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Environmental Effects on Coffee Seed

Biochemical Composition and Quality
Attributes: a Genomic Perspective
CONFERENCE PAPER SEPTEMBER 2014

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3 AUTHORS:
Thierry JOT

Benoit Bertrand

Institute of Research for Development

Cirad - La recherche agronomique pour le d

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Stphane Dussert
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Retrieved on: 29 October 2015

Index

Table of contents

Environmental Effects on Coffee Seed Biochemical Composition

and Quality Attributes: a Genomic Perspective
T. JOT1, B BERTRAND2, S. DUSSERT1
1
2

IRD, UMR DIADE, BP 64501, 34394 Montpellier, France,

CIRAD, UMR RPB, BP 64501, 34394 Montpellier, France

SUMMARY
Reserve compounds and secondary metabolites that accumulate in mature coffee seeds
contribute to a large extent directly or through roasting-induced chemical reactions to the
broad spectrum of aromas and flavours of the coffee brew. Although cultivation of coffee
trees under shade or at high elevation is known to favorably affect coffee quality, quantitative
data describing the influence of climatic conditions on the chemical composition of the seed
are still scarce. After a review of the relationships between coffee quality, seed chemical
composition and environmental effects, this report will focus on the last advances in the
understanding of environmental regulations of coffee seed metabolic pathways. Using
multienvironment designs displaying broad climatic variations, and parallel monitoring of
gene expression levels and metabolite accumulation profiles, we showed how growth
conditions - such as mean air temperature - influence, in a predictable way, fatty acid, sugar
and chlorogenic acid metabolisms and alter the chemical composition of the mature seed
through subtle transcriptional regulations during seed development. Progress in this area
could ultimately help in developing robust genomics/metabolomics fingerprints of coffee
bean origin and quality, and help in assisting breeding programs dedicated to coffee quality.
INTRODUCTION
The flavour of a cup of coffee is the final expression and perceptible result of a long chain of
chemical transformations from the seed to the cup. The reserve compounds and secondary
metabolites that accumulate during seed development contribute to a large extent directly or
through roasting-induced chemical reactions to the broad spectrum of aromas and flavours
of the coffee brew. In addition to common seed storage proteins (11S globulin), sugars
(mainly sucrose) and lipids (triacylglycerols), coffee seeds also synthesize spectacular
amounts of peculiar compounds not found in the seeds of model plants, such as
galactomannans, chlorogenic acids, caffeine and diterpenes. Each of these chemical classes
plays a crucial role in the complex roasting chemistry (Flament, 2002). For example, proteins
and amino acids are essential for the conversion of reducing sugars into aroma precursors
through Maillard reactions. Reducing sugars themselves result from the degradation of
sucrose and cell-wall polysaccharides. In addition, triacylglycerols are the major carriers of
aromas in the roasted bean. Their fatty acid composition determines the generation of
thermally-induced oxidation products, in particular aldehydes, which react readily with
Maillard intermediates giving rise to additional aroma compounds. Finally, chlorogenic acids
and caffeine are responsible for bitterness. Therefore, during the last decade, much research
has been devoted to elucidating the numerous factors that influence the chemical composition
of the seed. The seed content in aroma precursors may vary with genetic traits (Leroy et al.,
2006), agricultural practices (Vaast et al., 2006), post-harvest techniques (Selmar et al.,
2006), as well as climatic conditions. The first part of the present review aims at summarizing
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our knowledge on this latter aspect: the impact of the environment on the coffee seed
chemistry.
Aroma precursors are synthesized and/or transported in the endosperm (a triploid tissue that
constitutes most of the coffee seed volume) during seed development and maturation.
Therefore, a better understanding of the developmental processes that govern the
accumulation of these compounds, i.e. the identification of their biosynthetic genes, enzymes
and the characterization of the regulatory processes involved, may provide novel targets and
strategies for coffee quality breeding. Deciphering metabolic pathways that occur during
coffee seed development has benefited from several technical advances in transcriptome
analysis: real-time RT-PCR (Salmona et al., 2008), oligonucleotide microarrays (Privat et al.,
2011) and RNAseq (Denoeud et al., 2014). Using these techniques, the characterization of
gene expression profiles during seed development enabled to identify key quality-related
genes (for a review, see de Castro & Marraccini, 2006; Jot et al., 2012). In order to better
understand the influence of the environment on the chemical composition of the coffee seed,
these transcriptomic approaches have recently been employed in multilocation trials. The
second part of the present review provides the most relevant findings arising from these
