1H NMR Mixture Design-Fingerprints and ASCA Analysis in Ilex paraguariensis: Model Stability in Search of a Global Metabolome.

The effects of experimental repetitions and solvent extractors on the 1H NMR fingerprinting of yerba mate extracts, obtained from two genders and two light environments, were analyzed in-depth by ANOVA-simultaneous component analysis (ASCA). Different solvents were used according to a mixture design based on ethanol, dichloromethane, and hexane and their combinations. The number of experimental repetitions significantly affected the ASCA results. Increasing repetitions led to decreases in the percentage effect variance values and an increase in the percentage residual variance. However, secondary sexual dimorphism, light availability, and their interaction effects became more significant with decreasing p-values at or above the 95% confidence level. The choice of a solvent extractor significantly affects the chemical profile and can lead to distinct conclusions regarding the significance of effect values. Pure solvents yielded different conclusions about the significance of factorial design effects, with each solvent extracting unique metabolites and maximizing information for specific effects. However, the use of binary solvent mixtures, such as ethanol-dichloromethane, proved more efficient in extracting sets of compounds that simultaneously differentiate between different experimental conditions. The mixture design-fingerprint strategy provided satisfactory results expanding the range of extracted metabolites with high percentage of residual variances and low explained percentage effect variances in the ASCA models. Ternary and even higher-ordered mixtures could be good alternative extracting media for work-intensive procedures. Our study underscores the significance of experimental design and solvent selection in metabolomic analysis, improving the accuracy, robustness, and interpretability of metabolomic models, leading to a better understanding of the chemical composition and biological implications of plant extracts.

Stability of Leaf Yerba Mate (Ilex paraguariensis) Metabolite Concentrations over the Time from the Prism of Secondary Sexual Dimorphism.

The yerba mate leaf metabolic composition depends mainly on genetics, sex, plant and leaf age, light intensity, harvest time, climate, and fertilization. In yerba mate, the secondary sexual dimorphism (SSD), the leaf metabolic SSD association with the frequency of leaf harvests, and the stability of the metabolites in the two genders over the years is not known. It was hypothesized that (1) the SSD in the metabolite segregation would differ among the winter and summer growth pauses, (2) females would show lower metabolite concentrations, and (3) the metabolic concentrations would show stability over the years on the same plants, not obligatorily associated with the SSD stability expression. Variations in theobromine, caffeine, chlorogenic and caffeic acids were correlated to the increasing time since the previous harvest, especially in females. However, the frequency of the metabolic SSD were associated with the studied growth pauses, rejecting the first hypothesis. No regular gender superiority was expressed in the yerba mate leaf secondary metabolites, rejecting our second hypothesis, even though more cases of superior female metabolite accumulation were identified. The stability of the leaf protein was preserved over the four years, with no SSD cases observed. The leaf methylxanthines were time stable, while the decrease in the phenolic content occurred with tree aging, which was not associated with the SSD expression, partially proving our third hypothesis. The novelty was related to the time stability of the leaf metabolic SSD observed over the winter and summer growth pauses, and over the four consecutive years without a regular expression of the male- or female-biased concentrations in the studied metabolites. To demystify the random metabolic gender responses in yerba mate, gender-orientated experiments with a high number of tree repetitions must be conducted, including clonal plants grown in various environments, such as monoculture and agroforestry, or on plantations in different climates and altitudes.

Correlating Coffea canephora 3D architecture to plant photosynthesis at a daily scale and vegetative biomass allocation.

