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Early growth phase and caffeine content response to recent and projected increases in atmospheric carbon dioxide in coffee (Coffea arabica and C. canephora)

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While [CO2] effects on growth and secondary chemistry are well characterized for annual plant species, little is known about perennials. Among perennials, production of Coffea arabica and C. canephora (robusta) have enormous economic importance worldwide. Three Arabica cultivars (Bourbon, Catimor, Typica) and robusta coffee were grown from germination to ca. 12 months at four CO2 concentrations: 300, 400, 500 or 600 ppm. There were significant increases in all leaf area and biomass markers in response to [CO2] with significant [CO2] by taxa differences beginning at 122–124 days after sowing (DAS). At 366–368 DAS, CO2 by cultivar variation in growth and biomass response among Arabica cultivars was not significant; however, significant trends in leaf area, branch number and total above-ground biomass were observed between Arabica and robusta. For caffeine concentration, there were significant differences in [CO2] response between Arabica and robusta. A reduction in caffeine in coffee leaves and seeds might result in decreased ability against deterrence, and consequently, an increase in pest pressure. We suggest that the interspecific differences observed (robusta vs. Arabica) may be due to differences in ploidy level (2n = 22 vs. 2n = 4x = 44). Differential quantitative and qualitative responses during early growth and development of Arabica and robusta may have already occurred with recent [CO2] increases, and such differences may be exacerbated, with production and quality consequences, as [CO2] continues to increase.
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Early growth phase and caeine
content response to recent and
projected increases in atmospheric
carbon dioxide in coee (Coea
arabica and C. canephora)
Fernando E. Vega1*, Lewis H. Ziska2,7, Ann Simpkins1, Francisco Infante3, Aaron P. Davis4,
Joseph A. Rivera5, Jinyoung Y. Barnaby6 & Julie Wolf2
While [CO2] eects on growth and secondary chemistry are well characterized for annual plant species,
little is known about perennials. Among perennials, production of Coea arabica and C. canephora
(robusta) have enormous economic importance worldwide. Three Arabica cultivars (Bourbon, Catimor,
Typica) and robusta coee were grown from germination to ca. 12 months at four CO2 concentrations:
300, 400, 500 or 600 ppm. There were signicant increases in all leaf area and biomass markers in
response to [CO2] with signicant [CO2] by taxa dierences beginning at 122–124 days after sowing
(DAS). At 366–368 DAS, CO2 by cultivar variation in growth and biomass response among Arabica
cultivars was not signicant; however, signicant trends in leaf area, branch number and total above-
ground biomass were observed between Arabica and robusta. For caeine concentration, there were
signicant dierences in [CO2] response between Arabica and robusta. A reduction in caeine in coee
leaves and seeds might result in decreased ability against deterrence, and consequently, an increase
in pest pressure. We suggest that the interspecic dierences observed (robusta vs. Arabica) may be
due to dierences in ploidy level (2n = 22 vs. 2n = 4x = 44). Dierential quantitative and qualitative
responses during early growth and development of Arabica and robusta may have already occurred
with recent [CO2] increases, and such dierences may be exacerbated, with production and quality
consequences, as [CO2] continues to increase.
Because CO2 represents the sole source of carbon for photosynthesis, and because CO2 levels have been low
for the recent geological past (<800,000 years before present), recent (317–412 ppm since 1960) and projected
increases1 (450–600 ppm by 2050) represent a major shi in an essential resource needed for plant growth. e
biological role of rising atmospheric carbon dioxide concentration [CO2] is well recognized as altering physical
(e.g., growth rates, stomatal aperture), biochemical (e.g., carbon to nitrogen (C:N) ratios, photorespiration), phe-
nological (e.g., time to owering), and reproductive (e.g., seed yield) characteristics for a wide variety of plant
taxa, including agricultural crops26.