investigations.
ENVIRONMENTAL EFFECTS ON COFFEE SEED CHEMISTRY
Although environmental factors such as shade and altitude are empirically known to have
beneficial effects on coffee quality, the impact on the environment has hardly been
documented before the 2000s (Guyot et al., 1996). Macro-diagnostic surveys led at the
regional scale in Honduras, Costa Rica or Brazil, first established the relationships between
coffee quality and environmental factors (Decazy et al., 2003; Avelino et al., 2005; Barbosa
et al., 2012). These studies showed significant correlations between coffee quality and
geographic/topographic parameters such as latitude, altitude or slope exposure. However,
contradictory results were observed regarding the influence of the environment on the
chemical composition of green beans. These discrepancies may be due to the fact that these
surveys were not performed using experimental sites that were strictly designed to study
environmental effects: agricultural management and post-harvest treatments were not
controlled.
Further studies therefore focused on the effect of individual environmental factors using
dedicated experimental designs. Since elevation is the environmental factor most frequently
mentioned with respect to quality, and coffee grown at high elevations fetches a higher price
than that grown in lowland regions, this environmental parameter has received much
attention. It was thus demonstrated that coffee from higher elevations exhibits better beverage
quality (Bertrand et al., 2006). Elevation also impacts the chemical content of green beans, for
instance their lipid content and composition (Villareal et al., 2009). The percentages of the
two major fatty acids, namely linoleic and palmitic acids (3045% each), increased with
altitude and were negatively correlated with environmental temperature, while oleic and
stearic acids (5-10% each) were favored in warmer conditions. Shade is another
environmental factor frequently mentioned with respect to coffee quality. For instance,
agroforestry is empirically known to provide cooler conditions that delay berry ripening and
positively affect coffee quality. Several reports documented these effects using shade houses
specifically built to adjust growth irradiance at a fixed fraction of the global irradiance. This
approach was first described by Vaast and colleagues in Costa Rica (Vaast et al., 2006;
Geromel et al., 2008; Somporn et al., 2012). Shade was shown to increase seed size, to
positively affect coffee quality, as well as to influence chemical composition such as reducing
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sugar content (Geromel et al., 2008). Finally, the influence of rainfall was assessed in various
locations in Brazil through controlled water irrigation (Da Silva et al., 2005). However, the
influence of water availability was found to be rather limited.
QUANTITATIVE EFFECTS OF CLIMATIC FACTORS ON SEED CHEMICAL
COMPOSITION
A better understanding of the metabolic status of the seed at harvest in relation with climatic
factors requires an accurate measurement of the main climatic variables (temperature, rainfall,
irradiance, and evapotranspiration potential) experienced during seed development and a
comprehensive analysis of the quantitative relationships between these climatic variables and
coffee quality and chemical content. It also necessitates a large multilocation trial that
maximizes climatic gradients but lowers other sources of variations such as agricultural
practices.
Reunion Island, which associates a rich homogeneous volcanic soil, strong altitudinal and
rainfall gradients over very short distances, and a high density network of meteorological
stations, has recently been shown as an adequate area to address these issues (Jot et al.,
2010). In order to accurately assess climatic effects, coffee sensory quality and chemical
composition were analyzed from green coffee samples collected from 16 Arabica coffee plots
located throughout Reunion Island and encompassing a wide range of tropical climatic
conditions. All plots were planted the same year with the same cultivar and underwent
identical agricultural management and post-harvest treatments.
Of the climatic factors recorded, temperature played a paramount role on bean quality since it
was correlated with six (of the eight) sensory attributes measured. Aroma, acidity, fruitiness
and overall quality were all favored by cool climates, whilst the undesirable earthy and green
tastes were increasingly present as the temperature increased (Bertrand et al., 2012). The
other climatic variables played a minor role but it is worth noting that rainfall and potential
evapotranspiration were correlated with bitterness and green taste, respectively. Using the
same experimental plots, changes in lipid, chlorogenic acid, sugar and caffeine contents were
monitored throughout seed development (Jot et al., 2010). Surprisingly, none of the
environmental factors studied significantly influenced the accumulation of the four main
classes of storage compounds. Indeed, total cell-wall polysaccharides, total lipids, total free
sugars and total chlorogenic acids showed no significant correlation with any of the climatic
variables measured. In contrast, within a given chemical class (e.g. chlorogenic acids), several
compounds may be significantly influenced by the environment. Half of the 28 metabolites
