The Impact of Carbon Dioxide Concentrations and Low to Adequate Photosynthetic Photon Flux Density on Growth, Physiology and Nutrient Use Efﬁciency of Juvenile Cacao Genotypes

: Cacao ( Theobroma cacao L.) was grown as an understory tree in agroforestry systems where it received inadequate to adequate levels of photosynthetic photon ﬂux density (PPFD). As atmospheric carbon dioxide steadily increased, it was unclear what impact this would have on cacao growth and development at low PPFD. This research evaluated the effects of ambient and elevated levels carbon dioxide under inadequate to adequate levels of PPFD on growth, physiological and nutrient use efﬁciency traits of seven genetically contrasting juvenile cacao genotypes. Growth parameters (total and root dry weight, root length, stem height, leaf area, relative growth rate and net assimilation rates increased, and speciﬁc leaf area decreased signiﬁcantly in response to increasing carbon dioxide and PPFD. Increasing carbon dioxide and PPFD levels signiﬁcantly increased net photosynthesis and water-use efﬁciency traits but signiﬁcantly reduced stomatal conductance and transpiration. With few exceptions, increasing carbon dioxide and PPFD reduced macro–micro nutrient concentrations but increased uptake, inﬂux, transport and nutrient use efﬁciency in all cacao genotypes. Irrespective of levels of carbon dioxide and PPFD, intraspeciﬁc differences were observed for growth, physiology and nutrient use efﬁciency of cacao genotypes.


Introduction
Cacao (Theobroma cacao L) is native to the understory of the Amazonian forests of South America. As an understory plant, it has physiological characteristics similar to those of other shade-adapted species [1][2][3][4]. Growth and development of young cacao trees are better under shade; however, heavy shade is detrimental to growth and production of matured and older trees [5][6][7][8]. Cocoa is a C 3 species and prefers full sun, but is tolerant to moderate shading, due to its phenotypic plasticity for acclimatization in moderate shade conditions [9]. However, it does not tolerate dense shade, where pod production is low, even with adequate water levels and mineral nutrients availability in the soil. However, when the cacao tree is grown in full sun, there can be no limitations of water and mineral nutrients in the soil. In a long-term field study in Ghana, Amelonado cacao trees in full sun yielded three times as much as shaded trees; however, the economic life of unshaded trees did not last more than 10 years of intensive cropping due to infestation of diseases and insects and loss of needed soil nutrients [10]. There is no universal agreement on the iological traits and macro-micro nutrient uptake, influx and transport and use efficiency in seven genetically contrasting cacao genotypes.

Cacao Genotypes
In total, 7 cacao genotypes (Catongo, Coca 3370/5, CCN 51, Amaz 15, LCT EEN 37/A, Na 33 and SCA 6) were used for this study. Pods of these genotypes were received from MARS Center for Cocoa Science (MCCS) Almirante, Itajuipe, Bahia, Brazil. Catongo is from the lower Amazon region of Brazil; Amaz 15, NA 33 and SCA 6 are from the upper Amazon region of Peru; whereas LCT EEN 37A and Coca 3370/5 are from the upper Amazon region of Ecuador; and CCN 51 is a hybrid from Ecuador. These genotypes have been widely distributed in most of the cacao producing countries and some have been commonly used as parental or as cultivars in cacao breeding programs. Genetic background, origin and diseases resistance of these genotypes are covered in Bartley [56], Turnbull and Hadley [57], and Ahnert and Eskes [58]. Seeds were produced by self-pollination of plants. In the case of self-incompatible plants, they were obtained by the mixture of Herrania and cacao pollen, which helps to break self-incompatibility. Therefore, the self-pollinated family plants generated by such seeds have, on an average, similar traits to the parents, in this case, clonal cuttings. Findings of this study had a good scientific interest, showing differences between different genetic populations.