Because interspecic and intraspecic variation exists in response to resource changes, there has been a mer-
ited focus on quantifying intraspecic variation that could be used as a means of selection for adaptation to rising
[CO2] levels. For example, studies have conrmed that there is signicant intraspecic variation in the yield
response to future CO2 levels for cowpea (Vigna unguiculata (L.) Walp.)7; common bean (Phaseolus vulgaris L.)8,
1Sustainable Perennial Crops Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Beltsville,
MD, 20705, USA. 2Adaptive Cropping Systems Laboratory, U. S. Department of Agriculture, Agricultural Research
Service, Beltsville, MD, 20705, USA. 3El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, Mexico. 4Royal
Botanic Gardens, Kew, Richmond, Surrey, UK. 5Coee Intelligence, LLC, Pasadena, CA, 91105, USA. 6Dale Bumpers
National Rice Research Center, U. S. Department of Agriculture, Agricultural Research Service, Stuttgart, AR, 72160,
USA. 7Present address: Environmental Health Sciences, Mailman School of Public Health, Columbia University, New
York, NY, 10032, USA. *email: Fernando.Vega@ars.usda.gov
OPEN
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rice (Oryza sativa L.)911; wheat (Triticum aestivum L.)12,13 and soybean (Glycine max (L.) Merr.)14, such that
breeders could begin to select for CO2 responsiveness among currently available germplasm.
However, such eorts have been focused, in general, to annual crops, particularly those of global importance
(e.g., wheat, rice). Less attention, overall, has been given for CO2 selection among perennial crops. In that regard,
coee (Coea arabica L. (Arabica coee) and C. canephora Pierre ex A.Froehner (robusta coee)) is one of the
world’s most important perennial crops, and represents not only a widely traded agricultural commodity, but also
a social and economic foundation for numerous tropical developing countries, with approximately 125 million
people involved in coee growing15. Although there are 124 Coea species16, only two, Arabica and robusta are
associated with the bulk of global coee production17.
Arabica and robusta eld responses to ca. 550 ppm CO2, with an emphasis on photosynthetic metabolism, is
available18. Additional growth chamber studies evaluating temperature and [CO2] in the context of growth and
photosynthetic acclimation response (including transformations in stomatal characteristics) are also available for
coee1922. However, these data represent the growth and metabolic response of coee following transfer of 12 to
18-month-old coee plantlets into Free-Air CO2 enrichment (FACE) or [CO2] growth chambers. At present, any
dierential growth response within, or between Arabica and robusta to recent and projected increases in CO2
from germination through early growth (ca. 1 year) is not available. Yet, early exposure may be critical, as initial
vegetative growth may represent the temporal period of greatest physiological sensitivity to additional CO2, for
annuals23.
In addition to dierential growth, there is substantial evidence that supplementary CO2 may reduce protein
content and increase carbon to nitrogen (C:N) ratios for numerous plant taxa4,24,25 with potential eects on sec-
ondary compounds that have a high N content26. Caeine (C8H10N4O2; 1,3,7-trimethylxanthine; ca. 29% N by
molecular weight) may act as a defense against herbivores2729 and consequently, CO2-induced changes in leaf and
seed caeine concentration may be of ecological interest30 including unforeseen consequences for climate change
impact as a result of changes in plant-herbivore relationships31.
To determine the physiological impact of recent and projected increases in CO2 levels four Coea taxa, i.e.,
three Arabica cultivars (Bourbon, Catimor, Typica) and robusta coee, were grown from germination for approx-
imately one year at CO2 concentrations of 300, 400, 500 or 600 ppm, and measured growth (plant height, leaf area,
biomass, leaf weight, number of branches, dry weight), C: N ratio, and caeine concentration (mg g1).
Results
Comparisons of plant height indicate signicant increases at all sampling periods as a function of [CO2] above
the 300 ppm baseline (Table1; Fig.1). However, by the 12-month period (357–368 days aer sowing; DAS), there
was no signicant eect of [CO2] on plant height for robusta (Fig.1). Similarly, [CO2] stimulation of leaf area was
observed for all taxa at the 4 and 7-month period (122–124 and 203–211 DAS, respectively) in response to rising
[CO2]; however, by the 12-month period, robusta plants had stopped responding (Fig.2). Dierences in leaf area
as a function of [CO2] by Arabica/robusta were not signicant (P = 0.20; Table1).
