Food Chemistry 118 (2010) 693–701

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Food Chemistry

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Influence of environmental factors, wet processing and their interactionson the biochemical composition of green Arabica coffee beans

Thierry Joët a,*, Andréina Laffargue b, Frédéric Descroix c, Sylvie Doulbeau b, Benoît Bertrand d,Alexandre de kochko b, Stéphane Dussert b

a IRD, UMR DIA-PC, Pôle de Protection des Plantes, 97410 Saint Pierre, La Réunion, Franceb IRD, UMR DIA-PC, BP 64501, 34394 Montpellier, Francec CIRAD, UMR QualiSud, 97410 Saint Pierre, La Réunion, Franced CIRAD, UMR RPB, BP 64501, 34394 Montpellier, France

a r t i c l e i n f o

Article history:Received 12 March 2009Received in revised form 6 April 2009Accepted 18 May 2009

Keywords:CoffeaEnvironmentChlorogenic acidCaffeineFatty acidSoluble sugars

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.05.048

* Corresponding author. Tel.: +33 4 67 41 61 85; faE-mail address: thierry.joet@ird.fr (T. Joët).

a b s t r a c t

Although cultivation of Arabica coffee trees at high elevation is known to favourably affect the final qualityof the beverage, quantitative data describing the influence of climatic conditions on the chemical compo-sition of the seed are still lacking. Similarly, post-harvest treatments of the beans are known to affect thegeneration of flavour, but the chemical transformations that occur during wet processing are poorlyunderstood. To better characterise the effects of the environment, wet processing and their possible inter-actions, we quantified the changes in the main chemical components of the coffee seed (lipids, chlorogenicacids, sugars and caffeine) caused by wet processing, and analysed how these changes were affected by thevariations induced by the environment before harvest. Using 16 experimental plots in Reunion Islanddisplaying broad climatic variations, we showed that chlorogenic acids and fatty acids in the seed werecontrolled by the mean air temperature during seed development. By contrast, total lipid, total solublesugar, total polysaccharide and total chlorogenic acid contents were not influenced by climate. Glucosecontent was positively affected by altitude, while sorbitol content after wet processing depended directlyon the glucose content in fresh seeds.

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1. Introduction

The flavour of a freshly prepared cup of coffee is the finalexpression and perceptible result of a long chain of transforma-tions between the seed and the cup. The coffee seed contains allthe necessary precursors to generate coffee aroma during roasting.The major storage compounds of mature Coffea arabica seeds arecell-wall polysaccharides (CWP, 48–60% DM), mainly galacto-mannans and arabinogalactan-proteins, lipids (13–17% DM), pro-teins (11–15% DM), sucrose (7–11% DM) and chlorogenic acids(CGA, 5–8% DM) (for a review see De Castro & Marraccini, 2006).Each of these major storage compounds plays several crucial rolesin the complex roasting chemistry (Flament, 2002). For example,proteins and amino acids are essential for the conversion of reduc-ing sugars into aroma precursors through Maillard reactions.Reducing sugars themselves result from the degradation of sucroseand CWP. In addition, triacylglycerols are the major carriers of ar-oma in the roasted bean. Their fatty acid (FA) composition deter-mines the generation of thermally-induced oxidation products, inparticular aldehydes, which react readily with Maillard intermedi-

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ates, giving rise to additional aroma compounds. CGA and caffeineare responsible for bitterness. Therefore, during recent decades,much research has been devoted to elucidating the numerous fac-tors that influence the chemical composition of the seed. Indeed,seed levels in aroma precursors may vary with genetic traits (Leroyet al., 2006), soil–climatic conditions (Bertrand et al., 2006),agricultural practices (Vaast, Bertrand, Perriot, Guyot, & Genard,2006) and post-harvest techniques (Selmar, Bytof, Knopp, &Breitenstein, 2006).

Although shade and altitude are empirically known to havebeneficial effects on coffee quality, only a few scientific studieshave actually documented these effects. Both factors have a dra-matic influence on air temperature, and their positive effects havebeen mostly attributed to cooler climatic conditions. Low temper-atures have been suggested to slow down the ripening process,which in turns leads to higher accumulation of aroma precursors(Vaast, Bertrand, Perriot, Guyot, & Genard, 2006). It is generally ac-cepted that high altitude improves coffee quality (Avelino et al.,2005; Decazy et al., 2003; Guyot et al., 1996). However, contradic-tory results were obtained regarding its influence on the chemicalcomposition of green beans. For instance, total lipid content wasreported to either decrease with increasing altitude (Guyot et al.,1996) or to not be influenced by altitude at all (Avelino et al.,

694 T. Joët et al. / Food Chemistry 118 (2010) 693–701

2005). Similarly, chlorogenic acids were reported to accumulatewith an increase in altitude in one study (Avelino et al., 2005)but to remain stable in another (Guyot et al., 1996). These discrep-ancies may be due to the fact that previous surveys were not per-formed using experimental sites that were strictly designed tostudy the effect of altitude: i.e. the soil–climatic conditions mayalso have varied at the sites (soil type and solar exposure, forexample) as well as agricultural management. In addition, thestudies covered a low altitude range (6400 m). However, a recentstudy investigated the effect of altitude on coffee bean compositionusing eight experimental plots located at regular intervals withinan altitude range of 550 m on the same slope of the Poas volcanoin Costa Rica (Bertrand et al., 2006). Positive statistical relation-ships were found between altitude and caffeine, CGA (on the basisof the main CGA component, 5-CQA), and total lipids of greenbeans, estimated by NIRS. Unfortunately, mesoclimate parameterswere not recorded locally and it was therefore not possible to con-clude whether the effects of altitude were mainly related to a tem-perature gradient or to other soil–climatic variables. Consequently,a rigorous study is still required to comprehensively analyse therelationships between main climatic variables (temperature, rain-fall, irradiance and evapotranspiration potential) and the composi-tion of the coffee seed.

Concerning post-harvest treatments, green coffee is tradition-ally produced by either wet or dry processing. Both methods aimto remove the pericarp of the coffee cherry and to dry the raw cof-fee seed to a moisture content of 10–12% fresh weight. In the dryprocess, whole coffee fruits are dried in the sun and then hulledmechanically. In the wet process, ripe coffee cherries are mechan-ically depulped and the mucilaginous residues are degraded duringa ‘fermentation’ step and then washed off. The resulting beans arestill covered by the endocarp, termed parchment. The parchmentcoffee is then dried and subsequently hulled. The chemical compo-sition of wet- and dry-processed seeds may differ significantly, asobserved for free amino acids, organic acids, and non-structuralcarbohydrates (Bytof, Knopp, Schieberle, Teutsch, & Selmar, 2005;Knopp, Bytof, & Selmar, 2006). Dry-processed coffees are generallycharacterised by more body, whereas in most terroirs, wet-pro-cessed coffees have a better aroma, generally resulting in higheracceptance (Selmar, Bytof, & Knopp, 2002). In both treatments,the freshly processed coffee beans remain viable and exhibit activemetabolic processes (Bytof et al., 2007). This observation estab-lished a novel and stimulating bridge between two distant disci-plines in the coffee community: seed physiology and thechemistry of green bean technology.