Plants and Growth Medium
Growth medium was prepared containing sand: perlite: peat moss (2:2:1 volume) supplemented with essential nutrients (mg/kg) 600 N, 600 P, 240 K, 1012 Ca, 309 Mg, 500 S, 119 Fe, 0.7 B, 17.5 Mn, 7 Cu, 7 Zn, and 0.35 Mo. Nutrients were applied as Osmocote 18-6-12 (The Scotts Company, Marysville, Ohio, USA), triple superphosphate, urea, calcium sulphate, dolomitic lime and Scott's Micromix. Cacao seeds were removed from the pods, surface-sterilized with 10% bleach for 2 min, rinsed twice in Deionized-water, then soaked in 90% ethanol for 2 min and rinsed twice in DI water. Seeds were germinated on sterile moist filter paper for 48 h at 25 • C. Seeds with 2 mm radicle were planted in 3.8 L black plastic pots with adequate bottom drainage containing 2.2 kg of the growth mixture. One seedling was planted in each pot. Soil moisture was maintained near field capacity (−33kPa) by adding water every other day. An initial plant harvest was collected at 21 days after planting. The remaining plants were grown for additional 90 days.

CO 2 and PPFD Treatments
The experiment was conducted in two glasshouses (18 m 2 each) at Beltsville, MD and plants were grown with day/night temperatures of 30/28 • C. In the first glasshouse, ambient [CO 2 ] of 400 ± 50 µmol mol −1 was maintained and in the second glasshouse elevated [CO 2 ] of 700± 50 µmol mol −1 was maintained throughout the growth period. In the second glasshouse if [CO 2 ] fell below 700 µmol mol −1 a WMA4 CO 2 analyzer (PP Systems, Amesbury, MA, USA) injected the desired amount of CO 2 . After 55 days of growth, plants were swapped from one glasshouse to the other and [CO 2 ] levels were readjusted in each glasshouse as per the treatments. Within each glasshouse, electrical fans continuously circulated the air at an air speed of 0.5 m s −1 over the plants. Daytime air temperatures were maintained for 12h per day beginning at 6 AM. The greenhouses transmitted approximately 60% of the incident PPFD daily. A data logger (21x, Campbell Scientific, Logan, UT, USA) recorded the PPFD, temperature and [CO 2 ] in both glasshouses at 30-s intervals.
In both glasshouses, plants were grown at three levels of photosynthetic photon flux density (PPFD) (100 ± 20, 200 ± 20 and 400 ± 20 µmol m −2 s −1 ). To achieve these three levels of irradiance, mini-chambers were constructed with 2 cm (3/4 inch) diameter PVC pipe with overall dimensions of 114 cm W × 119 cm L × 81 cm H (45" × 47" × 32"). To achieve three different levels of PPFD, the tops and sides of the mini chambers were covered with three types of plastic mesh shade cloth: a single-ply of 70% smoke blue sun screen fabric (Easy Gardener, Waco TX) for low PPFD (100 µmol m −2 s −1 ), a single-ply of black fiberglass window screen (New York Wire, Mt. Wolf, PA, USA) for medium PPFD (200 µmol m −2 s −1 ) and a single-ply of 22% white shade cloth (National Tool Grinding, Inc, Erie, PA, USA) for high PPFD (400 µmol m −2 s −1 ). Each mini chamber was covered with mesh shade cloth so they have full air exchange with the environment. In each mini chamber the plants were rotated once per week to keep the light exposures consistent. The light levels in each mini chamber were measured at mid-day with a LI-190S quantum sensor (Li-Cor Inc., Lincoln, NE, USA). All experimental units were replicated three times and each experimental unit had a control pot with no plant in order to quantify evaporation. O m −2 s −1 ) is stomatal conductance. These parameters were obtained from CIRAS-2 portable photosynthesis system measurements.

Determination of Plant Growth Parameters
After a growth period of 90 days, plants were harvested. Shoots were divided into stems and leaves and weighed. Total leaf area (cm 2 ) was measured using a LI-3100 leaf area meter (Li-Cor Inc., Lincoln, NE, USA). Shoots were washed in deionized water, freezedried and dry weight was recorded. The roots were removed from the soil, washed, blotted dry and weighed. Root lengths (cm plant −1 ) were determined with a Comair Root Length Scanner (Hawker de Haviland, Melbourne, Victoria, Australia) and the roots were oven-dried at 70 • C for 5 days and the dry weights were recorded.
Additional growth parameters were calculated by the following formulas: Leaf area ratio (LAR, cm 2 /g) = [total leaf area, cm 2