Parameter [CO2]4 T A/R [CO2] × CV [CO2] × A/R [CO2] × 4 T
122–124 DAS
Leaf Area *** *** ** 0.15 0.68 0.47
Abv. Ground Wt. * *** *** (*) 0.12 **
211–213 DAS
Leaf Area *** *** *** 0.44 0.35 0.27
Abv. Ground Wt. *** *** *** 0.73 *0.21
366–368 DAS
% Nitrogen *** 0.12 *0.30 0.66 0.38
C:N *** ** *** 0.30 0.37 0.27
Caeine (mg g1)* * * 0.27 *0.26
Height (cm) *** *** *** 0.82 0.43 0.53
True Leaf No. 0.07 *** *** 0.99 0.63 0.91
Branch No. *** *** *** 0.27 * *
Leaf Area ** *** *** 0.89 0.20 0.60
Leaf Wt. ** *** *** 0.93 (*) 0.42
Branch Wt. ** *** *** 0.98 0.54 0.93
Stem Wt. ** *** *** 0.73 0.16 0.34
Total Wt. *** *** *** 0.97 (*) 0.57
Table 1. Statistical values for the three Arabica cultivars and robusta coee response to recent and projected
increases in atmospheric CO2 at three sampling periods (DAS, days aer sowing). A/R is Arabica vs. robusta;
[CO2] × CV is CO2 × Arabica cultivars only; [CO2] × 4 T is [CO2] × all four taxa. Total above ground weight
and vegetative characteristics are in g per plant. Leaf area is in cm2. (*)Indicates a P value between 0.05 and
0.10; *Indicates a P value between 0.05 and 0.01; **Indicates a P value between 0.01 and 0.001; ***Indicates a P
value < 0.001.
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For above-ground plant biomass, increasing [CO2] resulted in [CO2] by Arabica cultivar responses at the
4-month period (122–124 DAS), but not at 7 (203–211 DAS) or 12 months (357–368) (Table1; Fig.3). By the end
of the study (~12 months), no eect of [CO2] was evident for robusta (Fig.3); however, marginally signicant
dierences (P < 0.1) between Arabica and robusta for above ground dry weight were observed (Table1; Fig.4).
Overall, by 12 months, Arabica, on average, showed a signicant response to increasing [CO2] for several veg-
etative parameters; whereas robusta was insensitive to [CO2] for several vegetative parameters (Table1, Fig.4).
In addition to growth and vegetative response, [CO2] induced changes in qualitative parameters, e.g., % N,
carbon to nitrogen (C:N) ratio and caeine concentration are of interest.
For the nal harvest, when averaged for all taxa, signicant eects were noted for C:N ratio for [CO2] (Table1,
Fig.5A), and for Arabica vs. robusta (Table1, Fig.5B). Dierences for the Arabica cultivars were also noted for
C:N and caeine, but not for % N (P = 0.12) (Table1; Fig.5C). Interactions, [CO2] × Arabica cultivars only,
Arabica vs robusta or cultivar (all four taxa) were not signicant for % N or C:N ratio (Table1). When averaged
for all taxa, there were no signicant dierences in caeine (Table1, Fig.6A), in contrast to a signicant dierence
in reductions of caeine with increasing [CO2] for robusta but not Arabica (Table1, Fig.6B). No caeine concen-
tration (mg g1) dierences were observed among the three Arabica cultivars (Table1, Fig.6C),
Discussion
Plant growth and development, assuming physiologically relevant temperatures, relies on four environmental
(abiotic) resources: nutrients (macro- and micro-), light, water, and CO2. Any change in one (or more) of these
resources could lead to a change in tness among dierent genotypes32. In managed plant systems, there have
been numerous studies indicating intraspecic variation to [CO2] with respect to vegetative and physiological
characteristics, including yield, for a given crop species33. Sucient variation has been reported so that screening
or selecting for enhanced [CO2] responsive cultivars oers a potential means to increase crop yields and improve
nutrition, which are important steps to help adapt production to global climate change2,13,33,34.
0
2
4
6
8
10
12
14
Bourbon, Arabica
Catimor, Arabica
Typica, Arabica
Robusta
122-124 DAS
Plant Height (cm)
10
15
20
25
30
35
203-211 DAS
CO
2
concentration (ppm)
250300 350400 450500 550600 65
0
20
30
40
50
60
70
80
357-368 DAS
Figure 1. Change in plant height (Average + SE) as a function of days aer sowing (DAS) and [CO2] for three
Arabica cultivars and robusta coee.