It is now widely accepted that the metabolic reactions that oc-cur during wet-processing help generate pleasant cup quality attri-butes (Selmar et al., 2002). Based on the observation of the earlyinduction of the isocitrate lyase gene involved in the glyoxylate cy-cle, it was recently suggested that the germination process was ini-tiated during the course of wet processing (Selmar et al., 2006). Theconcomitant increase in the abundance of b-tubulin, a marker forcell division or elongation, corroborates this hypothesis (Bytofet al., 2007). While chemical differences between wet and dry-pro-cessed seeds are well documented, data on the changes induced bythe treatment itself (metabolic shift between fresh and processedseeds) are very scarce. Moreover, in contrast to the knowledge ac-quired on the metabolic processes that occur in the course of coffeeseed development (Joët et al., 2009), no data is available on howenvironmental factors and post-harvest processes interplay.

Considering the importance of terroirs and post-harvest treat-ments in the generation of flavour, a better understanding of thechemical transformations occurring during wet processing, andtheir relationships with the metabolic status of the seed at harvest,seems highly warranted. The present study was carried out usingsamples from 16 Arabica coffee plots located throughout Reunion

Island. Its rich homogeneous volcanic soil and various microcli-mates within short distances, combined with a dense network ofmeteorological stations, make Reunion Island a valuable experi-mental site to document the effects of environmental factors onthe final composition of the bean. In order to estimate these effectsprecisely, all plots were planted the same year with exactly thesame cultivar and underwent identical agricultural management.We quantified changes in lipid, chlorogenic acid, sugar and caffeinecontents caused by wet processing and analysed how thesechanges were affected by the chemical variations induced by theenvironment.

2. Materials and methods

2.1. Biological material

Experiments were performed in Reunion Island on seeds of C.arabica cv. ‘Laurina’ (a natural dwarf mutant of cv. ‘Bourbon’) pre-senting two specific traits identified so far: rather low caffeine con-tent and a typical oblong shape. The experimental plots wereplanted in 2003 without shade and were in their second (2006)production year (about 2 m high). Plant spacing was 2 m betweenrows and 1 m within rows. Fertilisers were applied and pests anddiseases were controlled according to locally recommended prac-tices. Among 107 experimental plots available throughout the is-land, sixteen plots that maximise variations in altitude (150–1032 m asl.) and climatic conditions (Table 1) were selected. Thesurvey unit was a compact plot of about 240 coffee trees.

2.2. Meteorological observations and date of harvests

Reunion Island hosts a dense meteorological network of morethan 50 automated stations dedicated to sugarcane agronomy (thisservice is under the supervision of Meteo France and Cirad). Cli-matic conditions (temperatures, rainfall, total irradiance, and po-tential evapotranspiration) were estimated for each plot fromrecords at the nearest meteorological stations (data from MeteoFrance and Cirad) and corrected taking into account the differencein altitude. Temperatures were also recorded locally (under thecoffee canopy) using portable temperature recorders. Dates of har-vest differed depending on the location (Table 1).

2.3. Berry harvest and processing

Coffee berries were collected at the harvest peak for each plot(from April to December 2006), when the coffee was of best qual-ity. Only just-ripe cherries (when pericarp turns purple) were har-vested. For each experimental plot studied, fruits were collected on20 trees randomly selected in the experimental plot. After cross-section, fresh seeds were separated from the pericarp and loculesand immediately frozen in liquid nitrogen in the field and thenstored at �80 �C. Seeds that were subjected to wet processing wereharvested in the morning (2 kg of coffee cherries), transported tothe CIRAD processing factory (Ligne Paradis, Saint Pierre), and di-rectly processed in the afternoon by the wet method (de-pulping,fermentation and drying) to obtain approximately 500 g of greencoffee beans (11% moisture). The temperature was recorded duringthe fermentation process which was divided into three successivesteps: ‘dry’ fermentation with only mucilage (24 h), maceration byadding water to cover the seeds (15 h) and, finally, washing bychanging the water three times during this step (11 h) (Table 1).The green coffee samples were screened through a size 15 sieveand the defective beans were discarded. For chemical analysis,seed tissues were freeze-dried and ground to a fine powder usingan analytical grinder (IKA A10, Staufen, Germany). Powdered seed

Table 1Geographic and climatic data for the last 5 months preceding harvest. Altitude (m); latitude and longitude (degrees); mean of minimum, average, and maximum dailytemperatures (�C) (Tmin, Taver and Tmax, respectively); TR, temperature range (calculated as Tmax�Tmin); RAIN, rainfall (mm d�1); IR, irradiance (J cm�2 d�1); ETP,evapotranspiration potential (mm d�1); Tferm, mean temperature during wet processing (�C). Cv = coefficient of variation.

Sites Harvest Alt Long Lat Tmin Taver Tmax TR RAIN IR ETP Tferm

1 04-2006 150 55 23 38 21 16 00 21.8 25.3 28.6 6.8 3.8 1728 4.6 24.02 04-2006 270 55 40 39 21 03 16 22.5 23.9 26.4 3.9 9.0 1783 2.1 25.33 07-2006 315 55 38 39 21 20 08 16.3 19.3 24.3 8.0 4.7 1284 2.5 19.64 08-2006 463 55 21 08 20 57 36 18.4 21.2 25.2 6.8 1.5 1488 2.5 20.35 05-2006 500 55 21 08 20 57 36 18.0 22.0 26.6 8.6 2.7 1150 2.9 21.66 08-2006 549 55 31 32 21 17 38 15.8 19.5 24.1 8.2 3.8 1374 3.4 19.77 07-2006 585 55 16 48 21 05 18 14.6 18.9 23.8 9.2 3.0 1043 2.2 18.78 09-2006 661 55 36 23 21 19 36 12.6 16.2 20.9 8.4 5.1 1199 2.4 21.39 09-2006 757 55 32 52 21 17 29 13.7 17.0 21.1 7.4 4.2 1148 2.6 20.310 10-2006 790 55 24 02 21 13 17 10.8 14.4 19.2 8.4 2.7 1164 2.7 23.011 08-2006 805 55 18 12 21 07 20 14.0 17.5 22.0 7.9 3.0 1031 2.2 19.512 09-2006 880 55 30 24 21 03 16 12.3 16.4 21.6 9.3 1.4 1270 1.9 18.913 09-2006 980 55 21 03 21 00 31 9.6 13.4 17.3 7.7 2.6 1023 1.9 20.814 10-2006 985 55 18 41 21 07 09 10.3 14.3 19.1 8.8 2.9 973 2.2 22.115 09-2006 1014 55 33 39 21 16 59 10.7 14.9 20.0 9.3 4.2 1133 2.6 19.716 12-2006 1032 55 25 02 21 12 35 9.9 14.0 17.7 7.8 2.0 1349 3.3 26.4Cv (%) 28.2 20.2 15.0 16.7 51.1 19.0 26.0 10.8

T. Joët et al. / Food Chemistry 118 (2010) 693–701 695

tissue from fresh and wet-processed seeds was stored over silica-gel in a hermetic plastic box at �20 �C until analysis.

