 0.25 −0.24 0.06 −0.27 0.17 0.21 −0.17LMR −0.07 −0.15 −0.33 0.03 −0.12 0.02 −0.33 −0.16 0.08 0.07 −0.80 −0.21 −0.79 0.11 −0.19 −0.53RMR −0.08 0.00 0.29 0.23 −0.16 −0.26 0.00 −0.09 −0.19 0.17 −0.77 −0.39 0.99 −0.17 0.21 0.81SMR 0.22 0.23 0.14 −0.38 0.39 0.30 0.51 0.37 0.13 −0.32 −0.58 −0.09 −0.40 0.09 −0.06 −0.52R/S −0.11 −0.03 0.29 0.24 −0.17 −0.04 −0.22 −0.11 −0.16 0.18 −0.73 −0.84 −0.37 0.00 −0.32 −0.72SLA 0.39 0.33 −0.19 −0.23 0.28 0.41 0.18 0.24 0.35 −0.43 0.05 −0.26 0.26 0.03 0.24 −0.20BGR 0.22 0.18 −0.06 0.22 0.30 0.28 0.59 0.42 0.44 −0.36 −0.16 −0.09 0.36 −0.14 0.23 0.22TB −0.26 −0.20 0.07 0.33 −0.33 −0.45 −0.55 −0.62 −0.15 0.43 −0.08 0.47 −0.47 −0.15 −0.19 −0.41

The PCA based on the relative decreases [(con-trol − WD)/control] in several traits was performed to definedrought responses and separate the clones we analyzed intogroups with varying tolerance to drought. Four PCs explained 73%of the total variation in the data and the first two PCs explained55% (PC1 = 36%; PC2 = 19%) of the variation. The PC1 was positivelycorrelated with A, gs, Ed, �13C, BGR, RMR and TB and negativelycorrelated with � pd, � md, Dw and LMR (Fig. 6). The PC2 waspositively correlated with A, gs, Dw, �13C and LMR and had astrong negative correlation with RMR (Fig. 6). The SLA had minoreffects on the first two PC analyzed. Four groups of coffee cloneswere identified (Fig. 6). (I) Two drought-tolerant clones (14 and120) had the least changes in traits related to carbon gain andwater use. (II) Four moderately drought-tolerant clones (02, 16, 22and 48) clustered near the center of the bi-plot, which reflectedtheir minimal changes in the biomass partitioning into leaves androots and intermediate photosynthetic performance. (III) Threedrought-susceptible clones (03, 109 and 201) were characterizedby sharp decreases in the traits related to carbon gain and wateruse. (IV) Finally, the most drought-susceptible clone (Apoatã) wasmostly localized to the PC1 but in the opposite direction of clones14 and 120. Overall, these data suggest that the coffee clonesthat postponed tissue dehydration (clones 14 and 120) were ableto maintain higher rates of water use and showed the lowestrelative decreases in traits related to carbon gain under the WDconditions.

4. Discussion

4.1. Functional divergence in C. canephora under FC conditions

The strong association between A and gs was accompanied byan increased Ci/Ca, which suggests that differences in the A of coffeeclones were not closely related to the stomatal limitations of photo-synthesis. Overall, our results suggest that the clones with a greaterability to use water (higher � md, Ed, KL and RMR and lower Dw)also showed improvements in carbon gain values (higher TB and A).These results suggest that a coordinated balance exists between thecoffee leaf photosynthetic capacity and the water supply capacityof the coffee root/stem system, which has also been demonstratedin other woody species (e.g., Jones et al., 2010; Poorter et al., 2010).Therefore, the relationship between the hydraulic supply and pho-tosynthesis should represent a functional divergence to be explored

in the selection of coffee clones for conditions of ample water avail-ability.

Despite the lack of significant correlations between the instan-taneous measurements of gas exchange and the integrated traitsof KL and Ed, we found positive correlations between Dw and A andgs, and the correlations between Dw and Ed and KL were also pos-itive. These data suggest that Dw, an easily measured trait, couldbe an integrator trait that describes the water transport capacitytoward the shoots of coffee plants. Dw has been negatively corre-lated with hydraulic efficiency in several studies (Meinzer et al.,2009; Poorter et al., 2010; Markesteijn et al., 2011), although nega-tive correlations between Dw and KL have not always been observed(e.g., Mitchell et al., 2008). Furthermore, the negative correlationbetween Dw and RMR circumstantially suggests that coffee clones(e.g., clones 02 and 03) with a lower Dw and higher hydraulic effi-ciency invested larger amounts of biomass into roots to supporthigher rates of water transport. This finding could translate intohydraulic safety against cavitation (Markesteijn et al., 2011), andthe larger carbon gain might compensate for the elevated costs ofbuilding a more robust root system. In contrast, a higher Dw wouldconstrain the hydraulic efficiency and the rates of water use in cof-fee plants, but a higher Dw would simultaneously reduce the needfor investments in a more robust root system (e.g., clones 14 and120).

