DOI: 10.5061/dryad.qrfj6q5r4
title: Elevated temperature and carbon dioxide alters growth and leaf-chemical composition in two important neotropical crops, Coffee (Coffea arabica) and Cacao (Theobroma cacao)

description: 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. 
description: <strong>Planting Material and Growing
 Conditions</strong> Coffee (<em>Coffea
 arabica</em> L., Rubiaceae, cv. Bourbon) saplings
 (<em>n</em> = 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<sup>®</sup>) to reduce possible transplanting shock.
 Cacao (<em>Theobroma cacao</em> 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 (<em>n </em>= 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 (<em>n</em>
 = 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 [CO<sub>2</sub>] until being moved into the glasshouse
 to initiate the experiment. <strong>Glasshouse
 Facility and Chamber Treatments</strong> 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
 [CO<sub>2</sub>] 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
 [CO<sub>2</sub>] 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 [CO<sub>2</sub>] was set to 400 ppm (i.e.,
 Temp<sub>amb</sub>
 [CO<sub>2</sub>]<sub>amb</sub>). In the second,
 air temperatures were set to track ambient + 4°C and
 [CO<sub>2</sub>]<sub> </sub>was set to 400 ppm
 (i.e., Temp<sub>elev</sub>
 [CO<sub>2</sub>]<sub>amb</sub>). In the third
 treatment, air temperatures were set to + 4°C above the ambient, and
 [CO<sub>2</sub>]<sub> </sub>was set to 800 ppm
 (i.e., Temp<sub>elev</sub>
 [CO<sub>2</sub>]<sub>elev</sub>). 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;
 [CO<sub>2</sub>]: GMP222, Vaisala, Helsinki, Finland), and
 regulated through a feedback control system implemented in the building
 management software. Air temperature (°C), [CO<sub>2</sub>]
 (ppm)<sub> </sub>and relative humidity (%) were recorded every
 5 minutes during the experiment. <strong>Seedling
 Growth Measurements, Harvesting Procedure and Tissue
 Sampling</strong> 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
 (cm<sup>2</sup>) 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<sup>−2</sup>)
 was calculated after weighing the dried leaf tissue, with this oven-dried
 material also used for subsequent nutrient and phenolic content
 analysis. <strong>Seedling Growth
 Indices</strong> 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<sup>−1</sup> d<sup>−1</sup>) of seedlings across
 time was calculated (See Supplementary Information) (Hoffmann and Poorter,
 2002), where m<sub>2</sub> and m<sub>1
 </sub>represent the population average dry biomass at the final and
 initial harvests, respectively, and where <em>t</em>
 represents the number of days between harvests.
 <strong>Gas Exchange Measurements</strong>
 Leaf gas exchange measurements were conducted prior to the final
 harvest, using the third newest fully expanded leaf of each seedling
 (<em>n =</em> 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 [CO<sub>2</sub>] of each treatment (i.e.,
 Temp<sub>amb</sub> 32 °C and Temp<sub>elev</sub>
 36 °C). Measurements were made at a photon flux of 1500 µmol
 m<sup>−2</sup> s<sup>−1</sup>, 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 Temp<sub>amb</sub> treatment and between
 35.4°C and 41.0°C for the Temp<sub>elev</sub>
 treatments. <strong>Leaf
 Phytochemistry</strong> 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<sup>−1</sup>). 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.