2.4. Lipid, sugar, caffeine and chlorogenic acid determination

Lipids, sugars, caffeine and chlorogenic acids were determinedas described in Joët et al. (2009). Roughly, fatty acid methyl esters(FAMEs) were prepared according to the ISO-5509 standard andmeasured by GC. Sugars were determined by High-PerformanceAnion Exchange Chromatography coupled with Pulsed Ampero-metric Detection (Dionex Chromatography Co., Sunnyvale, USA).The seed CWP content was estimated by measuring the defatedalcohol insoluble residue (DAIR). Chlorogenic acids and caffeinewere analysed by HPLC at 325 and 276 nm, respectively. All metab-olites were analysed in triplicate (from 3 different extractions)using a completely random experimental design.

2.5. Statistical analysis

Statistical significance of variations in metabolite content dueto wet processing was verified by analysis of variance (one-wayANOVA with fixed effect). Correlations between climatic variablesand metabolites levels as well as correlations among metaboliteslevels were analysed by linear regression using Pearson’s correla-tion coefficient. For both, ANNOVA and linear regression, a signifi-cance threshold of P = 0.01 was retained.

3. Results

3.1. Air temperature during seed development influences routingwithin sugar, chlorogenic acid and fatty acid metabolic pathways

CGA, caffeine, CWP, lipid and free sugar contents, as well asfatty acid composition, were measured in fresh mature seeds sam-pled in 16 experimental coffee plots. These plots showed very highclimatic variations, especially a very broad temperature gradient,which was highly correlated (R = 0.93, P = 0.0000 for Tmin and Taver,R = 0.92, P = 0.0000 for Tmax) with altitude, which ranged from 150to 1032 m (Table 1).

Relationships between metabolite contents and environmentalfactors were analysed by linear regression (Table 2). With theexception of caffeine, the overall lipid, sugar, and CGA compositionof fresh seeds (Table 3) did not differ from that previously observedin other varieties. No environmental factor significantly influenced

the accumulation of the four main classes of storage compoundsstudied. Indeed, total CWP, total lipids, total free sugars and totalCGA, did not show any significant correlation with any climaticvariable. Caffeine was not influenced by any of the environmentalfactors we measured. However, within a given chemical family(CGA, lipids or free sugars), some components were significantlyinfluenced by the environment. Out of the 28 metabolites analysed,half showed significant correlations with the mean average airtemperature during the last five months of seed development –i.e. the period when storage compounds accumulate in the seed(Joët et al., 2009). Significant correlations were also found withaltitude and with mean minimum and maximum daily tempera-tures (data not shown), but the highest correlation coefficientswere with the daily average temperature. In contrast, no significantcorrelation was found between rainfall or potential evapotranspi-ration and any of the compounds studied and only weak correla-tions were found with solar irradiance.

Within a given chemical family, when significant (P < 0.01), theslope of regression equations could differ among the compounds ofthe family concerned. For instance, 3-caffeoyl quinate (3-CQA) and4-CQA content were positively correlated with temperature whilethe reverse trend was observed for 5-CQA (Fig. 1A). Interestingly,the same phenomenon was found for di-CQA: i.e. di3.4-CQA anddi-4.5-CQA were positively influenced by temperature while a neg-ative correlation was observed for di3.5-CQA. These results suggestthat temperature may act directly on routing towards the differentisomers within the CGA metabolic pathway without affecting thefinal CGA content. A similar routing regulation was observed with-in the FA biosynthetic pathway. Indeed, the relative contents of thetwo major FA, namely linoleic and palmitic acids (35–45% each),were negatively correlated with environmental temperature, whilestearic acid (about 7%) accumulated more in warmer conditions(Fig. 1B). Similarly, oleic acid (about 7% of FA) was positively corre-lated with temperature, but the correlation coefficient was weaker(R = 0.57; P = 2.10�2, data not shown).

3.2. Wet processing affects storage compounds differently

Nine of the 28 metabolite variables analysed showed significantdifferences in fresh and wet-processed seeds, as tested by one-wayANOVA (P < 0.01; Table 3). At the level of the biochemical class,post-harvest treatment did not influence total free sugar, totalCGA or caffeine contents. In contrast, wet processing caused asignificant increase in total CWP and lipid contents, suggesting

Table 2Correlation matrix between metabolite contents in fresh and wet-processed seeds and mean environmental factors calculated during the last 5 months of fruit development.Numbers in brackets are correlations with wet-processed seeds. R = Pearson’s linear-correlation coefficients. R values in bold indicate P values < 0.01. Taver, average dailytemperatures; RAIN, rainfall; IR, irradiance; ETP, evapotranspiration potential; Tferm, mean temperature during wet processing. CGA, chlorogenic acid; CQA, caffeoyl quinic acid;FQA, feruloyl quinic acid.