Coffea canephora (C. canephora) has two botanical varieties, Robusta and Conilon. Intraspecific variability was hypothesized and projected for the selection of C. canephora plants able to maintain production in the context of global climate changes. For that, architectural, C-assimilation and biomass analyses were performed on 17-month-old Robusta (clones 'A1' and '3 V') and Conilon (clones '14' and '19') varieties grown in non-limiting soil, water and mineral nutrient conditions. Nondestructive coffee plant architecture coding, reconstruction and plant photosynthesis estimations were performed using a functional-structural plant modeling platform OpenAlea. 3D reconstructions and inclusion of parameters calculated and estimated from light response curves, such as dark respiration (Rd), maximum rate of carboxylation of RuBisCO and photosynthetic electron transport allowed the estimation of instantaneous and daily plant photosynthesis. The virtual orchard leaf area index was low, and light was not a limiting factor in early C. canephora development stages. Under such conditions, Robusta assimilated more CO2 at the plant and orchard scale and produced higher total biomass than Conilon. Lower plant daily photosynthesis and total biomass were correlated to higher Rd in Conilon than in Robusta. Among the architectural traits, leaf inclination, size and allometry were most highly correlated with plant assimilation and biomass. Relative allocation in leaf biomass was higher in '19' Conilon than in young Robusta plants, indicating intraspecific biomass partitioning. Similarly, variation in relative distribution of the root biomass and the root volume reflected clonal variation in soil occupation, indicating intraspecific variability in space occupation competitiveness. Coffea canephora denoted high root allocation in both Conilon and Robusta clones. However, relevant differences at subspecific levels were found, indicating the high potential of C. canephora to cope with drought events, which are expected to occur more frequently in the future, because of climate changes. The methodology developed here has the potential to be used for other crops and tree species. Highlights Functional-structural plant model was used to estimate photosynthesis on a plant and daily scales in Coffea canephora (C. canephora). Among the architectural traits, leaf shape and inclination had the most impact on photosynthesis and biomass. Under non-limiting conditions, Robusta had higher plant photosynthesis and biomass than Conilon. A higher leaf biomass allocation in Conilon clone '19' than in Robusta suggested variety-specific partitioning. Variation in the relative distribution of the root biomass indicated C. canephora intraspecific soil occupation variability.

Uncovering the wide protective responses in Coffea spp. leaves to single and superimposed exposure of warming and severe water deficit.

Climate changes boosted the frequency and severity of drought and heat events, with aggravated when these stresses occur simultaneously, turning crucial to unveil the plant response mechanisms to such harsh conditions. Therefore, plant responses/resilience to single and combined exposure to severe water deficit (SWD) and heat were assessed in two cultivars of the main coffee-producing species: Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153). Well-watered plants (WW) were exposed to SWD under an adequate temperature of 25/20°C (day/night), and thereafter submitted to a gradual increase up to 42/30°C, and a 14-d recovery period (Rec14). Greater protective response was found to single SWD than to single 37/28°C and/or 42/30°C (except for HSP70) in both cultivars, but CL153-SWD plants showed the larger variations of leaf thermal imaging crop water stress index (CWSI, 85% rise at 37/28°C) and stomatal conductance index (IG, 66% decline at 25/20°C). Both cultivars revealed great resilience to SWD and/or 37/28°C, but a tolerance limit was surpassed at 42/30°C. Under stress combination, Icatu usually displayed lower impacts on membrane permeability, and PSII function, likely associated with various responses, usually mostly driven by drought (but often kept or even strengthened under SWD and 42/30°C). These included the photoprotective zeaxanthin and lutein, antioxidant enzymes (superoxide dismutase, Cu,Zn-SOD; ascorbate peroxidase, APX), HSP70, arabinose and mannitol (involving de novo sugar synthesis), contributing to constrain lipoperoxidation. Also, only Icatu showed a strong reinforcement of glutathione reductase activity under stress combination. In general, the activities of antioxidative enzymes declined at 42/30°C (except Cu,Zn-SOD in Icatu and CAT in CL153), but HSP70 and raffinose were maintained higher in Icatu, whereas mannitol and arabinose markedly increased in CL153. Overall, a great leaf plasticity was found, especially in Icatu that revealed greater responsiveness of coordinated protection under all experimental conditions, justifying low PIChr and absence of lipoperoxidation increase at 42/30°C. Despite a clear recovery by Rec14, some aftereffects persisted especially in SWD plants (e.g., membranes), relevant in terms of repeated stress exposure and full plant recovery to stresses.

Ecometabolic mixture design-fingerprints from exploratory multi-block data analysis in Coffea arabica beans from climate changes: Elevated carbon dioxide and reduced soil water availability.