analyzed by conventional analytical chemistry methods were significantly correlated with the
average air temperature during the last five months of seed development i.e. the period
when storage compounds accumulate in the seed (Jot et al., 2009). However, no significant
correlation was found between rainfall or potential evapotranspiration and any of the
compounds studied and only weak correlations were found with solar irradiance.
The slope of regression lines may also differ among the compounds of a given chemical class.
For instance, within caffeoylquinic acids (CQAs), 3- and 4-CQA contents were positively
correlated with temperature while the reverse trend was observed for the major chlorogenic
acid, 5-CQA (Fig 1A). Interestingly, the same phenomenon was found for dicaffeoylquinic
acids (di-CQAs): i.e. di3.4-CQA and di-4.5-CQA were positively influenced by temperature
while a negative correlation was observed for di3.5-CQA. These results suggest that
temperature directly acts on routing towards the different branches within the chlorogenic
acid metabolic pathway without affecting the total seed chlorogenic acid content. A similar
regulation of routing was observed within the fatty acid biosynthetic pathway, as already
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reported by Villareal et al. (2009). The positive relationship observed between temperature
and stearic, oleic, arachidic and behenic acids showed that acyl chain elongation increased
with temperature. In contrast, linoleic and linolenic acid contents were negatively correlated
to temperature, showing that acyl desaturation increased at low temperatures (Villareal et al.,
2009; Jot et al., 2010). Finally, a similar trend was observed for free soluble sugars with
opposite temperature effects for glucose and stachyose.
In addition to major seed storage compounds, attention was paid to minor components that
may play a direct role on coffee aroma. Of the 44 volatile compounds quantified in green
coffee beans by gas-chromatography coupled to mass spectrometry, 21 displayed significant
variations among locations. The mean air temperature appeared again to be the predominant
causal factor since it was correlated with 16 volatile compounds (Bertrand et al., 2012).
Among the volatiles detected, most of alcohols, aldehydes, hydrocarbons and ketones were
positively correlated with temperature and solar radiation. These volatile compounds are
therefore possible indicators of unpleasant sensory attributes. For example, two alcohols
(butan-1,3-diol and butan-2,3-diol) were negatively correlated with aroma and acidity, and
positively correlated with earthy and green flavors.
CHARACTERIZATION OF ENVIRONMENTALLY-INDUCED VARIATIONS
DURING SEED DEVELOPMENT AND USE OF THESE VARIATIONS TO
DECIPHER TRANSCRIPTIONAL REGULATORY PROCESSES OF METABOLIC
PATHWAYS
So far, combining the use of multilocation trials, gene expression monitoring and metabolite
profiling constitutes the most advanced strategy to understand how environmental factors and
developmental programmes interplay to affect coffee seed quality. The metabolism of
chlorogenic acids and that of galactomannans are described in the present review to exemplify
the potential of this novel systems biology approach.
To investigate chlorogenic acid biosynthesis, the expression of selected phenylpropanoid
biosynthetic genes, together with the accumulation profile of chlorogenic acid isomers, was
monitored throughout seed development using the 16 locations in Reunion Island described
above, which maximize climatic variation. Environmental temperature was shown to have a
direct impact on the time-window for chlorogenic acid biosynthetic activity through subtle
transcriptional regulations (Jot et al., 2010b). The first steps of chlorogenic acid biosynthesis
involve the well-characterized key enzymes of the core phenylpropanoid pathway, namely
phenylammonialyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase
(4CL). Transcript profiling revealed the modulation of PAL and C4H gene expression by
temperature during endosperm development (Fig 1B). High temperatures first induced overaccumulation of mRNA encoding these two enzymes in early endosperm developmental
stage. The reverse situation (a negative correlation between the level of PAL and C4H
expression and temperature) was observed later, indicating a delay in the activation of
phenylpropanoid genes under cool climates. This finding provides a sound explanation for the
delay in the accumulation of 5-CQA observed at low temperatures. Moreover, the variability
in seed chlorogenic acid composition induced by environmental temperature constituted a
valuable system to test whether this accumulation is modulated at the transcriptional level
and, if so, to detect rate-limiting transcriptional steps for chlorogenic acid biosynthesis. Final
amount of 5-CQA, the major chlorogenic acid, was quantitatively correlated with early
expression of 4CL gene, but also with that of HQT, which encodes the enzyme thought to
catalyze the last step of 5-CQA biosynthesis (Fig 1C). These two genes thus are rate-limiting
transcriptional steps for chlorogenic acid biosynthesis and are referred as Quantitative Trait
Transcripts (QTTs) for chlorogenic accumulation (Jot et al., 2010b).
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Phenylalanine