description: # Elevated temperature and carbon dioxide alters growth and leaf-chemical
 composition in two important neotropical crops, coffee (Coffea arabica)
 and cacao (Theobroma cacao)
 [https://doi.org/10.5061/dryad.qrfj6q5r4](https://doi.org/10.5061/dryad.qrfj6q5r4) ## Description of the data and file structure Five separate .csv files are provided describing growth, biomass partitioning, gas exchange, and leaf biochemistry in the studies coffee and cacao seedlings. These data were 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 early growth stages, with destructive harvests occurring prior to treatment exposure, and at two subsequent harvest dates after treatment exposure.   ### Files and variables #### File: Biomass\_Data.csv **Description:** Data contains numerous variables describing plant/ seedling growth after exposure to treatments, at two harvest dates. ##### Variables * Plant_ID: The unique identification for each experimental plant (seedling). * Species: The plant (seedling) species. Coffee or cacao. * Plant_Number: The plant (seedling) replicate number. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * Treatment_*Description: The treatment applied to each unique Plant*_ID. LCLT: low CO2 low temperature, LCHT: low CO2 high temperature, HCHT: high CO2 high temperature. * Harvest_Number: Represents the first or final harvest date. * Harvest_Date: The date of destructive harvest. * Brown_Stem_Length: The length (cm) of the total stem that is brown (lignified). * Green_Stem_Length: The length (cm) of the total stem that is green (new and not yet very lignified). * Total_RootCollar_to_Meristem_Length_cm: The measured length from the seedling root collar to the apical meristem (converted to cm). * Stoutness.Stem: Total_RootCollar_to_Meristem_Length_cm / mass XYZ. * Prop.Green:  ((Brown_Stem_Length / Total_RootCollar_to_Meristem_Length_cm) *100) * Prop.Brown: ((Green_Stem_Length / Total_RootCollar_to_Meristem_Length_cm) *100) * RootCollar_Stem_Diameter(mm)1: The diameter of the seedling root collar (mm). * RootCollar_Stem_Diameter(mm)2: A second measurement of the diameter of the seedling root collar (mm). * Av.RC.Diam(mm): ((RootCollar_Stem_Diameter(mm)1 + RootCollar_Stem_Diameter(mm)2) / 2) * Mid_Stem_Diameter1: The diameter (mm) of the seedling stem, as measured at half of the Total_RootCollar_to_Meristem_Length_mm. * Mid_Stem_Diameter2: A second measurement of the diameter (mm) of the seedling stem, as measured at half of the Total_RootCollar_to_Meristem_Length_mm. * Av.MS.Diam(mm): ((Mid_Stem_Diameter1 + Mid_Stem_Diameter2) / 2) * Cotyledon_Mass_(g)dry.:The dry mass (g) of the collected cotyledons (cacao only). * Leaf_Thickness_1: Leaf thickness (µm) measurement 1. * Leaf_Thickness_2: Leaf thickness (µm) measurement 2. * Leaf_Thickness_3: Leaf thickness (µm) measurement 3. * Av_Leaf_Thickness: ((Leaf thickness (µm) measurement 1 + Leaf thickness (µm) measurement 2 + Leaf thickness (µm) measurement 3) /3) * WET_G.E.Leaf_Mass_g: Mass (fresh harvested mass, g) of leaf used for gas exchange measurements (target leaf). * DRY_G.E.Leaf_Mass_g: Mass (g) of leaf used for gas exchange measurements (target leaf) after drying. * PS.Leaf_Mass_g: Total mass (g) of photosynthetically active leaf biomass after drying. Excludes immature and senesced leaves. * Leaf_Number: The total number of mature leaves per plant. * Imm.Leaf_Mass_g: The total dry mass (g) of immature leaves harvested per plant. * Scenesced_Leaf_Mass_g: The total dry mass (g) of senesced leaves harvested per plant. * Total_DRY_Leaf_Biomass_g.(no.scenes.no.imm.): Same as PS.Leaf_Mass_g. * Stem_Mass_g: Total dry mass (g) of stem tissue. * Petiole_Mass_g: Total dry mass (g) of petiole tissue (cacao only, coffee petioles included in leaf biomass). * Root_Mass_g: Total dry mass (g) of root tissue. * Branch_Mass_g: Total dry mass (g) of branch tissue. * Number_of_Branches: The number of branches initiated per plant. * Total_AG_Biomass_g.(no.scenec.no.imm.no.cotyl): The total summed mass (g) of all dry tissues harvested above the root collar, per plant (stem, branches, leaves, petioles). Excludes immature and scenesced leaves and cotyledons. * Total_BG_Biomass_g: The total summed mass (g) of all dry tissues harvested below the root collar, per plant (root mass). * Total_Biomass_g: (Total_AG_Biomass_g.(no.scenec.no.imm.no.cotyl) + Total_BG_Biomass_g). dry biomass (g). * Additional_Stem_Number: If additional stems (chupons) grew in cacao, these were noted. * Architecture_Notes: Notes on the structural integrity of the plant, ie., if the plant was growing upright or appeared bent over by weight of leaf biomass. * Leaf.Area:  The total lamina area (m2) evaluated using a scanner (Canon CanoScan Flatbed Scanner LiDE120) and image processing in Image-J 1.52a Software. * Leaf.Area.m2: Leaf.Area * Leaf.Area.cm2: Leaf.Area converted to cm2. * LMA: Leaf mass per unit area (LMA, g m−2). * SLA.CM2.G: Specific leaf area. Note* cells containing 'NA' in this data set indicate where data is either not collected or not available. #### File: Height\_Time\_Series\_Data.csv **Description:** Data contains numerous variables describing plant height and growth across time of seedlings growing under chamber conditions.  ##### Variables * Plant_ID: The unique identification for each experimental plant (seedling). * Species: The plant (seedling) species. Coffee or cacao. * Mon.Week: Monitoring week (whole number). * Mon.Week_2: Monitoring week (time interval). * DIT: Days in treatment. * Sample_Date: The date of measurements. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * Treatment*Description: The treatment applied to each unique Plant*_ID. LCLT: low CO2 low temperature, LCHT: low CO2 high temperature, HCHT: high CO2 high temperature. * Leaf_Count: The number of attached leaves per plant. * Total_RootCollar_to_Meristem_Length_mm: The measured length from the seedling root collar to the apical meristem (mm). * Total_RootCollar_to_Meristem_Length_cm: The measured length from the seedling root collar to the apical meristem (converted to cm). * Av.RootCollar_Stem_Diameter_mm: The average diameter (mm) of the root collar. * Av.Mid_Stem_Diameter_mm: The average diameter (mm) of the seedling stem, as measured at half of the Total_RootCollar_to_Meristem_Length_mm. * Number_of_Branches: The total number of initiated branches observed per plant. Note* cells containing 'NA' in this data set indicate where data is either not collected or not available. #### File: Gas\_Exchange\_Data.csv **Description:** Gas exchange measured using a single 'target leaf' per plant 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., Temp amb 32 °C and Temp elev 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. ##### Variables * Plant_ID: The unique identification for each experimental plant (seedling). * Species: The plant (seedling) species. Coffee or cacao. * Plant_Number: The plant (seedling) replicate number. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * Treatmen_*Description: The treatment applied to each unique Plant*_ID. LCLT: low CO2 low temperature, LCHT: low CO2 high temperature, HCHT: high CO2 high temperature. * Harvest: Represents the first or final harvest date. * Photo: Photosynthetic rate (µmol CO2 m−2 s−1). * Cond: Conductance (mol m−2 s−1). * Ci: Intracellular CO2 (*µmol* CO2 *mol air-1*) * Trmmol:  Leaf transpiration (*E, mmol m-2 s-1*)  * VpdL:  Vapor pressure deficit based on Leaf temp (*kPa*)  * Tair: Air temperature (°C* *)  * Tleaf:  Temperature of the leaf thermocouple (* *°C)  * TBlk: Licor cooler block temperature. * RH_S: Relative Humidity in Licor chamber. * Flow: The leak rate from the Licor leaf chamber. * PARi: Internal incident light. * PARo: External light sensor. * Ci/Ca: Ratio of intercellular CO2 to sample CO2 concentrations. * A_T: (Photo / Trmmol) * A_C: (Photo / Cond) * Delta_T:  Leaf Δ temperature (*Tleaf* – *Tair*) Note* cells containing 'NA' in this data set indicate where data is either not collected or not available. #### File: Nitrogen\_Data.csv **Description:** Data contains results of Carbon and Nitrogen composition analysis 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, and Nitrogen and Carbon 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. ##### Variables * Plant_ID: The unique identification for each experimental plant (seedling). * Species: The plant (seedling) species. Coffee or cacao. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * 13C : Carbon 13. * \_15: Carbon 15. * C: Carbon * N: Nitrogen  * C_N.Mass.Ratio: Leaf Carbon to Nitrogen Mass Ratio. * C_mg*g: Leaf Carbon expressed as mg* per g. * N_mg_g: Leaf Nitrogen *expressed as mg* per g. Note* cells containing 'NA' in this data set indicate where data is either not collected or not available. #### File: Nutrients\_Data.csv **Description:** Data contains results of nutrient and phenolic composition analysis 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 and micronutrients via Inductively Coupled Plasma Optical Emission Spectrometer ICP-OES spectroscopy (Agilent 5100, Agilent Technologies Inc.). ##### Variables * Plant_ID: The unique identification for each experimental plant (seedling). * Species: The plant (seedling) species. Coffee or cacao. * Treatment_ID: The code (A,B,C) representing one of three temperature * CO2 treatment combinations. * Mg_ppm: Leaf Magnesium. * B_ppm: Leaf Boron. * P_ppm: Leaf phosphorus. * S_ppm: Leaf Sulphur. * K_ppm: Leaf Potassium. * Ca_ppm: Leaf Calcium. * Cu_ppm: Leaf Copper. * Fe_ppm: Leaf Iron. * Zn_ppm: Leaf Zinc. * Mn_ppm: Leaf Manganese. Note* cells containing 'NA' in this data set indicate where data is either not collected or not available.