Ecometabolic mixture design-fingerprinting in coffee cultivated under climate change was chemically explored using ComDim. Multi-blocks were formed using UV, NIRS, 1H NMR, SWV, and FT-IR data. ComDim investigated all these different fingerprints according to the extractor solvent and in virtue of atmospheric CO2 increase. Ethanol and ethanol-dichloromethane showed the best separations due to CO2 environment. 1H NMR loading indicate increases of fatty acids, caffeine, trigonelline, and glucose in beans under current CO2 levels, whereas quinic acid/chlorogenic acids, malic acid, and kahweol/cafestol increased in beans under elevated CO2 conditions. SWV indicated quercetin and chlorogenic acid as important compounds in coffee beans cultivated under current and elevated CO2, respectively. Based on the ethanol and ethanol-dichloromethane fingerprints, k-NN correctly classified the beans cultivated under different carbon dioxide environments and water availabilities, confirming the existence of metabolic changes due to climate changes. SWV proved to be promising compared with widely used spectrometric methods.

Biomass and Leaf Acclimations to Ultraviolet Solar Radiation in Juvenile Plants of Coffea arabica and C. canephora.

Despite the negative impacts of increased ultraviolet radiation intensity on plants, these organisms continue to grow and produce under the increased environmental UV levels. We hypothesized that ambient UV intensity can generate acclimations in plant growth, leaf morphology, and photochemical functioning in modern genotypes of Coffea arabica and C. canephora. Coffee plants were cultivated for ca. six months in a mini greenhouse under either near ambient (UVam) or reduced (UVre) ultraviolet regimes. At the plant scale, C. canephora was substantially more impacted by UVam when compared to C. arabica, investing more carbon in all juvenile plant components than under UVre. When subjected to UVam, both species showed anatomic adjustments at the leaf scale, such as increases in stomatal density in C. canephora, at the abaxial and adaxial cuticles in both species, and abaxial epidermal thickening in C. arabica, although without apparent impact on the thickness of palisade and spongy parenchyma. Surprisingly, C. arabica showed more efficient energy dissipation mechanism under UVam than C. canephora. UVam promoted elevated protective carotenoid content and a greater use of energy through photochemistry in both species, as reflected in the photochemical quenching increases. This was associated with an altered chlorophyll a/b ratio (significantly only in C. arabica) that likely promoted a greater capability to light energy capture. Therefore, UV levels promoted different modifications between the two Coffea sp. regarding plant biomass production and leaf morphology, including a few photochemical differences between species, suggesting that modifications at plant and leaf scale acted as an acclimation response to actual UV intensity.

Linking root and stem hydraulic traits to leaf physiological parameters in Coffea canephora clones with contrasting drought tolerance.

Knowing the key hydraulic traits of different genotypes at early seedling stages can potentially provide crucial information and save time for breeding programs. In the current study we investigated: (1) how root, stem and whole plant conductivities are linked to xylem traits, and (2) how the integrated hydraulic system impacts leaf water potential, gas exchange, chlorophyll a fluorescence and the growth of three coffee cultivars (clones of Coffea canephora Pierre ex Froehner cv. Conilon) with known differences in drought tolerance. The Conilon clones CL 14, CL 5 V and CL 109A, classified as tolerant, moderately tolerant, and sensitive to drought respectively, were grown under non-limiting soil-water supply but high atmospheric demand (i.e., high VPDair). CL 14 and CL 5 V displayed higher root and stem hydraulic conductance and conductivity, and higher whole plant conductivity than CL 109A, and these differences were associated with higher root growth traits. In addition, CL 109A exhibited a non-significant trend towards wider vessels. Collectively, these responses likely contributed to reduce leaf water potential in CL 109A, and in turn, reduced leaf gas exchange, especially during elevated VPDair. Even when grown under well-watered conditions, the elevated VPDair observed during this study resulted in key differences in the hydraulic traits between the cultivars corresponding to differences in plant water status, gas exchange, and photochemical activity. Together these results suggest that coffee hydraulic traits, even when grown under non-water stress conditions, can be considered in breeding programs targeting more productive and efficient genotypes under drought and high atmospheric demand.

Leaf gas exchange and bean quality fluctuations over the whole canopy vertical profile of Arabic coffee cultivated under elevated CO2.