4.2. Functional divergence in C. canephora under WD conditions

In this study, we analyzed coffee clonal performance under sim-ilar conditions of soil water availability. The imposed treatmentwas effective in changing phenotypic traits, particularly in the WD-susceptible clones (e.g., clones 03, 201 and Apoatã). In these clones,the � pd values were below −2.0 MPa, which was characterized asa moderate to severe WD in coffee plants (Pinheiro et al., 2005;Praxedes et al., 2006). In contrast, � pd was only slightly affectedin the most WD-tolerant coffee clones (clones 14 and 120), whichindicates that these clones were most likely operating well abovethe point of xylem failure (Markesteijn et al., 2011). This find-ing could translate into improved tissue hydration and, ultimately,improved physiological performance under the WD conditions.

In general, the partitioning of biomass was dramatically affectedby the WD treatment. We found the classic response of relativeincreases in the biomass of the root system at the expense of theshoot (especially in leaves) biomass (Poorter and Nagel, 2000). The

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Fig. 2. The predawn (� am) and midday (� md) leaf water potential, daily transpi-ration per unit leaf area (Ed) and apparent leaf-to-soil hydraulic conductivity (KL)for the 10 C. canephora clones under ample irrigation (filled bars) or WD condi-tions (empty bars). The values followed by the same letter do not significantly differamong the clones at the P ≤ 0.05 level of a Scott–Knott test; the capital and lowercaseletters denote significantly differing results from FC and WD conditions, respec-tively. Asterisks indicate significant differences between the irrigation treatmentsof a coffee clone at the P ≤ 0.05 level using a t-test. n = 6 ± S.E.

exceptions were clones 02 and Apoatã, which reflect a low pheno-typic plasticity for biomass partitioning in response to drought. Itshould be noted, however, that the RMR of Apoatã did not respondto water availability, whereas this clone showed the largest rel-ative increase in Dw (75%). In contrast, clone 109 displayed thelargest relative increase in RMR (75%) and did not exhibit anysignificant variation in Dw. These coffee clones also exhibited thegreatest decreases in KL (approximately 70%) under the WD con-ditions. Taken together, these data illustrate how differences inthe morphological traits of coffee clones may affect their hydrauliccapacities under WD conditions.

The negative correlation between RMR and � md suggests thatthe available soil water is rapidly depleted as the RMR increases.Differences in the � pd of coffee clones should reflect differencesin their ability to uptake and/or transport water from the roots tothe leaves because the clones were under similar soil water avail-ability. Thus, it is reasonable to suggest that more negative values

Fig. 3. The net carbon assimilation rate (A), stomatal conductance (gs), internal-to-ambient CO2 concentration ratio (Ci/Ca) and carbon isotopic composition ratio(�13C) for the 10 C. canephora clones under the ample irrigation (filled bars) or WDconditions (empty bars). The statistical tests and differences are as specified in Fig. 2.

of � pd and � md might be associated with cavitation. Indeed, moreconservative attributes describing the use of available water, suchas larger Dw, were positively correlated with � pd, � md, Ed and KL.Collectively, these relationships suggest that the coffee clones witha higher Dw exhaust the available water slowly and have a greaterhydraulic efficiency under the WD conditions due to a presum-able hydraulic safety against cavitation, which ultimately results inimproved hydration of their tissues. Based on the positive correla-tions between BGR and � pd, � md and Dw, improvement in tissuehydration should facilitate the maintenance of coffee clone growth.

The negative correlation between A and Ci/Ca suggests that sto-matal limitations could, to some extent, have contributed to theobserved reductions in A. However, it should be emphasized thatA and gs were negatively correlated with both � pd and � md undermild WD conditions (soil water availability equivalent to 66% ofthe FC) (data not shown), which was in sharp contrast with thedata shown in Table 2 for the conditions of soil water availabil-ity that were equivalent to 33% of the FC conditions. Collectively,this information suggests that the coffee clones with high wateruse rates perform well under well-watered or mild WD conditions.Therefore, these coffee clones should be recommended for use inareas that are subjected to short periods of drought. However, inregions where the WD can be severe, the exploitation of clones

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Fig. 4. The relationship between carbon isotopic composition ratio (�13C) and (A)the intrinsic water use efficiency (A/gs), (B) daily transpiration rate per unit leaf area(Ed) and (C) branch growth rate (BGR) for the 10 C. canephora clones under ampleirrigation (filled circles) or water deficit conditions (empty circles). n = 10 ± S.E.

with traits associated with high water use rates can result in thecomplete failure of the plantation because this exploitation wouldrapidly exhaust the available water. If the soil water reserves arenot replenished, an exacerbation of cavitation and impaired tissuehydration are expected (Markesteijn et al., 2011).