Determination of Nutrient Uptake Parameters
Dried stems and leaves were ground together to pass through a 1 mm sieve and sent to University of Florida, Indian River Research and Education Center (UF-IRREC), Fort Pierce, FL, USA. for macro-micro nutrient analysis. Plant samples of 0.4 g were digested in 5 mL of concentrated nitric acid (14 N), and macro-micro nutrient concentrations in the digested solutions were determined by using inductively coupled plasma optical emission spectrometry (ICPOES, Ultima JY Horiba Inc. Edison, NJ, USA) [59]. Total N in the plant tissue was analyzed by combustion method using a CN Analyzer (Vario MAX CN Elementar Analysensysteme GmbH, Hanau, Germany) [60].
Nutrient uptake (U), influx (IN), transport (TR) and nutrient use efficiency ratios (ER) were calculated using the following formulas: Uptake (U) = (Conc. of any given element) × shoot dry wt.
where U refers to elemental content in shoot (mmol/plant), T is time in seconds, Wr is root dry wt., and subscripts 1 and 2 refer to initial and final plant harvest times.
where Ws is shoot dry weight.
Nutrient Use Efficiency (NUE) = [mg of Ws/mg of any given element in shoot]

Statistical Analysis
Experiment was split plot design with [CO 2 ] as main plots, PPFD as subplots and genotypes as sub-sub plots and experimental units were replicated three times. All data were analyzed for statistical significance by ANOVA in SAS (Ver. 9.3, SAS Institute, Cary, NC, USA).

Growth Traits
Irrespective of [CO 2 ] and PPFD, significant intraspecific differences between cacao genotypes were observed for total and root wt., root length, stem height, leaf area, specific leaf area, relative growth rate (RGR) and net assimilation rates (NAR) ( Table 1). Overall, Amaz 15 genotype had higher total and root growth parameters than any of the other genotypes studied. Genetic, physiological and morphological determinants and their interactions with environmental variables such as levels of PPFD and [CO 2 ] profoundly influence the growth, development and nutrient use efficiency of cacao [9,30,35,46]. Variation in morphological characteristics among cacao genotypes has been reported [4,[53][54][55] and these morphological characteristics are known to be influenced by levels of PPFD and [CO 2 ] [1,13,35,45,46,61].
In the current study with exception of root/shoot ratio, all the growth traits of shoots and roots in cacao genotypes were significantly influenced by the level of [CO 2 ]. Irrespective of PPFD levels, increasing [CO 2 ] from 400 to 700 µmol mol −1 increased all growth traits except SLA which decreased with increasing [CO 2 ]. In many perennial tropical legume cover crops Baligar et al. [62,63] reported that increasing [CO 2 ] from ambient (400 µmol mol −1 ) to elevated (700 µmol mol −1 ) increased growth traits (dry biomass of shoot, leaf and roots, RGR and NAR). Generally, C 3 plants respond positively to increased [CO 2 ] above 370 µmol mol −1 [64][65][66]. In the current study, increasing [CO 2 ] significantly increased total leaf area in all the genotypes. Lahive et al. [46] reported increased leaf area in Amelonado cacao genotype grown at elevated [CO 2 ], however, in a recent study, Hebbar et al. [47] found no significant differences in leaf area between cacao grown at 400 and 700 µmol mol −1 [CO 2 ]. In the current study, increasing [CO 2 ] from 400 to 700 µmol mol −1 increased average root dry weight and root length by 0.97 to 2.32 g plant −1 and 2962 to 4974 cm plant −1 respectively. At elevated [CO 2 ], it seems that allocation of carbon fixed by photosynthesis to the roots is as high as that to the shoots. Elevated [CO 2 ] often increases the R/S ratio and fine-root proliferation [43].
In all the cacao genotypes studied, all the growth parameters were significantly influenced by levels of PPFD. Shade tolerant species including cacao are known to respond positively to elevated [CO 2 ], however such enhanced growth response is also governed by light levels [35,46,67,68]. Irrespective of levels of [CO 2 ], increasing PPFD from 100 to 400 µmol m −2 s −1 increased growth traits (total and root weight, stem height, root length, total leaf area, RGR and NAR). However specific leaf area (SLA) was reduced with increasing PPFD indicating that increasing PPFD increases the thickness of the leaves. In cacao genotypes, heavier shade may increase leaf area [19]. Such an adaptation seems to maximize the photon capture capacity of the leaves [45]. Irrespective of [CO 2 ], increasing PPFD from 100 to 400 µmol m −2 s −1 increased average root weight and root length by 1.34 to 1.84 g plant −1 and 3443 to 4357 cm plant −1 , respectively. This indicates an increased allocation of carbon fixed through photosynthesis to roots at higher PPFD. Aerial morphological characteristics could have great implications on the ability of plants to intercept and utilize solar radiation and these characteristics in cacao are influenced by level of irradiance [1,9,13,35,45,61].