Leaves in different positions respond differently to dynamic fluctuations in light availability, temperature and to multiple environmental stresses. The current hypothesis states that elevated atmospheric CO2 (e[CO2]) can compensate for the negative effects of water scarcity regarding leaf gas exchanges and coffee bean quality traits over the canopy vertical profile, in interactions with light and temperature microclimate during the two final stages of berry development. Responses of Coffea arabica L. were observed in the 5th year of a free air CO2 enrichment experiment (FACE) under water-limited rainfed conditions. The light dependent leaf photosynthesis curves (A/PAR) were modelled for leaves sampled from vertical profile divided into four 50-cm thick layers. e[CO2] significantly increased gross photosynthesis (AmaxGross), the apparent quantum yield efficiency, light compensation point, light saturation point (LSP) and dark respiration rate (Rd). As a specific stage response, considering berry ripening, all parameters calculated from A/PAR were insensitive to leaf position over the vertical profile. Lack of a progressive increase in AmaxGross and LSP was observed over the whole canopy profile in both stages, especially in the two lowest layers, indicating leaf plasticity to light. Negative correlation of Rd to leaf temperature (TL) was observed under e[CO2] in both stages. Under e[CO2], stomatal conductance was also negatively correlated with TL, reducing leaf transpiration and Rd even with increasing TL. This indicated coffee leaf acclimation to elevated temperatures under e[CO2] and water restriction. The e[CO2] attenuation occurred under water restriction, especially in A and water use efficiency, in both stages, with the exception of the lowest two layers. Under e[CO2], coffee produced berries in moderate- and high light level layers, with homogeneous distribution among them, contrasted to the heterogeneous distribution under actual CO2. e[CO2] led to increased caffeine content in the highest layer, with reduction of chlorogenic acid and lipids under moderate light and to raised levels of sugar in the shaded low layer. The ability of coffee to respond to e[CO2] under limited soil water was expressed through the integrated individual leaf capacities to use the available light and water, resulting in final plant investments in new reproductive structures in moderate and high light level layers.

Kaolin Film Increases Gas Exchange Parameters of Coffee Seedlings During Transference From Nursery to Full Sunlight.

Increases in water use efficiency (WUE) and the reduction of negative impacts of high temperatures associated with high solar radiation are being achieved with the application of fine particle film of calcined and purified kaolin (KF) on the leaves and fruits of various plant species. KF was applied on young Coffea arabica and Coffea canephora plants before their transition from nursery to full sunlight during autumn and summer. The effects of KF were evaluated through the responses of leaf temperature (Tleaf), net CO2 assimilation rate (A), stomatal conductance (g s), transpiration (E), WUE, crop water stress index (CWSI), index of relative stomatal conductance (Ig), initial fluorescence (F0), and photosynthetic index (PI) in the first 2-3 weeks after the plant transitions to the full sun. All measurements were performed at midday. In Coffea plants, KF decreased the Tleaf up to 6.7°C/5.6°C and reduced the CWSI. The plants that were not protected with KF showed lower A, g s, E, and Ig than those protected with KF. C. canephora plants protected with KF achieved higher WUE compared with those not protected by 11.23% in autumn and 95.58% in summer. In both Coffea sp., KF application reduced F0, indicating reduced physical dissociation of the PSII reaction centers from the light-harvesting system, which was supported with increased PI. The use of KF can be recommended as a management strategy in the transition of Coffea seedlings from the nursery shade to the full sunlight, to protect leaves against the excessive solar radiation and high temperatures, especially in C. canephora during the summer.

The main effects of elevated CO2 and soil-water deficiency on 1H NMR-based metabolic fingerprints of Coffea arabica beans by factorial and mixture design.

The metabolic response of Coffea arabica trees in the face of the rising atmospheric concentration of carbon dioxide (CO2) combined with the reduction in soil-water availability is complex due to the various (bio)chemical feedbacks. Modern analytical tools and the experimental advance of agronomic science tend to advance in the understanding of the metabolic complexity of plants. In this work, Coffea arabica trees were grown in a Free-Air Carbon Dioxide Enrichment dispositive under factorial design (22) conditions considering two CO2 levels and two soil-water availabilities. The 1H NMR mixture design-fingerprinting effects of CO2 and soil-water levels on beans were strategically investigated using the principal component analysis (PCA), analysis of variance (ANOVA) - simultaneous component analysis (ASCA) and partial least squares-discriminant analysis (PLS-DA). From the ASCA, the CO2 factor had a significant effect on changing the 1H NMR profile of fingerprints. The soil-water factor and interaction (CO2 × soil-water) were not significant. 1H NMR fingerprints with PCA, ASCA and PLS-DA analysis determined spectral profiles for fatty acids, caffeine, trigonelline and glucose increases in beans from current CO2, while quinic acid/chlorogenic acids, malic acid and kahweol/cafestol increased in coffee beans from elevated CO2. PLS-DA results revealed a good classification performance between the significant effect of the atmospheric CO2 levels on the fingerprints, regardless of the soil-water availabilities. Finally, the PLS-DA model showed good prediction ability, successfully classifying validation data-set of coffee beans collected over the vertical profile of the plants and included several fingerprints of different extracting solvents. The results of this investigation suggest that the association of experimental design, mixture design, PCA, ASCA and PLS-DA can provide accurate information on a series of metabolic changes provoked by climate changes in products of commercial importance, in addition to minimizing the extra work necessary in classic analytical approaches, encouraging the development of similar strategies.