Taver RAIN IR ETP Tferm

Total CWP �0.01 (�0.03) 0.18 (0.25) �0.01 (0.01) �0.11 (�0.51) (�0.19)Total soluble sugars 0.14 (�0.29) 0.31 (0.21) 0.14 (�0.20) �0.26 (�0.33) (0.41)Myo-inositol �0.49 (0.15) 0.00 (0.55) �0.20 (0.55) �0.01 (�0.05) (0.77)Glucose �0.71 (0.03) �0.41 (�0.14) �0.28 (0.03) �0.15 (�0.32) (�0.33)Fructose 0.18 (�0.03) �0.41 (�0.19) 0.12 (�0.05) 0.42 (�0.34) (�0.41)Sucrose 0.19 (�0.23) 0.36 (0.22) 0.15 (�0.16) �0.29 (�0.27) (0.43)Raffinose 0.02 (0.45) �0.08 (0.20) 0.11 (0.15) 0.16 (0.35) (0.13)Stachyose 0.59 (0.16) 0.48 (0.27) 0.63 (�0.05) 0.54 (�0.01) (�0.16)Sorbitol �0.19 (�0.89) 0.14 (�0.26) �0.09 (�0.58) �0.37 (�0.30) (0.05)Caffeine �0.06 (0.01) 0.07 (0.44) �0.28 (�0.21) �0.56 (�0.31) (0.05)Total CGA �0.31 (�0.24) 0.02 (0.10) �0.31 (�0.35) �0.53 (�0.27) (�0.13)3-CQA 0.80 (0.87) 0.53 (0.53) 0.60 (0.78) �0.03 (0.36) (0.41)4-CQA 0.71 (0.80) 0.53 (0.53) 0.53 (0.70) �0.14 (0.32) (0.38)5-CQA �0.64 (�0.59) �0.12 (�0.09) �0.49 (�0.61) �0.51 (�0.39) (�0.28)4-FQA 0.23 (0.61) 0.19 (0.61) 0.28 (0.42) �0.40 (�0.06) (0.15)5-FQA 0.10 (0.30) �0.26 (0.01) �0.17 (0.01) 0.07 (0.26) (0.27)di3.4-CQA 0.82 (0.85) 0.42 (0.42) 0.57 (0.67) 0.03 (0.35) (0.39)di3.5-CQA �0.72 (�0.73) �0.48 (�0.36) �0.73 (�0.73) �0.24 (�0.46) (�0.47)di4.5-CQA 0.76 (0.89) 0.38 (0.42) 0.60 (0.69) �0.03 (0.40) (0.41)Total lipids �0.29 (�0.34) �0.51 (�0.52) �0.24 (�0.27) 0.32 (0.42) (�0.03)Palmitic (16:0) �0.71 (�0.70) �0.44 (�0.28) �0.59 (�0.54) �0.50 (�0.54) (�0.26)Stearic (18:0) 0.89 (0.90) 0.35 (0.28) 0.64 (0.72) 0.39 (0.63) (0.25)Oleic (18:1) 0.57 (0.82) �0.13 (0.12) 0.11 (0.50) 0.21 (0.45) (�0.25)cis-Vaccenic (18:1-n7) �0.81 (�0.86) �0.18 (�0.31) �0.48 (�0.59) �0.44 (�0.54) (0.06)Linoleic (18:2) �0.70 (�0.85) �0.05 (�0.16) �0.28 (�0.56) �0.09 (�0.42) (0.07)Linolenic (18:3) 0.39 (0.49) 0.18 (�0.04) 0.14 (0.22) 0.02 (0.37) (�0.06)Arachidic (20:0) 0.90 (0.96) 0.54 (0.36) 0.72 (0.66) 0.27 (0.43) (0.19)Gondoic (20:1) �0.05 (�0.01) 0.37 (0.16) 0.02 (0.01) 0.17 (0.08) (0.16)Behenic (22:0) 0.81 (0.87) 0.49 (0.28) 0.65 (0.59) 0.22 (0.37) (0.30)Lignoceric (24:0) 0.09 (0.49) 0.40 (0.44) �0.03 (0.53) 0.13 (�0.04) (0.38)

Table 3Cell wall polysaccharide (CWP), soluble sugar, chlorogenic acid (CGA), caffeine and lipid composition of mature seeds before (fresh seeds) and after wet processing. Values areexpressed as percentages of dry mass (% DM) except for fatty acids which are expressed as relative contents (% of total fatty acids). CQA, caffeoyl quinic acid; FQA, feruloyl quinicacid. Effect of wet processing on chemical composition was tested using one-way ANOVA.

Fresh seeds Wet-processed seeds Effect F1,30 P value

Total CWP 62.479 ± 1.284 64.478 ± 1.145 ** 21.60 0.000063Total soluble sugars 8.653 ± 0.798 8.276 ± 0.536 2.464 0.126960Myo-inositol 0.142 ± 0.038 0.078 ± 0.020 ** 34.86 0.000002Glucose 0.261 ± 0.056 0.031 ± 0.033 *** 201.21 <10�6

Fructose 0.122 ± 0.051 0.031 ± 0.033 ** 35.85 0.000001Sucrose 8.081 ± 0.785 7.956 ± 0.532 0.28 0.602957Raffinose 0.013 ± 0.008 0.021 ± 0.016 3.43 0.073792Stachyose 0.015 ± 0.016 0.084 ± 0.023 *** 94.25 <10�6

Sorbitol 0.020 ± 0.015 0.074 ± 0.053 ** 15.52 0.000451Caffeine 0.736 ± 0.099 0.725 ± 0.066 0.13 0.721017Total CGA 7.359 ± 0.499 7.646 ± 0.655 1.95 0.1731913-CQA 0.310 ± 0.100 0.370 ± 0.116 2.45 0.1280564-CQA 0.527 ± 0.093 0.579 ± 0.110 2.08 0.1592105-CQA 4.962 ± 0.420 5.159 ± 0.609 1.13 0.2955794-FQA 0.056 ± 0.007 0.056 ± 0.008 0.00 0.9837875-FQA 0.414 ± 0.028 0.417 ± 0.048 0.03 0.861936di3.4-CQA 0.157 ± 0.049 0.189 ± 0.055 2.96 0.095590di3.5-CQA 0.816 ± 0.144 0.645 ± 0.144 ** 11.19 0.002219di4.5-CQA 0.117 ± 0.040 0.232 ± 0.058 *** 42.84 <10�6

Ratio CQA/diCQA 5.348 ± 0.415 5.747 ± 0.469 ** 6.46 0.016430Total lipids 16.022 ± 0.941 16.912 ± 0.707 ** 9.15 0.005063Palmitic (16:0) 35.637 ± 0.878 35.419 ± 1.027 0.42 0.523271Stearic (18:0) 7.022 ± 0.717 7.086 ± 0.980 0.04 0.834560Oleic (18:1) 7.438 ± 0.828 7.209 ± 1.027 0.48 0.493860cis-Vaccenic (18:1-n7) 0.442 ± 0.038 0.437 ± 0.043 0.15 0.703753Linoleic (18:2) 43.581 ± 1.359 44.024 ± 1.859 0.59 0.447809Linolenic (18:3) 1.898 ± 0.169 1.882 ± 0.136 0.09 0.762305Arachidic (20:0) 2.361 ± 0.353 2.372 ± 0.452 0.01 0.941386Gondoic (20:1) 0.284 ± 0.010 0.288 ± 0.011 1.25 0.273248Behenic (22:0) 0.522 ± 0.114 0.491 ± 0.111 0.60 0.443342Lignoceric (24:0) 0.209 ± 0.032 0.198 ± 0.026 1.10 0.302051

** P < 0.01.*** P < 10�6.

696 T. Joët et al. / Food Chemistry 118 (2010) 693–701

0.4

0.5

0.6

0.7

0.2

0.3

0.4

0.5

4.0

4.5

5.0

5.5

R = -0.71P = 2.20 10-3

4-C

QA

(%D

M)

Temperature (°C)

5-C

QA

(%D

M)

R = -0.64P = 7.91 10-3

3-C

QA

(%D

M)

R = -0.80P = 2.23 10-4

42

43

44

45

46

34

35

36

37

12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26 12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26

6.5

7.0

7.5

8.0

R = -0.71P = 2.10 10-3

Palm

itic

acid

(%FA

)

Temperature (°C)

Stea

ric a

cid

(%FA

)

R = 0.89P = 3.60 10-6

Lino

leic

aci

d (%

FA) R = -0.70

P = 2.52 10-3

A. CQA content B. FA content

Fig. 1. Effect of the mean average temperature during the last 5 months of fruit development on the CGA composition (A) and FA composition (B) of fresh seeds. R andP = Pearson’s linear correlation coefficient and probability of significance. Dotted lines represent the 95% confidence interval.