Physiological and Water Use Efficiency Traits
Significant intraspecific differences were observed for SPAD index, net photosynthesis (P N ), stomatal conductance (gs), internal CO 2 (Ci) and transpiration (E) irrespective of levels of [CO 2 ] and PPFD (Table 2). Amaz 15 had higher P N than any other cacao genotype at all levels of [CO 2 ] and PPFD evaluated. This genotype also had the highest leaf area per plant. Increasing [CO 2 ] from ambient to 700 µmol mol −1 has been shown to increase P N in C 3 plants [44]. In the current study irrespective of PPFD levels, increasing [CO 2 ] from 400 to 700 µmol mol −1 resulted in a significant increase in P N of all cacao genotypes from an average of 2.47 to 3.41 µmol CO 2 m −2 s −1 . In an earlier study with 1.5-year-old cacao plants, increasing [CO 2 ] from 370 to 680 µmol mol −1 resulted in a 33% increase in P N [30]. In cacao genotype Amelonado, increasing [CO 2 ] from 460 to 735 µmol mol −1 increased P N by 56% [46]. Recently Hebbar et al. [47] in an open top camber study with cacao reported an increase in P N of 29% by increasing [CO 2 ] from 400 to 700 µmol mol −1 . Increasing photosynthesis with increasing [CO 2 ] reported in these studies is typical of responses observed in other C 3 plants [69,70].
Levels of aerial [CO 2 ] have significant effects on gs activity. In all the cacao genotypes studied, irrespective of PPFD levels, increasing [CO 2 ] from 400 to 700 µmol mol −1 resulted in a significant reduction in gs from an average of 19.5 to 12.6 mmol H 2 O m −2 s −1 . In leaves of annual C 3 plants, doubling of [CO 2 ] reduced gs by 34% [69]. In an earlier study with cacao genotypes, Baligar et al. [30] reported around a 65% reduction in gs by increasing [CO 2 ] from 370 to 700 µmol mol −1 . Such a large decrease in gs led to a substantial reduction in E, which could improve cacao water status and drought resistance. Elevated [CO 2 ] has been shown to reduce E and gs in most C 3 plants [69]. However, Lahive et al. [46] reported that CO 2 concentrations of ambient (average of 466 µmol mol −1 ) and elevated (average of 725 µmol mol −1 ) did not have an effect on gs in cacao genotype Amelonado. Stomatal conductance (gs) plays a vital role in regulating P N , transpiration (E), leaf temperature and plant water stress tolerance [13,71,72].
Irrespective of the levels of PPFD, increasing [CO 2 ] from 400 to 700 µmol mol −1 resulted in a significant reduction in transpiration (E) from an average of 0.267 to 0.172 mmol m −2 s −1 . A large decrease in gs, as observed in the current study, with increasing [CO 2 ] could lead to reduced E and such changes could improve water status and drought resistance of cacao. Baligar et al. [30] reported that increasing [CO 2 ] from 85 to 850 µmol mol −1 significantly decreased E from 0.66 to 0.16 mmol m −2 s −1 in three cacao genotypes.
It has been widely reported that maximum photosynthesis (P N ) in cacao occurs at PPFD of 350 to 550 µmol m −2 s −1 [30][31][32][33]. The limited PPFD received at cacao canopy levels might be the reason for lower yields in agroforestry systems [73]. In the seven cacao genotypes in the current study, irrespective of [CO 2 ], increasing levels of PPFD from 100 to 400 µmol m −2 s −1 resulted in significant increases in P N from an average of 2.67 to 3.41 µmol m −2 s −1 . In an earlier study with three genetically differing cacao genotypes, Baligar et al. [30] reported that increasing PPFD from 50 to 400 µmol m −2 s −1 significantly increased P N . However, P N at 50 µmol m −2 s −1 of PPFD was about twothirds of the maximum 3 µmol CO 2 m −2 s −1 indicating that cacao needs very little radiant energy to support its P N . Higher rates of P N , thicker leaves and high rates of E have been observed in certain cacao genotypes when grown in full sunlight rather than under shade [1]. Increasing PPFD from 100 to 400 µmol m −2 s −1 reduced specific leaf area from an average of 284.7 to 227.7 cm 2 g −1 , such increases in leaf thickness might contribute to higher P N . However, exposure of leaves to extremely high light for longer periods may lead to photoinhibition and lower P N [34,41,42]. Baligar et al. [35] reported that PPFD of 1050 µmol m −2 s −1 was detrimental to shoot, root and leaf growth of cacao seedlings. In all the cacao genotypes studied irrespective of levels of [CO 2 ], increasing levels of PPFD from 100 to 400 µmol m −2 s −1 resulted in significant increases in gs and E from an average of 15.33 to 19.35 mmol H 2 O m −2 s −1 and 0.215 to 0.258 mmol H 2 O m −2 s −1 , respectively. In an earlier short-term study, Baligar et al. [30] reported that the gs was not significantly affected by PPFD over the observed range of 50 to 400 µmol m −2 s −1 ; however, there was a slight increase in E, but the relationship between E and PPFD was not significant. Under artificial shade, the quality of the PPFD that reaches the canopy of cocoa leaves is very different from the quality of the PPFD that reaches the canopy of cocoa trees leaves grown in field conditions and shaded by tree species. In field conditions, there is an attenuation of both the intensity and the quality of the light available for cocoa photosynthesis, depending on the greater or lesser absorbance and/or transmittance of electromagnetic light, mainly in the blue and red bands, which crosses canopy strata of different shade tree species. Depending on the photosynthetic characteristics of the shade tree canopy, different levels of PPFD blue and red light are absorbed and/or transmitted, which can affect net photosynthesis differently from cocoa grown under artificial shade. Therefore, obtained results of P N are based on cacao genotypes subjected to ambient and elevated levels of [CO 2 ] under various levels of artificial shade. Table 2. The effect of [CO 2 ] and photosynthetic photon flux density (PPFD) on photosynthesis and its components, and water use efficiency of seven cacao genotypes.  Intraspecific differences in WUE traits (WUE Total , WUE Inst and WUE Intr ) between the cacao genotypes were observed but the differences were not significant ( Table 2). Amaz 15 had the highest WUE Inst and WUE Intr , which is a reflection of its high P N compared to the other cacao genotypes. In eight contrasting cacao genotypes, variations in WUE Inst and WUE Intr were negatively related to specific leaf area [4]. In the current study, all three WUE traits increased with decreasing specific leaf area. In the seven cacao genotypes studied, increasing PPFD and [CO 2 ], increased WUE traits. Increasing [CO 2 ] from 400 to 700 µmol mol −1 caused significant increases in all three water use efficiency traits (WUE Total , WUE Inst and WUE Intr ). Such significant increases in WUE Inst and WUE Intr traits at elevated [CO 2 ] could be related to increased P N and reduced gs and E [13,74]. Lahive et al. [46] reported significantly greater intrinsic water use efficiency (WUE Intr ) in plants grown at elevated CO 2 (average of 725 µmol mol −1 ) and related such an increase to higher P N , as there was no difference in the measured gs between ambient and elevated CO 2 . In open top chambers, elevated [CO 2 ] up to 700 µmol mol −1 increased P N by 27% and resulted in high cacao biomass accumulation, and thus improved whole plant WUE [47]. Further Hebbar et al. [47] concluded that higher WUE at elevated [CO 2 ] was due to high P N rather than reduced water loss through stomata (E). In the current research with seven contrasting cacao genotypes, increasing [CO 2 ] from 400 to 700 µmol mol −1 significantly increased P N but gs and E were reduced significantly. Based on these findings, it is concluded that increasing P N and decreasing gs and E at elevated [CO 2 ] substantially contributes to the significant increases in WUE Inst and WUE Intr [30,46,74]. Enhanced WUE Intr at elevated [CO 2 ] is related to maintenance of higher plant water potential (Ψ) through reduced gs and greater fine root production [43]. Reduced gs in elevated [CO 2 ] may alter plant responses to drought and improve WUE [75].
Irrespective of levels of [CO 2 ], increasing levels of PPFD from 100 to 400 µmol m −2 s −1 increased WUE Total , but there were no changes in WUE Inst . Increasing PPFD from 100 to 400 µmol m −2 s −1 slightly reduced WUE Intr from an average of 0.22 to 0.19 µmol CO 2 mmol H 2 O −1 . This is a reflection of increases of gs from average of 15.33 to 19.35 mmol H 2 O m −2 s −1 and moderate increases in P N from average of 2.67 to 3.41 µmol m −2 s −1 . In other crops, it has been reported that relationships between WUE Total and WUE Inst may be either positive or negative [76]. Increases or decreases in WUE traits with varying PPFD and [CO 2 ] are determined by increases or decreases of P N , gs, and E [4,13,46]. As occurrences of drought episodes are becoming more common in tropical cacao regions [77][78][79], selection of cacao genotypes with high WUE under increasing levels of [CO 2 ] would be beneficial in sustaining yield potential of cacao in current and future drought prone areas.