Factorial design fingerprint discrimination of Coffea arabica beans under elevated carbon dioxide and limited water conditions.

Coffee is one of the most important commodities, showing sensitivity to environmental variations. The main effects and their interaction for two levels of atmospheric CO2 concentrations and two water regimes of a factorial design were investigated for the metabolic profiles of Coffea arabica raw beans using UV fingerprint analysis from a mixture design. UV fingerprint results obtained from pure ethanol and binary ethanol-dichloromethane mixtures showed the largest metabolic discriminations between CO2 levels and their extracts were investigated in detail. The biosynthesis of major metabolites, chlorogenic acids, cafestol, kahweol and caffeine were altered owing to environmental conditions. Higher amounts of chlorogenic acids and kahweol were observed in beans from unirrigated plants grown with enriched CO2 and irrigated ones at the current CO2 level. Water availability and CO2 concentration interaction affects the metabolite amounts. Besides a significant CO2 atmospheric effect water availability was a limiting factor for metabolite content only at current CO2 level, suggesting the successful metabolic coping of CO2 enriched Arabic coffee beans suffering future droughts.

Sequential mixture design optimization for divergent metabolite analysis: Enriched carbon dioxide effects on Coffea arabica L. leaves and buds.

The first metabolic study of the impact of elevated CO2 (590 µL CO2 L-1) levels on the leaves and buds of Coffea arabica L. plants is reported. A novel sequential statistical mixture design strategy allowed optimization of both the extraction and mobile phase solvent systems to increase differences detected in metabolites of Coffea arabica L. plants and buds. Factor analysis showed that the 227 and 273 nm bands of the 1:1:1 ternary ethyl ether - dichloromethane - methanol mixture spectra resulted in discrimination of elevated CO2 extract samples from those obtained from leaves grown in a current level CO2 atmosphere (390 µL CO2 L-1) of leaf sample extracts. DAD-HPLC spectral peak evidence showed a 32% increase in absorbance of the 273 band for the enriched CO2 leaf extracts. This band has been assigned to caffeine-like substances and confirmed by the mass spectral signal at m/z 195 ([M + H]+). No enrichment band increases were found for kahweol, kaempferol and quercetin that had presence confirmed by mass spectral analysis. No epigenetic effect of this metabolic profile was found in new leaves after the addition of CO2 stopped. Enriched CO2 perturbation of the bud metabolite were much smaller than for the leaf samples. Absorbance increases in the 228 nm and decreases in the 235 nm bands play a prominent role in the discrimination of enriched CO2 buds from the controls in the pure dichloromethane extracting solvent. This global metabolome strategy allows the monitoring of chemical groups of plants susceptible to environmental changes as well as elucidate metabolic variations in complex matrices of biochemical responses.

PARAFAC HPLC-DAD metabolomic fingerprint investigation of reference and crossed coffees.

In this study two cultivars of Coffea arabica L., Bourbon (reference) and IPR101 (crossing) were analyzed. The extracts were prepared according to a simplex centroid design with four components, ethanol, ethyl acetate, dichloromethane, and hexane. Multiway data were obtained by HPLC-DAD analysis of the fifteen different mixtures for each cultivar. The PARAFAC methodology was used to investigate the chromatographic fingerprint. For both cultivars, Factor 1 was able to discriminate mixtures containing ethyl acetate as solvent. Factor 2 indicated that mixtures in pure ethanol and binary mixtures containing ethanol were the most efficient in extracting chlorogenic acids and factor 3 identified methylxanthines through spectrophotometric profile in all mixtures. Higher concentrations were obtained by the ethanol, dichloromethane and hexane ternary mixture for the Bourbon cultivar and by the quaternary mixture of these solvents with ethyl acetate for the IPR101 cultivar. Trigonelline and cafestol were extracted in both cultivars. The reference coffee showed higher relative abundances of cafestol ester, chlorogenic acids and trigonelline whereas the crossed coffee showed higher levels of caffeine. To confirm these results, UPLC-MS was used as a complementary method to confirm the presence of the metabolites in these extracts. The three way PARAFAC strategy determines correlations of HPLC-DAD chromatographic and spectral data simultaneously with samples permitting a more unambiguous assignment of metabolic groups than can be obtained treating chromatographic and spectral data separately by two way methods. This can provide higher quality chromatographic fingerprints for food chemistry.