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Eorts have been made to identify variation in productivity responses to elevated [CO2] for forest tree spe-
cies35. Such studies have found considerable intraspecic variation in photosynthesis, stem biomass and volume
for poplar, pine, birch, eucalyptus, etc., at elevated [CO2], suggesting that under non-limiting environmental
conditions, (e.g., temperature, nutrients, water), intraspecic variation could be used to select for increased pro-
ductivity as atmospheric CO2 increases36,37. However, similar eorts for intraspecic or interspecic selection to
[CO2] among tree crops (e.g., apples, cacao) are, at present, unavailable, despite experiments showing that trees
can be more responsive than herbaceous plants to elevated CO234.
In the current study, while Arabica cultivars showed a signicant response to rising [CO2] above the mid-20th
century baseline (i.e., 317 ppm) for leaf area and growth parameters, signicant variation among Arabica cultivars
was not evident for any DAS harvest. In contrast, robusta coee was consistently less responsive to rising [CO2]
for growth biomass traits. Accordingly, there is a clear interspecic (between species) dierence between Arabica
and robusta to rising CO2 with respect to the degree of [CO2] stimulation. Such divergence is evident in leaf
weight, number of branches, and above ground biomass (Fig.4).
In addition to dierential growth response to rising [CO2], numerous reports have indicated CO2 induced
changes in secondary plant chemistry4. Of ubiquitous note in these observed changes is the CO2 induced decline
in protein and N, with subsequent increases in C:N ratio26. In the current study, similar N declines were observed,
but no interspecic or intraspecic dierences were recorded. However, caeine concentration (mg g1) when
averaged for all Arabica cultivars and for robusta combined, declined with additional [CO2], and this decline was
signicantly more for robusta vs. Arabica. Whether such declines may improve or reduce beverage quality in the
future remains to be determined.
If caeine acts as a deterrent against herbivores2729, a reduction in caeine in coee leaves and seeds might
result in decreased ability against deterrence, and consequently, increase pest pressure on the plants. Even though
the projected eects of climate change on the coee berry borer (Hypothenemus hampei), coee leaf miner
(Leucoptera coeella), coee white stem borer (Monochamus leoconotus), root-knot nematode (Meloidogyne
incognita), and coee leaf rust have been examined38, none of these studies considers possible changes in caeine
levels, and other chemistry, as a result of increasing CO2 levels.
0
50
100
150
Bourbon, Arabica
Catimor, Arabica
Typica, Arabica
Robusta
122-124 DAS
Leaf Area (cm
2
plant
-1
)
200
400
600
800
1000
203-211 DAS
CO
2
concentration (ppm)
250300 350400 450500 550600 65
0
2000
3000
4000
5000
6000
7000
8000
9000
357-368 DAS
Figure 2. Change in leaf area (cm2 per plant, average + SE) for three Arabica cultivars and robusta coee at
three dierent sampling times (days aer sowing, DAS) in response to [CO2].
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e results presented here indicate no signicant intraspecic variation in response to [CO2] among Arabica
cultivars and hence, no clear indication as to whether recent or projected changes in atmospheric CO2 could be
used as a selection factor for Arabica coee adaptation. However, there appear to be clear interspecic dierences
between Arabica and robusta in relation to both growth and caeine concentration at [CO2] above a 300 ppm
baseline. Such dierences suggest potential for dierential selection in tness as CO2 continues to increase.
ere are some obvious challenges in analyzing these data in the larger context of whether recent or projected
[CO2] can be used to select for more CO2 responsive coee cultivars or coee species. For example, vegetative
development is known to be the most sensitive stage of growth in relation to rising [CO2]23 and has been sug-
gested as a means to select for cultivar responsiveness in annual crops9,39. However, for tree crops, with slower
relative growth, rst year assessments may be useful in assessing initial response, but insucient to discern
longer-term dierential eects on seed production (i.e., crop yield and quality). ere are additional interspe-
cic and intraspecic issues related to environmental shis likely to change in parallel to rising [CO2] such as
precipitation and/or temperature that, in turn, will also inuence selection and adaptation of coee to climate
change. Yet, as indicated by these initial data, it seems unlikely that Arabica and robusta will respond similarly to
increasing [CO2] and such potential dierences may have long-term qualitative and quantitative consequences
for Arabica and robusta production globally. In addition, it will be of interest to compare interspecic dierences
between Arabica with C. eugenioides, the other parent of Arabica coee in a future study to determine if a similar
response pattern is observed for C. eugenioides.