T. Joët et al. / Food Chemistry 118 (2010) 693–701 697

intense metabolic reconversion during this treatment. At the com-pound level within each class, free sugars (including sugar alcoholsand oligosaccharides) were the most affected by wet processingsince five out of the seven soluble sugars studied showed signifi-cant variations (Table 3). Moreover, while glucose, fructose andmyo-inositol contents decreased during the post-harvest treat-ment, stachyose and sorbitol contents increased. In contrast,although lipid content increased, FA composition of total lipidswas stable. Finally, CGA were relatively unaffected, as only twominor isomers underwent significant changes (a slight drop indi-3,5CQA and an increase in di-4,5CQA). However, the ratio be-tween CQA and di-CQA increased significantly during wetprocessing.

3.3. Correlations between compound balances are possible indicatorsof metabolic inter-conversion

To better understand the reactions that occur during wet pro-cessing, the net gain or loss of each compound (difference betweencontents in wet-processed and fresh seeds) was calculated and cor-relations between the variations in the compounds were analysedby linear regression. Among the 45 relationships studied, only fourwere highly significant (P < 0.01). The increase in CWP induced bywet processing was highly correlated with loss of sucrose (Fig. 2)suggesting a link between sucrose mobilisation during wet pro-cessing and CWP metabolism. Similarly, the extent of sorbitol

accumulation during wet processing was highly correlated withglucose and myo-inositol losses, suggesting that their variationswere tightly inter-connected in the reactions that took place dur-ing wet processing. Indeed, the only one positive correlation wasfound between glucose and myo-inositol losses, most probablylinked to the process of sorbitol accumulation (Fig. 2).

Relationships between metabolite contents and mean tempera-ture during the fermentation process were also analysed by linearregression (Table 2). Myo-inositol was the only compound whosecontent showed a significant correlation with Tferm. Similarly, whenconsidering variations in compounds (data not shown), only netgains of stachyose (R = �0.55, P = 0.027), 3-CQA (R = 0.55,P = 0.028) and di3.4-CQA (R = 0.57, P = 0.021) showed significantcorrelations with Tferm, underlining the modest role played by fer-mentation temperature on the metabolic interconversions that oc-cur during post-harvest processing.

3.4. The metabolic status of the seed at harvest plays a primordial rolein variations in soluble sugars during wet processing

Since CGA and fatty acids were almost unaffected by wetprocessing (Table 3), compounds of these two families which weresignificantly influenced by temperature in fresh seeds were, asexpected, also significantly correlated with temperature in wet-processed seeds (Table 2). In contrast, among the soluble sugars,three compounds – glucose, stachyose and sorbitol – showed a

-0.15 -0.10 -0.05

0.00

0.05

0.10

0.15

-0.35 -0.30 -0.25 -0.20 -0.15

0.00

0.05

0.10

0.15R = -0.75P = 8.38 10-4

Glucose balance (%DM)

Sor

bito

l bal

ance

(%

DM

)

Myo-inositol balance (%DM)

Sor

bito

l bal

ance

(%

DM

) R = -0.68P = 3.42 10-3

-1.0 -0.5 0.0 0.5 1.0

-1

0

1

2

3

4

5

Sucrose balance (%DM)

CW

P b

alan

ce (

%D

M)

R = -0.81P = 1.33 10-4

-0.35 -0.30 -0.25 -0.20 -0.15

-0.15

-0.10

-0.05

0.00

Glucose balance (%DM)

Myo

-inos

itolb

alan

ce (

%D

M)

R = 0.66P = 5.41 10-3

Fig. 2. Correlations between increases and decrease in free sugars caused by wet-processing. R and P = Pearson’s linear correlation coefficient and probability ofsignificance. Dotted lines represent the 95% confidence interval.

698 T. Joët et al. / Food Chemistry 118 (2010) 693–701

significant correlation with temperature in fresh seeds but not inprocessed seeds (or the reverse). For instance, the glucose contentof fresh seeds was highly influenced by field temperature duringdevelopment (Fig. 3A), but since glucose was almost completelydegraded during post-harvest treatment (Table 3), the correlationbetween the two variables was lost in processed seeds (Fig. 3B).Interestingly, the reverse situation occurred for sorbitol (Fig. 3Aand B). Since the glucose and sorbitol contents of the bean didnot depend on the fermentation temperature (Table 2), one couldlogically hypothesise that during post-harvest treatment, sorbitolsynthesis was directly influenced by glucose content (its precursor)in unprocessed seeds. This is a valuable example of the influence ofthe seed metabolic status at harvest on the interconversions thatoccur during wet processing.

4. Discussion

Through comprehensive metabolite profiling of samples from16 experimental coffee plots, we showed for the first time that nei-ther altitude, temperature, rainfall, irradiance nor evapotranspira-tion had a significant direct influence on final total CWP, CGA, lipidand caffeine contents of fresh (Laurina) coffee seeds. The onlystudy which provides comparable data is that performed in CentralAmerica by Bertrand et al. (2006). These authors found that totallipid content of the seed was highly significantly correlated withaltitude in the traditional variety Caturra but not in recent F1 hy-brids. These authors hypothesised that trees of the variety Caturrasuffered from a decreased leaf lifespan at low altitudes resulting inan impaired leaf-to-fruit ratio and therefore in a lower carbon sup-ply to developing seeds (Vaast, Angrand, Franck, Dauzat, & Genard,2005). By contrast, F1 hybrids exhibited high vigour in the wholerange of altitudes tested, and thus no excessive fruit charge. Wedid not observe any noticeable differences in the leaf-to-fruit ratiowith varying altitude in our Laurina field trials during the experi-ment. The relationships between altitude/temperature and finaltotal CWP, CGA, lipid and caffeine contents thus appear to behighly dependent on the variety, even if a common trend towards5-CQA accumulation with altitude was observed in both studies.However, it is not possible to ascertain whether the positive effectof altitude on seed lipid content observed by Bertrand et al. (2006)in Caturra was directly linked to temperature or to another cli-matic parameter. Clearly, the effects of climate on allocations tothe main pools of storage compounds in the developing coffee seedrequire further investigations using a broad panel of C. arabicavarieties.