Nutrient Concentrations and Uptake
Cacao genotypes, irrespective of levels of [CO 2 ] and PPFD, showed significant differences in macro-micro nutrient concentrations (Table 3). Overall, LCT EEN 37A, compared to the other genotypes, had the highest concentrations of P, K, Ca, Cu and Fe. The concentrations of P, Ca, Mg and Mn were slightly higher, but concentrations of other macro and micronutrients were comparable to the concentrations reported in the literature [50,80,81]. In all the genotypes tested, irrespective of PPFD, increasing [CO 2 ] from 400 to 700 µmol mol −1 significantly reduced macro-micro nutrient concentrations; however, the effect of increasing [CO 2 ] on Zn concentration was non-significant. This is a reflection of increased dry matter in the shoots (Table 1) of all cacao genotypes with increasing [CO 2 ] which created dilution effects on the nutrient concentrations. The decline in concentrations of all macro-micro nutrients in cacao genotypes with increasing levels of [CO 2 ] differed slightly from the conclusion drawn by Dong et al. [82] from meta-analysis of vegetable crops. They concluded that elevated [CO 2 ] enhanced yield in vegetable crops but decreased the concentration of nitrate, Mg, Fe, and Zn by 18.0, 9.2, 16.0 and 9.4%, respectively, and increased the concentration of Ca by 8.2%. However, the concentration of P, K, S, Cu and Mn in that study were not affected by elevated [CO 2 ]. In Amelonado cacao, Lahive et al. [46] reported that leaf N content decreased at elevated [CO 2 ]. In mango leaves, elevated levels of [CO 2 ] reduced concentrations of several minerals [83]. With the exception of N, Cu and Mn, and irrespective of levels of [CO 2 ], increasing PPFD from 100 to 400 µmol m −2 s −1 reduced concentrations of the other macro and micronutrients. However, the effects were only significant for concentrations of N, K, Ca, Mg and Mn. In several tropical perennial cover crop legumes, increasing [CO 2 ] from 400 to 700 µmol mol −1 slightly decreased all the macro-micro nutrient concentrations. However, increasing PPFD from 100 to 450 µmol m −2 s −1 only slightly decreased concentrations of K, Ca and Fe [62]. In another study with perennial legume cover crops, Baligar et al., [84] reported that increasing PPFD from 200 to 400 µmol m −2 s −1 significantly decreased the concentrations of most of the micronutrients and they attributed this to increased dry matter at the slightly higher PPFD which caused dilution effects.
Uptake of all macro-micro nutrients were significantly influenced by genotypes and Amaz 15 had the highest nutrient uptake (Table 4). Overall, increasing levels of [CO 2 ] from 400 to 700 µmol mol −1 and PPFD from 100 to 400 µmol m −2 s −1 significantly increased uptake of all the macro-micro nutrients. In cacao genotype comum, Baligar et al. [35] reported that increasing [CO 2 ] from 380 to 700 µmol mol −1 increased uptake of all essential nutrients and further stated that such an increase in nutrient uptake at higher [CO 2 ] is due to increased demand for mineral nutrients due to enhanced dry matter accumulation. The overall nutrient accumulation in the current study was in the order of N > Ca >K >Mg > P for macro nutrients and Mn > Zn > Fe > B > Cu for micronutrients.