Daily heliotropic movements assist gas exchange and productive responses in DREB1A soybean plants under drought stress in the greenhouse.

Drought stress is one of the most severe environmental constraints on plant production. Under environmental pressures, complex daily heliotropic adjustments of leaflet angles in soybean can help to reduce transpiration losses by diminishing light interception (paraheliotropism), increase diurnal carbon gain in sparse canopies and reduce carbon gain in dense canopies by solar tracking (diaheliotropism). The plant materials studied were cultivar BR 16 and its genetically engineered isoline P58, ectopically overexpressing AtDREB1A, which is involved in abiotic stress responses. We aimed to follow the movements of central and lateral leaflets in vegetative stages V7-V10 and reproductive stages R4-R5, integrating the reversible morphogenetic changes into an estimate of daily plant photosynthesis using three-dimensional modeling, and to analyze the production parameters of BR 16 and P58. The patterns of daily movements of central leaflets of BR 16 in V7-V10 and R4-R5 were similar, expressing fewer diaheliotropic movements under drought stress than under non-limiting water conditions. Daily heliotropic patterns of lateral leaflets in V7-V10 and R4-R5 showed more diaheliotropic movements in drought-stressed P58 plants than in those grown under non-limiting water conditions. Leaf area in R4-R5 was generally higher in P58 than in BR 16. Drought significantly affected gas exchange and vegetative and reproductive architectural features. DREB1A could be involved in various responses to drought stress. Compared with the parental BR 16, P58 copes with drought through better compensation between diaheliotropic and paraheliotropic movements, finer tuning of water-use efficiency, a lower transpiration rate, higher leaf area and higher pod abortion to accomplish the maximum possible grain production under continued drought conditions.

Variations in leaf growth parameters within the tree structure of adult Coffea arabica in relation to seasonal growth, water availability and air carbon dioxide concentration.

Background and Aims: Dynamics in branch and leaf growth parameters, such as the phyllochron, duration of leaf expansion, leaf life span and bud mortality, determine tree architecture and canopy foliage distribution. We aimed to estimate leaf growth parameters in adult Arabica coffee plants based on leaf supporter axis order and position along the vertical profile, considering their modifications related to seasonal growth, air [CO2] and water availability. Methods: Growth and mortality of leaves and terminal buds of adult Arabica coffee trees were followed in two independent field experiments in two sub-tropical climate regions of Brazil, Londrina-PR (Cfa) and Jaguariúna-SP (Cwa). In the Cwa climate, coffee trees were grown under a FACE (free air CO2 enrichment) facility, where half of those had been irrigated. Plants were observed at a 15-30 d frequency for 1 year. Leaf growth parameters were estimated on five axes orders and expressed as functions of accumulated thermal time (°Cd per leaf). Key Results: The phyllochron and duration of leaf expansion increased with axis order, from the seond to the fourth. The phyllochron and life span during the reduced vegetative seasonal growth were greater than during active growth. It took more thermal time for leaves from the first- to fourth-order axes to expand their blades under irrigation compared with rainfed conditions. The compensation effects of high [CO2] for low water availability were observed on leaf retention on the second and third axes orders, and duration of leaf expansion on the first- and fourth-order axes. The second-degree polynomials modelled leaf growth parameter distribution in the vertical tree profile, and linear regressions modelled the proportion of terminal bud mortality. Conclusions: Leaf growth parameters in coffee plants were determined by axis order. The duration of leaf expansion contributed to phyllochron determination. Leaf growth parameters varied according the position of the axis supporter along the vertical profile, suggesting an effect of axes age and micro-environmental light modulations.

Structural and functional changes in coffee trees after 4 years under free air CO2 enrichment.