e basis for dierential responses to rising [CO2] between Arabica and robusta is uncertain. ey may be
related to: (1) interspecic variation, due to physical (morphological) or physiological dierences, or a combina-
tion of both; (2) the eect of polyploidy, which amongst other features, inuences cell size, genomic stability, gene
expression and evolution rates40. All species of coee are diploid (2n = 2x = 22), except Arabica coee, which is an
allotetraploid (2n = 4x = 44)41,42. One of the recorded features of polyploidy in coee is that higher ploidy results
in fewer but larger stomata43,44, and this may be linked to the dierent CO2 eects we record in coee. Another
0.0
0.5
1.0
1.5
Bourbon, Arabica
Catimor, Arabica
Typica, Arabica
Robusta
122-124 DAS
Plant biomass (g plant
-1
)
0
2
4
6
8
10
12
203-211 DAS
CO
2
concentration (ppm)
250 300 350 400 450 500 550 600
650
0
20
40
60
80
100
357-368 DAS
Figure 3. Change in total plant biomass (grams per plant, average + SE) for three Arabica cultivars and robusta
coee at three dierent sampling times (days aer sowing, DAS) in response to [CO2].
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well-known consequence of polyploidy and specically allopolyploids, is self-compatibility (self-fertilization)4547.
ere are numerous evolutionary consequences for self-compatibility, including the reduction in genetic diver-
sity48; for cultivated (farmed) Arabica coee, this would be compounded by the severe genetic bottleneck created
through the domestication process49. is may explain the lack of dierence in CO2 response in the three Arabica
cultivars we have examined, although our sample size is not large enough to make any meaningful assessment.
ere would appear to be potential for [CO2] to be used as a selective factor in adaptation and yield response
for tree and perennial crops. Such eorts, however, are still in their infancy. e current study, the rst to examine
Arabica and robusta responses to recent and projected levels of CO2, from germination through the rst year of
growth, is suggestive of either interspecic dierences or polyploidy level, but additional, long-term information
will be needed to adequately determine how, and to what extent, recent and ongoing increases in [CO2] and/or
climate change may act as a selection factor among Arabica cultivars. Moreover, it will be necessary to consider
drought stress (reduced water availability), which so far has received scant attention in CO2 enrichment inu-
ences for coee with regard to climate change50. It has been argued that the inuence of climate change on coee
production has been overestimated, although work so far has focused on elevated air temperatures22. Indeed,
mitigation of elevated temperatures due to elevated CO2 does seem to oer potential where there is adequate
soil water availability (e.g., at eld capacity)19,20 but in many circumstances it is soil water availability (including
temporal availability), and its relationship with other climatic variables (including temperature), that is the main
limiting factor when considering climate change induced morbidity and mortality50. e interaction between
elevated CO2 and abscisic acid signaling, stomatal closure and CO2 inux, as well as other physiological and
chemical processes involved with drought51, require careful investigation.
Methods
Seeds. Three 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 coee cultivars grown
worldwide and are believed to have originated from coee grown in Yemen of Ethiopian origin54,55. Arabica coee
grown in Indonesia originated from Yemen, and seeds taken from Java (Indonesia) to Amsterdam and then to
Leaf Wt. (g)
0
20
40
60
Arabica
Robusta
No. Branches
0
5
10
15
20
[CO
2
]
300400 500 600
Abv. Grd. Dry Wt. (g)
0
20
40
60
80
100
120
Figure 4. Dierential changes between Arabica and robusta coee (average + SE) for above ground dry weight,
number of branches, and leaf weight in response to [CO2] at 357–368 DAS.
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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 coee cultivars: cv. ‘Caturra’ and cv. ‘Híbrido de Timor’ or ‘Timor Hybrid’ (a natural polyploid hybrid origi-
nating 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 coee leaf rust (Hemileia vastatrix) and gained their
resistance genes from robusta coee52,53.
Mature coee 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 caeine compared to Arabica (ca. 1.7% vs. 1%, respectively)56 and is adapted to growth at lower eleva-
tions 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.