However, our results evidenced for the first time in coffee thatduring seed development, environmental temperature had a directimpact on routing within several major metabolic pathways. With-out affecting total CGA, lipid and soluble sugar contents, tempera-ture directly influenced the composition of each of these threepools. For instance, the percentage of linoleic acid increased withdecreasing temperatures, while the percentage of its precursor,oleic acid, decreased. Temperature has previously been shown tohave a dramatic effect on the FA composition of the seed in manycold-tolerant crops such as soybean or rapeseed (Byfield, & Up-church, 2007; Deng, & Scarth, 1998), and in the model plant Arabid-opsis thaliana (Blodner, Goebel, Feussner, Gatz, & Polle, 2007). Bycontrast, to our knowledge, the influence of climatic conditionson seed FA has only very rarely been studied in cold-sensitive spe-cies such as coffee. Moreover, the response of each individual FA totemperature variations in coffee did not differ from that observedin the cold-tolerant species cited above. For instance, an increase inpolyunsaturated FA (PUFA) and a decrease in oleic acid withdecreasing temperature are mechanisms shared by all these spe-cies. It is therefore plausible that the effects of temperature onthe FA composition of the bean are governed by mechanismsshared by this wide diversity of species. Temperature-mediatedmodulation of PUFA membrane content is believed to enable cellsurvival across a broad range of growth temperatures throughthe control of membrane fluidity (Kodama, Hamada, Horiguchi,Nishimura, & Iba, 1994). The modulation of FA composition in stor-age lipids may be controlled at both transcriptional and post-tran-scriptional levels (Byfield, & Upchurch, 2007; Tang, Novitzky,Griffin, Huber, & Dewey, 2005), but also through the O2 concentra-tion of the seed and the stability of the FA biosynthetic enzyme,which both depend directly on environmental temperature (Rollet-schek et al., 2007). Interestingly, a recent study demonstrated thatin cotton, another cold-sensitive subtropical crop, the leaf linoleicacid content also increased at low temperatures, together with ahigher transcription of the gene encoding the enzyme that converts

0.00

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12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26 12 14 16 18 20 22 24 26

12 14 16 18 20 22 24 26

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0.25

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0.01

0.02

0.03

0.04

Sorb

itol c

onte

nt (%

DM

)

Temperature (°C) Temperature (°C)

A. Fresh seed B. Wet processingR = -0.71P = 1.88 10-3

R = -0.89P = 4.44 10-6

R = 0.028P = 0.92

R = -0.19P = 0.49

Glu

cose

con

tent

(%D

M)

Sorb

itol c

onte

nt (%

DM

)G

luco

se c

onte

nt (%

DM

)

Fig. 3. Relationships between the mean average environmental temperature during the last 5 months of fruit development and seed glucose and sorbitol contents before (A)and after (B) wet-processing. R and P = Pearson’s linear correlation coefficient and probability of significance. Dotted lines represent the 95% confidence interval.

T. Joët et al. / Food Chemistry 118 (2010) 693–701 699

oleic acid into linoleic acid, D12 desaturase (Kargiotidou, Deli, Gal-anopoulou, Tsaftaris, & Farmaki, 2008). Similarly, changes in phe-nolic compounds in the seed with variations in temperaturecould be a cold acclimation mechanism. For example, accumula-tion of anthocyanins and flavonoids was observed in A. thaliana un-der cold conditions (Leyva, Jarillo, Salinas, & Martinezzapater,1995). Since changes in CGA isomer composition as a function oftemperature is reported here for the first time, additional work isnecessary to better understand its physiological role.

The chemical analysis of fresh and wet-processed seeds clearlyconfirmed the significant influence of post-harvest treatments onthe final composition of green beans. The measurement of netgains and losses during wet processing illustrated the occurrenceof an important catabolism, such as that of glucose and fructose,but also revealed the neo-synthesis of other non-structural carbo-hydrates, such as sorbitol, raffinose and stachyose. More impor-tantly, we showed for the first time, that non-negligible amountsof CWP and lipids accumulated during this short period of time.Our results therefore reinforce the assumption that significantmetabolism occurs within coffee seeds during wet processing. Thiswas previously shown for free amino-acids and c-aminobutyricacid on the one hand, and for reducing sugars on the other hand,whose contents respectively significantly increased (Bytof et al.,2005) and massively decreased (Knopp et al., 2006) during post-harvest treatment. In the latter study, variations in glucose andfructose contents were in the same range as those described inthe present study. Moreover, as previously reported (Menezes,1994), we showed that the CQA/diCQA ratio, reported as a markerof bean maturity and quality, was positively affected by wet pro-cessing. Our data are therefore in agreement with most previousreports and suggest that these metabolic processes are indepen-dent of the variety. Metabolic reactions that occur during wet pro-cessing are targeted towards particular metabolites, mainly non-structural carbohydrates and CWP, while most of the other com-pounds we studied, i.e. caffeine, most CGA isomers, and FA wereunaffected by post-harvest treatment.

The major decrease in glucose and fructose contents that oc-curred during wet processing has previously been interpreted asa consequence of the glucose turnover due to anaerobic fermenta-tion, which enables ATP biosynthesis in the coffee seed under thehypoxia conditions conferred by flooding (Knopp et al., 2006).However, the overexpression of the glyoxylate cycle-specific isoci-trate lyase (ICL) gene during wet processing was also recentlyinterpreted as an early mobilisation of reserves: in other words,the early induction of germination processes (Selmar et al.,2006). This assumption was reinforced by the observation usingflow cytometry of a peak in cell division (4-C nuclei), concomitantwith an increase in the abundance of b-tubulin, a marker for celldivision or elongation (Bytof et al., 2007). One of the main featuresof germination metabolism is the mobilisation of reserves. Sincefree amino acids are typical products of storage protein hydrolysis,their increase during wet processing was interpreted as reflectingthe mobilization of reserves (Bytof et al., 2005). Similarly, duringseed germination, soluble carbohydrates are among the first com-ponents which are affected by the resumption of active metabo-lism. However, taken together, our results do not support thehypothesis of the mobilisation of reserves during wet processing.Conversely, we detected a significant accumulation of the twomajor storage compounds in coffee, namely CWP and lipids. More-over, we observed the concomitant biosynthesis of oligosaccha-rides such as raffinose and stachyose. Finally, the decrease inglucose content was highly correlated to sorbitol biosynthesis, sug-gesting the conversion of glucose to sorbitol in addition to itscatabolism through the ethanol fermentative pathway. Instead,our results argue for the induction of a stress metabolism linkedto the hypoxia and the osmotic shock caused by flooding duringwet processing. In this respect, the recent report of early inductionof ICL gene expression under hypoxic conditions in submerged riceseedlings is worth mentioning (Lu, Wu, & Han, 2005). In floodedrice seedlings, the glyoxylate cycle appeared to be unrelated tothe mobilisation of storage lipids but rather served to replenishcarbons for the Krebs cycle (Fan, Lane, & Higashi, 2003). Similarly,

700 T. Joët et al. / Food Chemistry 118 (2010) 693–701

accumulation of the b-tubulin transcript has been linked to osmo-tic priming in tomato seeds (De Castro, Zheng, Bergervoet, De Vos,& Bino, 1995) and could be triggered in coffee seeds by the hypo-osmotic conditions that occur during wet processing.