Background and Aims: Climate forecasts suggest that [CO2] in the atmosphere will continue to increase. Structural and ecophysiological responses to elevated air [CO2] (e[CO2]) in tree species are contradictory due to species-dependent responses and relatively short-term experiments. It was hypothesized that long-term exposure (4 year) to e[CO2] would change canopy structure and function of Coffea arabica trees. Methods: Coffee plants were grown in a FACE (free air CO2 enrichment) facility under two air [CO2]: actual and elevated (actual + approx. 200 µL CO2 L-1). Plants were codified following the VPlants methodology to obtain coffee mock-ups. Plant canopies were separated into three 50 cm thick layers over a vertical profile to evaluate their structure and photosynthesis, using functional-structural plant modelling. Key Results: Leaf area was strongly reduced on the bottom and upper canopy layers, and increased soil carbon concentration suggested changes in carbon partitioning of coffee trees under e[CO2]. Increased air [CO2] stimulated stomatal conductance and leaf photosynthesis at the middle and upper canopy layers, increasing water-use efficiency. Under e[CO2], plants showed reduced diameter of the second-order axes and higher investment in the youngest third to fifth-order axes. Conclusions: The responses of Arabica coffee grown under long-term exposure to e[CO2] integrated structural and functional modifications, which balanced leaf area loss through improvements in leaf and whole-plant photosynthesis.

Strategies to reconstruct 3D Coffea arabica L. plant structure.

Accurate model of structural elements is necessary to model the foliage and fruit distributions in cultivated plants, both of them being key parameters for yield prediction. However, the level of details in architectural data collection could vary, simplifying the data collection when plants get older and because of the high time cost required. In the present study, we aimed at reconstructing and analyzing plant structure, berry distributions and yield in Coffea arabica (Arabica coffee), by using both detailed or partial morphological information and probabilistic functions. Different datasets of coffee plant architectures were available with different levels of detail depending on the tree age. Three scales of decomposition-plant, axes and metamers were used reconstruct the plant architectures. CoffePlant3D, a software which integrates a series of mathematical, computational and statistical methods organized in three newly developed modules, AmostraCafe3D, VirtualCafe3D and Cafe3D, was developed to accurately reconstruct coffee plants in 3D, whatever the level of details available. The number of metamers of the 2nd order axes was shown to be linearly proportional to that of the orthotropic trunk, and the number of berries per metamer was modeled as a Gaussian function within a specific zone along the plagiotropic axes. This ratio of metamer emission rhythm between the orthotropic trunk and plagiotropic axes represents the pillar of botanical events in the C. arabica development and was central in our modeling approach, especially to reconstruct missing data. The methodology proposed for reconstructing coffee plants under the CoffePlant3D was satisfactorily validated across dataset available and could be performed for any other Arabica coffee variety.

Simulating the grazing of a white clover 3-D virtual sward canopy and the balance between bite mass and light capture by the residual sward.

BACKGROUND AND AIMS: The productivity and stability of grazed grassland rely on dynamic interactions between the sward and the animal. The descriptions of the sward canopies by standard 2-D representations in studies of animal-sward interactions at the bite scale need to be improved to account for the effect of local canopy heterogeneity on bite size and regrowth ability. The aim of this study was to assess a methodology of 3-D digitized canopies in order to understand the balance between bite mass and light interception by the residual sward. METHODS: 3-D canopy structures of four white clover swards were recorded using a POLHEMUS electromagnetic digitizer and adapted software (POL95). Plant components were removed after digitizing to determine aerial dry matter. Virtual canopies were synthesized and then used to derive canopy geometrical parameters, to compute directional interception and to calculate bite mass. The bit masses of cattle and sheep were simulated according to their form, depth and placement on the patch, taking account of explicit sward architecture. The resulting light interception efficiency (LIE) of each organ was then calculated using a projective method applied to the virtual residual sward. This process enabled an evaluation of light interception based on Beer's law at the bite scale. KEY RESULTS: The patterns of the vertical profiles of LAI appeared as bimodal, triangular or skewed parabolic functions. For a single bite of similar area and depth, the lowest mass was observed with half-spherical form and the highest for the cylindrical form, whatever the initial sward structure. The differences between the actual LIE and that calculated by Beer's law were marked for residual swards shorter than 8 cm. Bite mass and LIE values after grazing were more strongly affected by the initial structure of the sward than by bite form and placement. CONCLUSIONS: 3-D digitizing techniques enabled a definition of the geometry of each component in sward canopies and an accurate description of their vertical and horizontal heterogeneities. The discrepancy between Beer's law results and actual light interception was reduced when the sward regrew rapidly and if the rest period was long. Studies on the biting process would greatly benefit from this method as a framework to formulate and test hypotheses in a quantitative manner.