Environmental cues & gene expression

Early endosperm development

PAL

C4H
p-Coumarate

C4H

(B)

PAL2

Expression

Cinnamate

Expression

PAL2

C4H

5-CQA (final amount)

QTTs
4CL

(C)

14 16 18 20 22 24

Temperature

14 16 18 20 22 24

Temperature

p-Coumaroyl CoA
4CL8

HQT
Environmental cues & metabolites
p-Coumaroyl Quinate

HQT
Expression
(early dvt)

(A)
C3'H

0.5
0.4

3-CQA

0.3
0.2
12 14 16 18 20 22 24 26

5-Caffeoyl quinate
5-CQA

5.5
5.0
4.5
4.0
12 14 16 18 20 22 24 26

Temperature

0,7

4-CQA

0,6
0,5
0,4
12 14 16 18 20 22 24 26

Temperature

Figure 1. System analysis of chlorogenic acid metabolism in the developing coffee seed.
The different boxes highlight highly significant correlations obtained between climatic,
genomic, and metabolite datasets. Abbreviations: C3'H, p-coumaroyl CoA 3hydroxylase; C4H, trans-cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase;
HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase; QTT,
quantitative trait transcripts; PAL, phenylalanine ammonia lyase. Adapted from Jot et
al. (2010b).
A similar strategy was chosen to elucidate the biosynthesis of galactomannans, which are the
major cell-wall storage polysaccharide in coffee (Jot et al., 2014). The expression profile of
28 genes related to carbohydrate metabolism and galactomannan biosynthesis was measured
during seed development. This enabled to detect two types of environmental effect on
transcriptional activity. First, a modulation of transcript accumulation by temperature was
revealed for several genes involved in the synthesis of activated nucleotide sugars, i.e. GDPmannose and UDP-galactose, which primarily serve as building blocks for galactomannan
synthesis. For example, the expression level of MGT (mannose 1P-guanyl transferase), and
UG4E (UDP-glucose 4 epimerase) was quantitatively affected by environmental
temperature. Second, the environmental temperature impacted the timing of transcription for
several key genes encoding galactomannan biosynthetic enzymes, such as galactomannangalactosyl transferase (GMGT). The variability induced by the environment was also
evidenced to be a useful tool to detect significant transcript-transcript quantitative
correlations, hence enabling the characterization of gene transcriptional modules
(quantitatively co-expressed genes). A dense module of nine genes was detected at the onset
of galactomannan accumulation. This module included the five genes of the core
galactomannan synthetic machinery, which encode the enzymes needed to assemble the
mannan backbone (e.g. ManS), introduce the galactosyl side chains (e.g. GMGT), modulate
the post-depositional degree of galactose substitution (e.g. -Gal). This module also included
the sucrose synthase gene SUSY1, stressing the tight transcriptional coordination between the
sucrolytic activity required for nucleotide sugar production and galactomannan assembly.
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Finally, the module contained two other genes, GolS3 (galactinol synthase) and SDH (sorbitol
dehydrogenase), revealing a link between galactomannan synthesis and raffinose
oligosaccharide and sorbitol metabolisms, suggesting these metabolites play a role as transient
carbohydrate reservoirs during peak galactomannan synthesis (Jot et al., 2014).
PERSPECTIVES
For now, only a few recent studies have coupled transcriptomics and metabolomics to
investigate the metabolism of developing coffee seeds. These new system biology approaches,
using multienvironment designs, offer outstanding opportunities to unravel the regulation of
the coffee seed biosynthetic processes and to revisit the effects of the environment, such as
those of shade and drought. Coupled transcriptome-metabolome surveys in larger
experimental designs may also be helpful for a better prediction of climate change impact on
seed quality. Furthermore, using multigenotype designs, such approaches could also enable
the fine characterization of Genotype X Environment interactions. Progress in these areas will
ultimately help in developing robust genomics/metabolomics fingerprints of coffee bean
origin (Bertrand et al., 2008) and quality, and help in assisting breeding programs dedicated
to coffee quality.
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