Elevated temperature and carbon dioxide alters growth and leaf-chemical composition in two important neotropical crops, Coffee (Coffea arabica) and Cacao (Theobroma cacao)

This dataset contains data collected from a 6 month climate-controlled glasshouse experiment using coffee (Coffea arabica) and cacao (Theobroma cacao) seedlings subject to increases in temperature in the presence and absence of elevated [CO2]. Seedlings of each species were subject to temperatures of ambient +4 degrees celcius and increased [CO2] levels of 800ppm. Observations were taken throughout the duration of their growth, with destructive harvests occurring prior to treatment exposure, and at two subsequent harvest dates. By measuring parameters of height, partitioned biomass, gas exchange, leaf chemical compound composition and more, this dataset allows exploration of the effects and possible interactions of elevated temperature and increased [CO2] on the early growth, physiology and biochemistry these species. 

Methods
Planting Material and Growing Conditions

Coffee (Coffea arabica L., Rubiaceae, cv. Bourbon) saplings (n = 100, approximately 3 months old), grown in plastic planting tubes (50 x 50 mm at top, 120 mm tall, 250 ml volume) were obtained from the Skybury nursery in the Atherton Tablelands. The saplings were grown from seeds planted in late August 2016 and had received only water during their development. Sixty coffee saplings, selected for physical homogeneity, were transplanted into 25 L pots on 3 December 2016, and each provided with 500 mL of liquid seaweed fertilizer (Seasol®) to reduce possible transplanting shock. Cacao (Theobroma cacao L., Malvaceae) seeds were obtained from the Goodman Cacao Estate in Killaloe, North Queensland, Australia. Ten ripe cacao pods were harvested from a single cacao tree (SG2 hybrid variety, Papua New Guinea origin), the seeds were extracted, de-pulped, and primed in water for 24 hours. Large, round and healthy seeds were planted into 25 L pots (n = 60) on 6 December 2016, with five seeds, spaced evenly, per pot. All the planted cacao seeds had germinated 10 days after planting (DAP). At 14 DAP, four of the five cacao saplings per pot were removed by harvesting at the root collar, producing a homogeneous subset of saplings (n = 60) for experimental trial. The pots used for both coffee and cacao saplings were free-draining black plastic pots (30 cm diameter, 50 cm depth), each filled with a 10:1 (w/w) soil and perlite mix. The soil was a potting mix obtained from a local landscaping company and a 25mm layer of river stones was placed in the base of each pot to assist drainage. Coffee and cacao saplings were maintained in a shade house under 75% shade and ambient [CO2] until being moved into the glasshouse to initiate the experiment.

Glasshouse Facility and Chamber Treatments

This study was conducted in a climate-controlled glasshouse facility, located at the James Cook University’s Environmental Research Complex, Cairns, Australia. The glasshouse is divided into three independently regulated and parallel growth chambers each 5(L) × 3(W) × 4(H) m. The chambers are arranged in a N-S orientation, with independent temperature and [CO2] regulation, receiving natural sunlight at about 50 % full sunlight, due to SOLARO 5220 D O FB climate screening (Ludvig Svensson Inc.). Fifty-four seedlings per species were translocated into the glasshouse on 8 December 2016 for coffee, and on 12 December 2016 for cacao. For each species, the seedlings were randomly allocated to one of three chambers (i.e., eighteen seedlings per species per chamber).

To quantify the effects and potential interactive effects of elevated temperature and [CO2] on the seedlings of coffee and cacao, I subjected seedlings to one of three treatments. In the first, air temperatures were set to track the ambient conditions of Cairns, North Queensland and [CO2] was set to 400 ppm (i.e., Tempamb [CO2]amb). In the second, air temperatures were set to track ambient + 4°C and [CO2] was set to 400 ppm (i.e., Tempelev [CO2]amb). In the third treatment, air temperatures were set to + 4°C above the ambient, and [CO2] was set to 800 ppm (i.e., Tempelev [CO2]elev). Seedlings and their associated treatments were rotated between chambers monthly to reduce possible chamber effects unrelated to the imposed treatments. All seedlings were watered daily to field capacity for the duration of the experiment and given 20 g of slow-release granular fertilizer (Scotts Osmocote® Plus Trace Elements: Native Gardens) once per month. To improve micronutrient availability, a one-off application of micronutrient foliar spray (Manutec Trace Elements) was applied to all cacao seedlings.

Chamber conditions were monitored with sensors (Temp/ RH: QFM2160, Siemens, Bayswater, VIC, Australia; [CO2]: GMP222, Vaisala, Helsinki, Finland), and regulated through a feedback control system implemented in the building management software. Air temperature (°C), [CO2] (ppm) and relative humidity (%) were recorded every 5 minutes during the experiment.