The biosynthesis of sorbitol during wet processing, which wasdirectly correlated with the decrease in the glucose and myo-inositol pools, is reported here for the first time. Sorbitol couldbe synthesised either from glucose-6P by sorbitol-6-phosphatedehydrogenase (S6PDH), as it is the case in fruit trees of the Rosa-ceae family, or directly from glucose via aldose reductase activity,as already evidenced in germinating soybean seeds (Kuo, Doehlert,& Crawford, 1990). Interestingly, an increase in the sorbitol poolwas associated with myo-inositol depletion in transgenic tobaccooverexpressing S6PDH (Sheveleva et al., 1998), suggesting thatmyo-inositol and sorbitol were both synthesised from the sameglucose-6P pool in processed coffee seeds. The myo-inositol deple-tion observed in processed seeds could be due to the decrease of itsturnover from glucose-6P in favour of sorbitol biosynthesis. How-ever, the sorbitol biosynthetic pathway in coffee seed remains tobe characterised at the molecular level as no candidate gene hasbeen found for S6PDH and aldose reductase in coffee EST dat-abases. From this point of view, it is worth mentioning that sorbitolwas also shown to transiently accumulate during coffee seeddevelopment (Joët et al., 2009). This transient rise in polyol contentwas concomitant with the peak of expression of a gene encoding asorbitol dehydrogenase (SDH), an enzyme that catalyses thereversible oxidation of sorbitol to fructose. This enzyme could bealso involved in sorbitol biosynthesis (Ohta, Moriguchi, Kanahama,Yamaki, & Kanayama, 2005). We thus suggest that the accumula-tion of sorbitol during wet processing could be related to theinduction of stress-related metabolism induced under the hypoxicand highly reductive conditions caused by flooding. Sorbitol andother polyols play important roles as osmoprotectants. Many pre-vious reports indicated that marginal accumulation of polyols in-creases stress tolerance, likely through the stabilisation ofmacromolecular structures (Tarczynski, Jensen, & Bohnert, 1993).Alternatively, the reduction of glucose to sorbitol could act as asafety redox valve, consuming NAD(P)H and supporting the storageof reducing power under the highly reductive conditions encoun-tered during flooding.

Our results also revealed a clear negative relationship betweenvariations in sucrose content during wet processing and variationsin CWP. This trade-off can be easily interpreted by considering theroles played by sucrose synthase (SUSY), a key enzyme controllingcarbon partitioning in sink tissues. SUSY catalyses the reversibleinterconversion of sucrose to UDP-glucose and fructose. A mem-brane-bound isoform of SUSY is directly involved in CWP synthesisby channelling UDP-glucose to cellulose synthase (Amor, Haigler,Johnson, Wainscott, & Delmer, 1995). Interestingly, SUSY wasshown to be regulated at both the transcriptional and post-tran-scriptional levels by oxygen depletion caused by flooding (Dejar-din, Sokolov, & Kleczkowski, 1999).

5. Conclusions

Using samples from 16 plots with considerable variations intheir climatic characteristics, the present work established forthe first time the effects of temperature on the final compositionof the coffee seed. Similarly, we demonstrated the influence ofwet processing on the chemical composition of beans and its inter-actions with the metabolic status of seeds at harvest. This studyalso highlighted the fact that most changes can be interpreted byphysiological processes decrypted in other plants with regard tothe effects of temperature during seed development and of hypoxicconditions during wet processing. One of the most important

outcomes of this study is therefore to support the developmentof approaches coupling genomics and metabolomics to improveour understanding of the influence of the environment on coffeebean ‘quality’.

However, despite the very wide range of metabolites covered, itseems unlikely that any of them explains to a large extent the bet-ter flavour and aroma of high-altitude coffees. Indeed, among com-pounds exhibiting a significant correlation with temperature, noneshowed an absolute variation high enough to be involved in theconsiderable quality differences reported elsewhere betweenlow- and high-altitude Arabica coffees. Similarly, none of the vari-ations revealed during wet processing are likely to play a majorrole in the generation of aroma and flavour. Obviously, futurechemical targets of metabolomics approaches cannot be the com-pounds that are classically reported in the coffee literature (chlor-ogenic acids, caffeine, soluble sugars, fatty acids, etc.). Instead, wesuggest focusing such studies on other compounds whose contentis non-negligible (diterpenes, amino acids, organic acids) and onvolatile molecules that could be more directly involved in beveragequality attributes.

Acknowledgments

This work was supported by the European Union, Région Réu-nion and IRD (DOCUP Réunion 2000-2006).

References

Amor, Y., Haigler, C. H., Johnson, S., Wainscott, M., & Delmer, D. P. (1995). Amembrane-associated form of sucrose synthase and its potential role insynthesis of cellulose and callose in plants. Proceedings of the NationalAcademy of Science U S A, 92(20), 9353–9357.

Avelino, J., Barboza, B., Araya, J. C., Fonseca, C., Davrieux, F., Guyot, B., et al. (2005).Effects of slope exposure, altitude and yield on coffee quality in two altitudeterroirs of Costa Rica, Orosi and Santa Maria de Dota. Journal of the Science ofFood and Agriculture, 85(11), 1869–1876.

Bertrand, B., Vaast, P., Alpizar, E., Etienne, H., Davrieux, F., & Charmetant, P. (2006).Comparison of bean biochemical composition and beverage quality of Arabicahybrids involving Sudanese-Ethiopian origins with traditional varieties atvarious elevations in Central America. Tree Physiology, 26(9), 1239–1248.

Blodner, C., Goebel, C., Feussner, I., Gatz, C., & Polle, A. (2007). Warm and coldparental reproductive environments affect seed properties, fitness, and coldresponsiveness in Arabidopsis thaliana progenies. Plant Cell and Environment,30(2), 165–175.

Byfield, G. E., & Upchurch, R. G. (2007). Effect of temperature on delta-9 stearoyl-ACP and microsomal omega-6 desaturase gene expression and fatty acidcontent in developing soybean seeds. Crop Science, 47(4), 1698–1704.