Planting. Twelve 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 aer 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 rst run, seeds were planted on August
10, 2016 and the rst germination occurred on September 5, 2016. For the second run, seeds were planted on
September 12, 2017 and the rst germination occurred on October 11, 2017. Rates of germination did not vary
as a function of [CO2].
8
9
10
11
12
All Taxa
Carbon : Nitrogen (leaf)
8
9
10
11
12
Arabica
Robusta
CO
2
(ppm)
300400 50
06
00
8
9
10
11
12
Bourbon
Catimor
Typica
A.
B.
C.
Figure 5. Response of carbon to nitrogen ratio (+SE) for: (A) all taxa; (B) Arabica and robusta coee; and (C)
all Arabica cultivars in response to [CO2].
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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 nal harvest. An iron chelate micronutrient (Sprint 330, Becker
Underwood, Ames, IA, USA) was sprayed as needed. e growth medium/soil was maintained at, or close to,
eld capacity.
Environmental chambers. Providing pre-ambient [CO2] concentrations is not possible in situ; therefore,
controlled environment chambers (Bio-Chambers, Incorporated, Winnipeg, Canada) were used. e temperature
for each chamber was kept constant at 25 °C, day/night. Light, quantied as photosynthetically active radiation
(PAR), was maintained at 400 µmol mol1. e daily light period was 12 h light was supplied by height-adjustable,
dimmable banks of metal halide and high-pressure sodium bulbs (400 µmol m2 s1).
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 day1. ese
[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
rst run, and 303 ± 23.2, 409 ± 29.6, 499 ± 20.4, and 596 ± 23.0 in the second run.
Harvests. Destructive harvests were performed at three dierent 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) aer
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.
0
5
10
15
20
All Taxa
Caffeine Concentration (mg g
-1
)
0
5
10
15
20
Arabica
Robusta
CO
2
(ppm)
300400 50
06
00
0
5
10
15
20
Bourbon
Catimor
Typica
A.
B.
C.
Figure 6. Caeine concentration as a function of [CO2] (average +SE) for: (A) all taxa; (B) Arabica and robusta
coee; and, (C) all Arabica cultivars in response to [CO2].
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C:N ratios and caeine analysis. For 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 caeine, leaves within a replicate were ash 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. e slurry
was centrifuged at 5,000 rpm for 10 min before being diluted (1:20), ltered, 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 Scientic Instruments, Columbia, MD, USA) using a mobile phase of 80% methanol/
water and 15 mM phosphate buer at pH 6.2. Separation was conducted using a ermo Scientic Aquasil reverse
phase C18 column (4.6 × 250 mm, 5 µm particle size; ermo Fisher Scientic, Waltham, MA, USA) at a ow
rate of 0.550 ml/min. Detection and quantication was done using a UV detector at 275 nm and determined using
a calibration curve. e caeine calibration curve was created using an HPLC grade caeine standard (99.7%
purity; ACROS Organics #10816-5000; ermo Fisher Scientic, Waltham, MA, USA) across ve concentrations
2.5, 5, 10, 20, and 25 ppm. e tted 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%.
e caeine concentration in leaves can also be used as a proxy for concentrations in coee beans, based on a
correlation between caeine 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 signicant dif-
ferences in caeine content between the last pair of fully expanded leaves and all remaining leaves combined (coty-
ledons excluded; using March 2017 samples, i.e., rst year, second sampling; 7 months and 18 days post-planting).
Based on these results, we pooled all leaves at each sampling date for caeine analysis. Mazzafera and Magalhães63
found no correlation between leaves and seeds, but these were collected from mature plants, not seedlings.
Statistical analysis. ree replicate bins for each Arabica cultivar and for robusta coee (i.e., 12 bins per
chamber) were present for each of four [CO2] treatments. Within each chamber [CO2], bins were randomized;
and randomized again aer the rst two harvests at 4 and 7 months to avoid edge eects. Aer the rst run of
the experiment (i.e., one year), the chambers were randomly reassigned [CO2] treatments and the experiment
repeated. Humidity, PAR, and temperature were quantied 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 Soware, Cary, NC, USA).