Time series in analysis of yerba-mate biennial growth modified by environment.

To assess differences in the lag-effect pattern in the relationship between yerba-mate biennial growth and environmental factors, a time-series analysis was performed. A generalized Poisson regression model was used to control time trends, temperature, growing degree days (GDD), rainfalls and night length (NL). It was hypothesized that the active growth and growth pauses in yerba-mate are controlled endogenously and modified by environment, and that genders would respond differently to environmental modifications. The patterns in the lag effect from the distributed-lag models were similar to those of time-series models with meteorological data means with lag = 0. GDD and NL were principal factors affecting biennial yerba-mate shoot elongation and the number of green leaves of females grown in monoculture, besides their significant effects on metamer emission and leaf area in males grown in monoculture. NL also had a significant influence on shoot elongation and leaf area of both genders grown in forest understorey (FUS), indicating that yerba-mate growth is synchronized by an internal clock sensitive to temperature adjustments. The morphological plasticity and the adaptation efforts of yerba-mate were more pronounced in monoculture than in FUS. Sexual dimorphism was expressed-males were more sensitive to environmental changes than females, especially in monoculture. Growth modifications were much more intense when plants were grown in a cultivation system that is less like yerba-mate natural habitat (monoculture) than in one resembling its natural habitat (FUS). Our data support the ecological specialization theory.

Intensity of bitterness of processed yerba mate leaves originated in two contrasted light environments

The bitterness intensity of beverage prepared from the leaves produced on the males and females of yerba mate (Ilex paraguariensis), grown in the forest understory and monoculture, was evaluated. The leaves were grouped by their position (in the crown and on the branch tips) and by the leaf age. The leaf gas exchange, leaf temperature and photosynthetic photon flux density were observed. Inter and intra-specific competition for light and self-shading showed the same effect on yerba mate beverage taste. All the shading types resulted in bitterer taste of the processed yerba mate leaves compared to the leaves originated under the direct sun exposure. The leaves from the plants grown in the monoculture showed less bitterness than those grown in the forest understory. This conclusion was completely opposite to the conventionally accepted paradigm of the yerba mate industries. The leaves from the tips (younger leaves) of the plants grown in the monoculture resulted a beverage of softer taste; the males produced less bitter leaves in any light environment (forest understory or in the crown in monoculture). The taste was related to the photosynthetic and transpiration rate, and leaf temperature. Stronger bitterness of the leaves provided from the shade conditions was related to the decreased leaf temperature and transpiration in the diurnal scale.

Mediu-se a intensidade de amargor da bebida preparada a partir de folhas da erva-mate (Ilex paraguariensis) de diversas idades, situadas em duas posições na copa (interior e ponteiras), produzidas por plantas masculinas e femininas cultivadas na floresta antropizada e em monocultura. As trocas gasosas foliares, a temperatura de folhas e a densidade de fluxo de fótons fotossinteticamente ativos também foram medidas. Com isso verificou-se que a idéia corrente de que o sombreamento está diretamente relacionado ao sabor suave do chimarrão é completamente equivocada, já que as competições inter- e intra-específicas por luz e auto-sombreamento provocaram o mesmo efeito no sabor do chimarrão, isto é, todos os tipos de sombreamento resultaram em sabor mais amargo de folhas processadas do de pleno sol. Assim, as folhas de plantas cultivadas em monocultura mostraram-se menos amargas do que aquelas de plantas manejadas sob a floresta. Além disso, as folhas de ponteiras (mais jovens) da monocultura originaram o chimarrão mais suave e os machos produziram folhas menos amargas que as fêmeas em ambos ambientes luminosos (floresta e monocultura). O sabor mostrou-se relacionado à fotossíntese, à transpiração e à temperatura de folha. Em sombreamento a modificação de sabor foi direcionada ao aumento de amargor e, aparentemente, relacionada ao decréscimo da temperatura de folhas e da transpiração na escala diurna.