Bytof, G., Knopp, S. E., Kramer, D., Breitenstein, B., Bergervoet, J. H. W., Groot, S. P. C.,et al. (2007). Transient occurrence of seed germination processes during coffeepost-harvest treatment. Annals of Botany, 100(1), 61–66.

Bytof, G., Knopp, S. E., Schieberle, P., Teutsch, I., & Selmar, D. (2005). Influence ofprocessing on the generation of gamma-aminobutyric acid in green coffeebeans. European Food Research and Technology, 220(3–4), 245–250.

De Castro, R. D., & Marraccini, P. (2006). Cytology, biochemistry and molecularchanges during coffee fruit development. Brazilian Journal of Plant Physiology,18(1), 175–199.

De Castro, R. D., Zheng, X., Bergervoet, J., De Vos, C., & Bino, R. J. (1995). [beta]-Tubulin accumulation and DNA replication in imbibing tomato seeds. PlantPhysiology, 109(2), 499–504.

Decazy, F., Avelino, J., Guyot, B., Perriot, J. J., Pineda, C., & Cilas, C. (2003). Quality ofdifferent Honduran coffees in relation to several environments. Journal of FoodScience, 68, 2356–2361.

Dejardin, A., Sokolov, L. N., & Kleczkowski, L. A. (1999). Sugar/osmoticum levelsmodulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis. Biochemical Journal, 344(2),503–509.

Deng, X., & Scarth, R. (1998). Temperature effects on fatty acid compositiondevelopment of low-linolenic oilseed rape (Brassica napus L.). Journal of theAmerican Oil Chemists Society, 75(7), 759–766.

Fan, T. W. M., Lane, A. N., & Higashi, R. A. (2003). In vivo and in vitro metabolomicanalysis of anaerobic rice coleoptiles revealed unexpected pathways. RussianJournal of Plant Physiology, 50(6), 787–793.

Flament, I. (2002). Coffee flavour chemistry. Chichester, UK: John Wiley and Sons.Guyot, B., Gueule, D., Manez, J. C., Perriot, J. J., Giron, J., & Villain, L. (1996). Influence

de l’altitude et de l’ombrage sur la qualité des cafés Arabica. Plantations,Recherche, Développement, 3, 272–280.

T. Joët et al. / Food Chemistry 118 (2010) 693–701 701

Joët, T., Laffargue, A., Salmona, J., Doulbeau, S., Descroix, F., Bertrand, B., et al. (2009).Metabolic pathways in tropical dicotyledonous albuminous seeds: Coffeaarabica as a case study. New Phytologist, 182(1), 146–162.

Kargiotidou, A., Deli, D., Galanopoulou, D., Tsaftaris, A., & Farmaki, T. (2008). Lowtemperature and light regulate delta 12 fatty acid desaturases (FAD2) at atranscriptional level in cotton (Gossypium hirsutum). Journal of ExperimentalBotany, 59(8), 2043–2056.

Knopp, S., Bytof, G., & Selmar, D. (2006). Influence of processing on the content ofsugars in green Arabica coffee beans. European Food Research and Technology,223(2), 195–201.

Kodama, H., Hamada, T., Horiguchi, G., Nishimura, M., & Iba, K. (1994). Geneticenhancement of cold tolerance by expression of a gene for chloroplast omega-3-fatty-acid desaturase in transgenic tobacco. Plant Physiology, 105(2),601–605.

Kuo, T. M., Doehlert, D. C., & Crawford, C. G. (1990). Sugar metabolism ingerminating soybean seeds: Evidence for the sorbitol pathway in soybeanaxes. Plant Physiology, 93(4), 1514–1520.

Leroy, T., Ribeyre, F., Bertrand, B., Charmetant, P., Dufour, M., Montagnon, C., et al.(2006). Genetics of coffee quality. Brazilian Journal of Plant Physiology, 18(1),229–242.

Leyva, A., Jarillo, J. A., Salinas, J., & Martinezzapater, J. M. (1995). Low-temperatureinduces the accumulation of phenylalanine ammonia-lyase and chalconesynthase messenger-RNAs of Arabidopsis thaliana in a light-dependentmanner. Plant Physiology, 108(1), 39–46.

Lu, Y., Wu, Y. R., & Han, B. (2005). Anaerobic induction of isocitrate lyase and malatesynthase in submerged rice seedlings indicates the important metabolic role ofthe glyoxylate cycle. Acta Biochimica et Biophysica Sinica, 37(6), 406–414.

Menezes, H. C. (1994). The relationship between the state of maturity of raw coffeebeans and the isomers of caffeoylquinic acid. Food Chemistry, 50(3), 293–296.

Ohta, K., Moriguchi, R., Kanahama, K., Yamaki, S., & Kanayama, Y. (2005). Molecularevidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant.Phytochemistry, 66(24), 2822–2828.

Rolletschek, H., Borisjuk, L., Sanchez-Garcia, A., Gotor, C., Romero, L. C., Martinez-Rivas, J. M., et al. (2007). Temperature-dependent endogenous oxygenconcentration regulates microsomal oleate desaturase in developingsunflower seeds. Journal of Experimental Botany, 58(12), 3171–3181.

Selmar, D., Bytof, G., Knopp, S. E., & Breitenstein, B. (2006). Germination of coffeeseeds and its significance for coffee quality. Plant Biology (Stuttg), 8(2), 260–264.

Selmar, D., Bytof, G., & Knopp, S. (2002). New aspects of coffee processing: Therelation between seed germination and coffee quality. In Proceedings of theinternational congress of ASIC, (p. 19).

Sheveleva, E. V., Marquez, S., Chmara, W., Zegeer, A., Jensen, R. G., & Bohnert, H. J.(1998). Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco.High amounts of sorbitol lead to necrotic lesions. Plant Physiology, 117(3),831–839.

Tang, G. Q., Novitzky, W. P., Griffin, H. C., Huber, S. C., & Dewey, R. E. (2005). Oleatedesaturase enzymes of soybean: Evidence of regulation through differentialstability and phosphorylation. Plant Journal, 44(3), 433–446.

Tarczynski, M. C., Jensen, R. G., & Bohnert, H. J. (1993). Stress protection oftransgenic tobacco by production of the Osmolyte Mannitol. Science, 259(5094),508–510.

Vaast, P., Angrand, J., Franck, N., Dauzat, J., & Genard, M. (2005). Fruit load andbranch ring-barking affect carbon allocation and photosynthesis of leaf and fruitof Coffea arabica in the field. Tree Physiology, 25(6), 753–760.

Vaast, P., Bertrand, B., Perriot, J. J., Guyot, B., & Genard, M. (2006). Fruit thinning andshade improve bean characteristics and beverage quality of coffee (Coffeaarabica L.) under optimal conditions. Journal of the Science of Food andAgriculture, 86(2), 197–204.