Although displaying only small reductions, Ci/Ca paralleled thesignificant decreases in A, what was enough to be reflected in lessnegative values of �13C in most of the coffee clones under theWD conditions, which suggests an increase in the long-term WUE(Farquhar et al., 1989). However, we found a negative correlationbetween �13C and instantaneous intrinsic WUE (A/gs) under theWD conditions (Fig. 4A), suggesting that A/gs did not match thelong-term WUE. This result could be explained taking into accountthat A/gs was estimated at the end of a drought period, whereas�13C represents a more integrative trait because it reflects the dis-crimination against 13CO2 during the entire phase of leaf formation,which occurred over the period of WD imposition (Erice et al.,2011). In any case, the univariate analysis evidenced significant andnegative associations between �13C and traits associated with boththe water use (e.g., gs, Ed, KL) and carbon gain (e.g., A) under theWD conditions, suggesting that less negative �13C (higher WUE)was achieved at the expense of lower water use rates, which areexpected to compromise the carbon gains. As a whole, these rela-tionships further strength the inverse pattern between carbon gain

Fig. 5. The first two axes of a PCA of all 10 C. canephora clones under ample irrigation.(A) The correlation coefficients for all traits are represented by Eigenvectors (solidarrows). (B) The segregation of the 10 studied clones based on several morpho-logical and physiological traits. Four groups of coffee clones were observed. Theabbreviations are defined in the Abbreviation list.

and water use found in this study. In any case, the PCA suggeststhat the changes in A, gs and TB of coffee clones were accompaniedby alterations in �13C (Fig. 6). In this context, the �13C might be apromising alternative for identifying coffee clones with improvedperformance under WD conditions. Nonetheless, our results indi-cate that the performance of coffee clones with greater WUE underthe WD conditions could not be predicted from the analysis of �13Cunder the conditions of ample irrigation (Table 2, Fig. 4A and B).This finding is in sharp contrast to the results of an analysis of fivegenotypes of arabica coffee by Meinzer et al. (1990), who reportedthat the �13C obtained from irrigated coffee plants could be usedto predict the genotypic performance under WD conditions.

Our PCA identified a large variation in the WD-tolerance of thecoffee clones studied. Four groups of coffee clones that differedin drought tolerance were identified, and the morphophysiologi-cal performance in 9 of the 10 studied clones was consistent withempirical observations of drought tolerance in coffee plants underfield conditions. The exception was clone 03, which has empiricallybeen considered a drought-tolerant coffee clone, although this tol-erance might be linked to key traits that were not measured inthis study. Therefore, although the data were obtained from pottedclones, our results can be compared to coffee clone performancein the field. Our data also suggest that the screening and selectionof WD-tolerant coffee clones should be performed under drought

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Fig. 6. The first two axes of a PCA of all 10 C. canephora clones. When constructingthe bi-plot, the PCA scores were based on the relative difference, i.e., (control − waterdeficit)/control in the data sets. (A) The correlation coefficients for all traits as repre-sented by Eigen vectors (solid arrows). (B) The segregation of the 10 studied clonesaccording to several morphological and physiological traits. Four groups of coffeeclones were observed. The abbreviations are defined in the Abbreviation list.

conditions because the performance of clones grown under droughtconditions cannot be accurately predicted from the performance ofclones grown under conditions of ample water supply.

Although focused on some physiological and morphologicaltraits, the present study could be further complemented withresearch of other parameters that might constitute useful tools forstress tolerance screening in coffee, e.g., qualitative changes of thelipid matrix of membranes and parameters related to the control ofoxidative stress. Changes in these parameters are usually triggeredunder environmental stress conditions, and apparently constitute acommon acclimation response of coffee plants to several stresses,namely high irradiance, cold and drought (Ramalho et al., 1998;DaMatta and Ramalho, 2006; Pinheiro et al., 2004; Fortunato et al.,2010; Pompelli et al., 2010; Partelli et al., 2011).

5. Concluding remarks

Our results suggest that, under conditions of ample irrigation,clones with superior water use ability displayed enhanced carbongains. In contrast, under the WD conditions, clones that postponeddehydration via more conservative water use rates showed lowerrelative decreases in gs, A and TB. Our results also suggest that

combining useful morphological (e.g., Dw and allometric traits) andphysiological (e.g., A, gs, Ed, �13C and � w) traits facilitates the suc-cessful assessment of coffee clonal performance in response to WDat the seedling stage. This strategy may be valuable when exploringa large number of genotypes in coffee-breeding programs becauseit saves time and resources that would otherwise be wasted onpotentially undesirable genotypes. In this context, we believe thatour data provide valuable resources for traits of morphophysiolog-ical importance that can be used in coffee-breeding programs torank clones with improved performance in drought-prone regions.

Acknowledgments

This research was partially supported by the Foundation forResearch Assistance of the Minas Gerais State (Fapemig) and anaward from the National Council for Scientific and TechnologicalDevelopment (CNPq) to F.M. DaMatta.

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