Received: 6 September 2019; Accepted: 16 March 2020;
Published: xx xx xxxx
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Acknowledgements
We thank H. Vázquez Álvarez and L. Vázquez López in Chiapas, Mexico, for giving us permission to collect coee
fruits from their plantations; two reviewers for comments on a previous version of this paper; D. Baxam (USDA-
ARS) for assistance in running the growth chambers; and R. Erdman (USDA-ARS) for help in sampling coee
plants.
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Author contributions
F.E.V., L.H.Z. and A.S. conceived the project and designed the study; F.E.V., A.S. and J.W. ran the experiments;
F.E.V., L.H.Z. and A.P.D. did the literature review; L.H.Z. did the statistical analysis; L.H.Z., F.E.V. and A.P.D.
wrote the report. F.E.V., L.H.Z., A.S., F.I., A.P.D., J.A.R., J.Y.B. and J.W. interpreted the results, commented on the
dra version of the report, and approved the submission dra.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to F.E.V.
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... Concluyendo que hay diferencias en el crecimiento de plántulas de café según las condiciones climáticas donde se estén desarrollando, por cuanto se recomiendan localidades con menor altitud para el establecimiento de almácigos dado el mayor crecimiento obtenido, siendo las hojas la sección de mayor influencia. crop (Melo and Piñeros, 2015;Rodríguez et al., 2015;Robiglio et al., 2017;García et al., 2017;Borjass et al., 2018;Milla et al., 2019;Vega et al., 2020). ...
... Conversely, the other three municipalities showed lower mean values for these climatic variables. Additionally, studies based on comparisons of leaf area across robusta and arabica coffee plants report significant increases between four-and seven-month periods in response to temperature (Vega et al., 2020). The highest mean value for LA was obtained in La Unión (244.05cm 2 ), followed by La Florida (125.05cm 2 ), Consacá (112.84cm 2 ), and Sandoná (75.21cm 2 ). ...
... Additionally, studies based on comparisons of leaf area across robusta and arabica coffee plants report significant increases between four-and seven-month periods in response to temperature (Vega et al., 2020). ...
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The peach aphid Myzus persicae (Sulzer) and cotton aphid Aphis gossypii (Clover) (Hemiptera: Aphididae) are considered to be key pests affecting greenhouse pepper crops in Argentina as a result of their frequent occurrence and the seriousness of the damage caused by their feeding behavior and the transmission of virus. The goal of this research was to determine the efficiency of botanical products to control aphids and their side effects on parasitoids in this crop. Thus, three biorational pest control formulations derived from essential oils (EO) and plant extracts (Es) were tested, namely (i) neem EO, cinnamon EO, clove EO, oregano EO and American marigold EO (formulation 1); (ii) garlic EO and cinnamon EO (formulation 2); (iii) garlic E and rue E (formulation 3); and a soy lecithin adjuvant (lecithin), and finally, a control (water spray method). For this research, a completely randomized design was replicated 3 times. These treatments were applied directly to the foliage by means of a backpack sprayer on a weekly basis until the end of this trial. Subsequently, the total number of healthy aphids and parasitized aphids (mummies) on every leaf was recorded in the field and the laboratory through repeated measures Analysis of Variance (ANOVA) and LSD Fisher method. The results showed that formulation 1 and formulation 3 recorded a lower number of aphids and mummies compared to the other treatments. This evidence would demonstrate that these formulations repel aphids and parasitoids without the lethal effects caused by the use of broad spectrum insecticides.
... The leaf content can also be used as an alternative for coffee bean concentration. Spinoso-Castillo et al. [31] and Vega et al. [37] found a correlation among caffeine, trigonelline, and 5-CQA metabolites concentration in young leaves and beans. ...
... The first significant (P < 0. (Table 1). coffee, Vega et al. [37] found a reduction in caffeine in coffee leaves and seeds due to a decreased ability against deterrence and increased pest pressure. Also, in Arabica coffee leaves, Chen et al. [38] described values of 5-CQA (most abundant chlorogenic acid in Arabica coffee) ranging from 0.78 ± 0.04 to 21.39 ± 0.51 mg g − 1 of leaf. ...
