Early growth phase and caffeine content response to recent and projected increases in atmospheric carbon dioxide in coffee (Coffea arabica and C. canephora)
SeedsThree Arabica cultivars widely grown throughout Latin America were tested: cv. ‘Bourbon’, cv. ‘Catimor’, and cv. ‘Typica’52,53. Typica and Bourbon are the progenitors of most Arabica coffee cultivars grown worldwide and are believed to have originated from coffee grown in Yemen of Ethiopian origin54,55. Arabica coffee grown in Indonesia originated from Yemen, and seeds taken from Java (Indonesia) to Amsterdam and then to the American continent led to the denomination Typica53. Seeds taken from Yemen and grown in Île de Bourbon (Bourbon Island; present day La Réunion) led to the denomination Bourbon18. Catimor is the result of crossing of two coffee cultivars: cv. ‘Caturra’ and cv. ‘Híbrido de Timor’ or ‘Timor Hybrid’ (a natural polyploid hybrid originating in Timor, an island in the Malay Archipelago, and resulting from a crossing between Arabica and robusta). Híbrido de Timor and the derived Catimor are resistant to coffee leaf rust (Hemileia vastatrix) and gained their resistance genes from robusta coffee52,53. Mature coffee fruits for the Arabica cultivars were collected in August 2016, and again in September 2017 from plants at Rancho El Porvenir (869 masl; N 15.13229, W 92.20151) in Chiapas, Mexico. Robusta has higher levels of caffeine compared to Arabica (ca. 1.7% vs. 1%, respectively)56 and is adapted to growth at lower elevations in Guineo-Congolian forests57 and thus warmer and mostly wetter conditions relative to Arabica, which originates from high altitudes forest in Ethiopia and South Sudan and is adapted to a cooler, more seasonal environment58. Robusta fruits were collected in 2016 and again in 2017 from plants at Ejido Salvador Urbina (693 masl; N 15.04415 W 92.18578) in Chiapas, Mexico. Fruits were depulped, fermented, washed, and dried (ca. 12% moisture) and sent to the USDA-ARS Beltsville laboratory for germination.
PlantingTwelve plastic bins measuring ca. 60 cm × 50 cm × 33 cm deep (ca. 99 L by volume) were used to provide three monocultures of the four (three Arabica and one robusta) taxa for each [CO2] treatment (four chambers). Each bin was perforated with 12 holes (1 cm diam.) to allow for water drainage. A screen mesh was placed at the bottom of each bin prior to adding the growing medium (Pro-Mix BX; Premier Horticulture Inc., Quakertown, CA, USA) to minimize growing medium loss after watering. Seeds were soaked in water 24 h prior to planting, to promote germination. Each bin was moistened before planting 72 seeds per tub, ca. 2.5 cm deep, and ca. 5 cm apart. For the first run, seeds were planted on August 10, 2016 and the first germination occurred on September 5, 2016. For the second run, seeds were planted on September 12, 2017 and the first germination occurred on October 11, 2017. Rates of germination did not vary as a function of [CO2]. For both trials, nutrients were initially provided at sowing and again at two months post-planting using a complete nutrient solution59. MiracleGro 24-8-16 (Marysville, OH) was provided at ca. 3 months following planting and given at 2–3 weeks’ intervals until final harvest. An iron chelate micronutrient (Sprint 330, Becker Underwood, Ames, IA, USA) was sprayed as needed. The growth medium/soil was maintained at, or close to, field capacity.
Environmental chambersProviding pre-ambient [CO2] concentrations is not possible in situ; therefore, controlled environment chambers (Bio-Chambers, Incorporated, Winnipeg, Canada) were used. The temperature for each chamber was kept constant at 25 °C, day/night. Light, quantified as photosynthetically active radiation (PAR), was maintained at 400 µmol mol−1. The daily light period was 12 h light was supplied by height-adjustable, dimmable banks of metal halide and high-pressure sodium bulbs (400 µmol m−2 s−1). CO2 concentrations were maintained by injection of either CO2 or CO2-free air using a TC-2 controller that monitors [CO2] in real time as measured by an infrared gas maintained in absolute mode. To maintain a range of recent and projected atmospheric CO2, concentrations were set at 300, 400, 500 and 600 ppm, 24 h day−1. These [CO2] values represent the measured Mauna Loa values from 1915 to 2015, and those projected by the end of the current century60. Actual mean [CO2] values (+SD, in [ppm]), from measurements recorded every three minutes throughout the experiments in each of the chambers, were 326 ± 38.6, 430 ± 42.7, 511 ± 26.2, and 607 ± 27.9 in the first run, and 303 ± 23.2, 409 ± 29.6, 499 ± 20.4, and 596 ± 23.0 in the second run.