Nutrient Influx (IN) and Transport (TR)
In most of the cacao growing regions, cacao is often grown in infertile acidic soils and is subjected to the high temperature and radiation common with low soil moisture levels. Such climatic stresses could have major effects on the ability of plants to influx (IN) nutrients from soil through the roots and to transport (TR) these essential nutrients to shoots. In addition to these stresses, increasing atmospheric concentrations of [CO 2 ] could aggravate rates of IN and TR by increasing transpiration losses and photosynthesis. However, very limited information is available on how increasing levels of [CO 2 ] and low to adequate levels of PPFD affect IN and TR of macro-micro nutrients in cacao. In the current study, IN for all macro and micro nutrients were significantly influenced by genotypes, [CO 2 ] and PPFD ( Table 5). Irrespective of levels of [CO 2 ] and PPFD, cacao genotype SCA 6 had higher IN of all macro-micro nutrients. Based on these findings SCA 6 could be a superior genotype to use as rootstock in establishing new plantations in infertile soils under changing climatic conditions. In the current study, irrespective of levels of PPFD, IN for all macro-micro nutrients increased significantly by increasing [CO 2 ] from 400 to 700 µmol mol −1 . It has been previously reported in cacao genotype comum that increasing [CO 2 ] from 380 to 700 µmol mol −1 tended to increase IN for many of the essential nutrients [35]. In the current study, increasing PPFD from 100 to 400 µmol m −2 s −1 significantly increased IN for all nutrients irrespective of levels of [CO 2 ]. Baligar et al. [35] found a similar result, but also that increases in PPFD to 1050 µmol m −2 s −1 tended to decrease IN for N, K, Ca, Mg, P, S, Cu and Fe. Increased plant influx (IN) of more nutrients from the growth medium helps meet increased demand by increased shoot biomass accumulation.
With the exceptions of K, Ca and Cu, transport (TR) for the other macro-micro nutrients were significantly influenced by cacao genotypes (Table 6). SCA 6 was superior in transport of N, Ca, Fe, and Mn and Coca 3370 was superior in TR for Mg, B and Mn. Overall, with a few exceptions, TR for all the macro-micro nutrients were significantly increased by increasing [CO 2 ] from 400 to 700 µmol mol −1 and PPFD from 100 to 400 µmol m −2 s −1 . In cacao genotype comum, Baligar et al. [35] reported that increasing [CO 2 ] from 380 to 700 µmol mol −1 decreased TR of N, Ca, and Zn, and increased TR for other elements. Such variations in IN and TR at varying levels of [CO 2 ] and PPFD could be related to nature of genotypes and their interactions with levels of [CO 2 ] and PPFD.

