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EnvironmentalEffectsonCoffeeSeedBiochemicalCompositionandQualityAttributes:aGenomicPerspective

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42

Environmental Effects on Coffee Seed Biochemical Composition

and Quality Attributes: a Genomic Perspective

T. JOËT1, B BERTRAND2, S. DUSSERT1

1 IRD, UMR DIADE, BP 64501, 34394 Montpellier, France, 2 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

43

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; Joët 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 (30–45% 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

44

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 (Joët 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 (Joët 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 (Joët 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

45

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; Joët 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 (Joët 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 over-

accumulation 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 (Joët et al., 2010b).

46

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 3-

hydroxylase; 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 Joët et

al. (2010b).

A similar strategy was chosen to elucidate the biosynthesis of galactomannans, which are the

major cell-wall storage polysaccharide in coffee (Joët 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. GDP-

mannose 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 galactomannan-

galactosyl 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. α-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.

HQT

C3'H

p-Coumaroyl Quinate

5-Caffeoyl quinate

p-Coumarate

4CL

C4H

Phenylalanine

PAL

Cinnamate

p-Coumaroyl CoA

Early endosperm development

14 16 18 20 22 24

PAL2

C4H

Temperature

PAL2

14 16 18 20 22 24

C4H

Temperature

Exp

ressio

n

Exp

ressio

n

5-C

QA

(fi

nal a

mo

un

t)

HQT

4CL8

Expression

(early dvt)

Environmental cues & gene expression

QTTs

(C)

(B)

12 14 16 18 20 22 24 26

4.0

4.5

5.0

5.5

12 14 16 18 20 22 24 26

0.2

0.3

0.4

0.5

12 14 16 18 20 22 24 26

0,4

0,5

0,6

0,7

3-CQA

4-CQA

Temperature Temperature

5-C

QA

(A)

Environmental cues & metabolites

47

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 (Joët 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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