Seedling Growth Measurements, Harvesting Procedure and Tissue Sampling

For coffee and cacao, six seedlings were destructively harvested prior to the initiation of the experimental treatments (initial harvest, 8 and 12 December 2016, respectively) to determine initial plant biomass and leaf area. After growth in treatment conditions, nine seedlings per species and per treatment were destructively harvested on two harvesting events on 07 – 08 March (first harvest) and 24 – 25 April 2017 (final harvest). During the experiment, weekly measurements of stem height (root collar to apical meristem, mm) and the number of branches (coffee only) were made until and upon the first harvest, and upon the final harvest. Upon destructive harvest, each seedling was separated into cotyledons (cacao only), leaves, petioles (cacao only, coffee leaves had petioles included), main stems, branches, and roots. The harvesting procedure was to first remove the above-ground biomass by cutting the stem at the root collar. The leaves (and their petioles) were then detached and classified as either fully expanded, immature or senescent, before petiole removal (cacao only). Expanded leaves were defined as leaves that were fully developed and contributing to photosynthesis whilst immature leaves were young, small, soft leaves that were typically light green (coffee) or translucent pink/light green (cacao) and still expanding. After leaf and petiole removal, the remainder of the above-ground biomass was separated into branch and stem tissues. The below ground root systems were obtained by gently washing the roots free from all soil with water before processing for fresh and dry mass. All tissue types (cotyledons, leaves, petioles, main stems, branches and roots) were oven dried at 60°C for 14 days before being re-weighed for dry mass (g).

Leaves used for gas exchange measurements (see gas exchange below) were processed separately to determine leaf morphological and biophysical characteristics. Leaf thickness (µm) was measured before determination of total lamina area (cm2) using a scanner (Canon CanoScan Flatbed Scanner LiDE120) and image processing in Image-J 1.52a Software (Schneider et al., 2012). Leaf mass per unit area (LMA, g m−2) was calculated after weighing the dried leaf tissue, with this oven-dried material also used for subsequent nutrient and phenolic content analysis.

Seedling Growth Indices

The total dry biomass (g) of seedlings was calculated as the combined dry mass (g) of all harvested tissues (leaves, petioles, main stems, branches and roots but excluding senesced leaves and cotyledons). The relative growth rate (RGR, g g−1 d−1) of seedlings across time was calculated (See Supplementary Information) (Hoffmann and Poorter, 2002), where m2 and m1 represent the population average dry biomass at the final and initial harvests, respectively, and where t represents the number of days between harvests.

Gas Exchange Measurements

Leaf gas exchange measurements were conducted prior to the final harvest, using the third newest fully expanded leaf of each seedling (n = 9), hereafter referred to as the ‘target’ leaves. Gas exchange was measured between 08:00 and 12:00 using a portable photosynthesis system (Li-Cor 6400, Li-Cor Inc., Lincoln, NE, USA) with Licor-cuvette conditions set to mimic typical midday glasshouse chamber temperature and [CO2] of each treatment (i.e., Tempamb 32 °C and Tempelev 36 °C). Measurements were made at a photon flux of 1500 µmol m−2 s−1, supplied by an artificial LED light source (6400-02B, Li-Cor Inc.), with leaf vapour pressure deficit maintained between 1.46 and 2.36 kPa. Leaf temperatures during the gas exchange measurements for both species were between 33.4°C and 34.7°C for the Tempamb treatment and between 35.4°C and 41.0°C for the Tempelev treatments.

Leaf Phytochemistry

I evaluated the nutrient and phenolic composition of leaf tissues of both species at the final harvest using the dried target leaves. Dried leaf tissue was ground using a Qiagen TissueLyser II, digested using a nitric acid and peroxide microwave digest (Bergof SW-4) and analysed for macronutrients (i.e., P, K, Ca, Mg) and micronutrients (i.e., Fe, Zn; see Supplementary Table 2, 3) via Inductively Coupled Plasma Optical Emission Spectrometer ICP-OES spectroscopy (Agilent 5100, Agilent Technologies Inc.).

Nitrogen (N) and Carbon (C) concentration were determined using a Costech 4010 Elemental Analyser and following the Dumas method (Buckee, 1994). Elemental concentrations were used to calculate C:N and N:P mass ratios. Phenolic extraction and analysis of leaf phenolic diversity (i.e., the number of different phenolic compounds) and the total phenolic concentration (mAU) were conducted following procedures described by Uesugi and Kessler (2016) and with modifications as per Forbes et al., (2020) using HPLC analysis. Furthermore, two microlitres for coffee samples, and five microlitres for cacao samples, of the supernatant liquid were injected into the HPLC for analysis. The different injection volumes were standardized by dividing the peak area by the volume injected for each species and converted to absorption units per microlitre (AU μl−1). Standard curves for compound quantification were unavailable therefore I interpreted absorbance (the area under the curve of each compound peak) as equivalent to concentration. Given the differing HPLC injection amounts for each species, any differences in phenolic composition between species should be interpreted with caution. The major phenolic component of the coffee phenolic profile was also identified and measured separately.