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Background Coffee quality is an important selection criterion for coffee breeding. Metabolite profiling and Genome-Wide Association Studies (GWAS) effectively dissect the genetic background of complex traits such as metabolites content (caffeine, trigonelline, and 5-caffeoylquinic acid (5-CQA)) in coffee that affect quality. Therefore, it is important to determine the metabolic profiles of Coffea spp. genotypes. This study aimed to identify Single Nucleotide Polymorphisms (SNPs) within Coffea spp. genotypes through GWAS and associate these significant SNPs to the metabolic profiles of the different genotypes. Methods and results A total of 1,739 SNP markers were obtained from 80 genotypes using the DArTseq™ method. Caffeine, trigonelline, and 5-CQA content were determined in coffee leaves using Ultra-Performance Liquid Chromatography/tandem mass spectrometry (UPLC-MS/MS) analyses. The GWAS was carried out using the Genome Association and Prediction Integrated Tool (GAPIT) software and a compressed mixed linear model. Finally, a total of three significant SNP markers out of ten were identified. One SNP, located in the coffee chromosome (Chr) 8, was significantly associated with caffeine. The two remaining SNPs, located in Chr 4 and 5, were significantly associated with trigonelline and six SNPs markers were associated with 5-CQA in Chr 1, 5 and 10, but these six markers were not significant. Conclusions These significant SNP sequences were associated with protein ubiquitination, assimilation, and wall receptor kinases. Therefore, these SNPs might be useful hits in subsequent quality coffee breeding programs.
... From an ecological, food-web perspective, rising [CO 2 ] can compromise plant chemical defenses against invasive insects [53]. The basis for these changes is unclear, and it has been suggested that the increase in carbon relative to other elements is reflected in secondary chemistry as a resource-use hypothesis; however, such a response is not consistently evident [54]. ...
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... Finland is the largest per capita consumer of coffee, while China consumes the most coffee by volume [6]. Coffea arabica and C. canephora (robusta) are the two most-grown coffee species in the world [7], accounting for 60% and 40% of global production, respectively [8]. ...
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Coffee is grown in more than 80 countries as a cash crop and consumed worldwide as a beverage and food additive. It is susceptible to fungal infection during growth, processing and storage. Fungal infections, in particular, can seriously affect the quality of coffee and threaten human health. The data for this comprehensive review were collected from the United States Department of Agriculture, Agricultural Research Service (USDA ARS) website and published papers. This review lists the fungal species reported on coffee based on taxonomy, life mode, host, affected plant part and region. Five major fungal diseases and mycotoxin-producing species (post-harvest diseases of coffee) are also discussed. Furthermore, we address why coffee yield and quality are affected by fungi and propose methods to control fungal infections to increase coffee yield and improve quality. Endophytic fungi and their potential as biological control agents of coffee disease are also discussed.
... Recently, the effect of increasing [CO 2 ] on caffeine concentration was investigated in leaves of C. arabica and C. canephora grown under field conditions (Vega et al., 2020). In this work, caffeine amount was negatively correlated with e[CO 2 ] in C. canephora (cv. ...
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... Notably, these alterations on the gene expression profile of Icatu under eCO 2 are not reflected on bean physical and chemical characteristics, which was previously found to show marginally, if at all, changes in response to eCO 2 [24]. This was also suggested for caffeine in other C. arabica genotypes, using leaf caffeine contents as a proxy for concentrations in the beans [52]. ...
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Elevated atmospheric CO2 concentration (e[CO2]) can stimulate the photosynthesis and productivity of C3 species including food and forest crops. Intraspecific variation in responsiveness to e[CO2] can be exploited to increase productivity under e[CO2]. However, active selection of genotypes to increase productivity under e[CO2] is rarely performed across a wide range of germplasm, because of constraints of space and the cost of CO2 fumigation facilities. If we are to capitalise on recent advances in whole genome sequencing, approaches are required to help overcome these issues of space and cost. Here, we discuss the advantage of applying prescreening as a tool in large genome × e[CO2] experiments, where a surrogate for e[CO2] was used to select cultivars for more detailed analysis under e[CO2] conditions. We discuss why phenotypic prescreening in population-wide screening for e[CO2] responsiveness is necessary, what approaches could be used for prescreening for e[CO2] responsiveness, and how the data can be used to improve genetic selection of high-performing cultivars. We do this within the framework of understanding the strengths and limitations of genotype-phenotype mapping.
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