Nutrient Use Efficiency
With the exception of Mn, all cacao genotypes in this study, irrespective of levels of [CO 2 ] and PPFD, showed significant differences for NUE of all the other essential nutrients ( Table 7). The existence of interspecific variations in NUE of macro and micro nutrients have been well documented for field, horticultural and perennial legume crops [62,63,[85][86][87][88]. Variations in the growth and uptake and nutrient use efficiency among crop cultivars have been related to absorption, translocation, shoot demand, and dry matter production potentials per unit of nutrient absorbed [85,86]. In agroforestry systems, cacao is grown as an understory plant and subjected to rising [CO 2 ] and low levels of PPFD. Under such situations cacao genotypes that have high nutrient use efficiency for essential nutrients might be able to grow well and produce higher yields. Deficiencies of P, Ca, Mg, Zn and Fe have been widely reported in soils of cacao growing regions of the world [11,48,50]. Cabala-Rosand et al. [50] state that under field conditions the most common deficiencies noted in cacao are N, K, Zn, Fe and B. P is also a limiting nutrient in almost all soils under cacao [49]. Genotypes that have high NUE for any of these nutrients could improve the sustainability and productivity of cacao grown in nutrient deficient soils under agroforestry systems. Amaz 15 was most efficient in NUE for N, K, Ca, B, Cu and Fe and SCA 6 was most efficient for P and K. Since Amaz15 had the longest root length among the cacao genotypes tested, this probably helped it to acquire more nutrients. Barber [89] states that the quantity of a nutrient taken up by a plant depends on the configuration and growth rate of the roots. Irrespective of levels of PPFD, increasing [CO 2 ] from 400 to 700 µmol mol −1 significantly increased NUE for all nutrients. In cacao Comum, Baligar et al. [35]

Conclusions
Under glasshouse conditions, elevated [CO 2 ] increased growth, physiology, nutrient uptake and use efficiency; however, low light decreased growth, photosynthesis and nutrient uptake of cacao genotypes. Intraspecific differences were found in the genotypes such that AMAZ 15 was the highest for many parameters and LCT EEN 37A was often the lowest. Na 33 had high Fe uptake which could be a problem on Fe limited soils, but further testing is needed. Higher WUE in increasing levels of [CO 2 ] should be considered in selection of cacao genotypes useful for drought prone areas to maintain cacao sustainability and improve yields.