HarvestsDestructive harvests were performed at three different times, ca. 4, 7, and 12 months post-planting. At each harvest, 3–5 plants within a bin (for all taxa and [CO2] treatments) were removed from the tubs, height determined (cm), then separated into leaf laminae, branches, stems, and roots. Leaf (cm2) area was determined photometrically using a leaf area meter (Li-Cor 3100, Lincoln, NE, USA). All plant material was weighed (g) after drying at 65 °C until dry weight was constant. Root binding did not occur as indicated by visual examination at the conclusion of the experiment when plants were removed from tubs.
C:N ratios and caffeine analysisFor each sample, all leaves, per plant were pooled and oven-dried (65 °C) until the sample was completely dry. Each dried sample was ground using a Wiley Mill with a mesh size #20. Total C and N contents were determined using a Vario Max CN (Elementary Americas, Inc., Ronkonkoma, NY, USA). Nitrogen and carbon content were determined as a percentage of the dry weight of the sample. For extraction and determination of caffeine, leaves within a replicate were flash frozen in liquid N and stored at −80 °C until lyophilized. Leaves were then pulverized using an A11 Basic Analytical Mill (IKA Works Inc., Wilmington, NC, USA). A total of 100 mg of pulverized leaf material was added into 15 ml centrifuge tubes with 5.0 mL of a 70% methanol/water mixture. Tubes were then vortexed for 30 s and sonicated for 60 min. The slurry was centrifuged at 5,000 rpm for 10 min before being diluted (1:20), filtered, and ultimately stored in 1.5 mL HPLC vials. All reagents used for the analysis were of HPLC grade purity and prepared fresh on each day of the analysis. Instrumental analysis was performed using a Shimadzu Prominence High Performance Liquid Chromatograph (Shimadzu Scientific Instruments, Columbia, MD, USA) using a mobile phase of 80% methanol/water and 15 mM phosphate buffer at pH 6.2. Separation was conducted using a Thermo Scientific Aquasil reverse phase C18 column (4.6 × 250 mm, 5 µm particle size; Thermo Fisher Scientific, Waltham, MA, USA) at a flow rate of 0.550 ml/min. Detection and quantification was done using a UV detector at 275 nm and determined using a calibration curve. The caffeine calibration curve was created using an HPLC grade caffeine standard (99.7% purity; ACROS Organics #10816-5000; Thermo Fisher Scientific, Waltham, MA, USA) across five concentrations 2.5, 5, 10, 20, and 25 ppm. The fitted curve showed excellent linear responsivity as demonstrated by an r2 of 0.998. In addition, there was negligible variation between replicate injections at 10 ppm using the same standard as measured by its percent relative standard deviation of 0.385%. The caffeine concentration in leaves can also be used as a proxy for concentrations in coffee beans, based on a correlation between caffeine concentration in seedling leaves and seeds61,62. Dias Chaves et al.61 focused on the 1st and 3rd pair of leaves in the seedlings, while de Moraes et al.62 used the 3rd and 4th pair. We found no significant differences in caffeine content between the last pair of fully expanded leaves and all remaining leaves combined (cotyledons excluded; using March 2017 samples, i.e., first year, second sampling; 7 months and 18 days post-planting). Based on these results, we pooled all leaves at each sampling date for caffeine analysis. Mazzafera and Magalhães63 found no correlation between leaves and seeds, but these were collected from mature plants, not seedlings.
Statistical analysisThree replicate bins for each Arabica cultivar and for robusta coffee (i.e., 12 bins per chamber) were present for each of four [CO2] treatments. Within each chamber [CO2], bins were randomized; and randomized again after the first two harvests at 4 and 7 months to avoid edge effects. After the first run of the experiment (i.e., one year), the chambers were randomly reassigned [CO2] treatments and the experiment repeated. Humidity, PAR, and temperature were quantified before and at the end of each harvest to determine within chamber and among chamber variability. Values for each parameter were consistent between experimental runs. All measured parameters were based on tub averages (3–4 plants per tub) for both runs. All measured and calculated parameters were analyzed using analysis of variance including [CO2], Arabica cultivars, Arabica vs. robusta, and harvest time (Statview Software, Cary, NC, USA).
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