In order to produce tradeable standard green coffee, processed beans must be dried. The drying procedure affects the abundance of relevant aroma substances, e.g. carbohydrates. Using molecular tools, the corresponding metabolic basis is analyzed. A decrease in water potential of the still living coffee seeds induces massive drought stress responses. As a marker for these stress reactions, accumulation of a general stress metabolite, GABA ( γ -aminobutyric acid), and associated gene expression of drought stress-associated dehydrins were monitored. The results of this study indicate that metabolism in drying coffee beans is quite complex since several events trigger accumulation of GABA. The fi rst peak of GABA accumulation during drying is correlated with expression of isocitrate lyase and thus with ongoing germination processes in coffee seeds. Two subsequent peaks of GABA accumulation correspond to maxima of dehydrin gene expression and are thought to be induced directly by drought stress in the embryo and endosperm tissue, respectively. Apart from the signifi cance for understanding basic seed physiology, metabolic changes in coffee seeds during processing provide valuable information for understanding the role and effect of the steps of green coffee processing on the quality of the resulting coffee. Keywords: γ -Aminobutyric acid (GABA) • Coffea arabica • Dehydrins • Drought stress • Isocitrate lyase . Abbreviations : ACN acetonitrile ICL , isocitrate lyase DMSO , dimethylsulfoxide GABA , γ -aminobutyric acid OPA , o -phthaldialdehyde RT–PCR , reverse transcription–PCR THF , tetrahydrofuran. Introduction Traditionally, green coffee is produced by either wet or dry pro- cessing of coffee cherries. Processing is required to remove the fruit fl esh (exo- and mesocarp) and the parchment (endocarp) as well as to dry the coffee beans (seeds) morphological details are given in Fig. 1 . In wet processing, coffee cherries are mechan- ically depulped—a process in which the major part of the fruit fl esh is removed by squeezing. Considerable parts of the fruit fl esh remain sticking on the parchment. These mucilaginous residues are degraded by fermentation subsequently the resulting parchment coffee is dried. Before shipping, the dry parchment and the testa (seed coat, Fig. 1 ) are removed by hulling. In contrast, in dry processing, the entire coffee fruits are dried before all seed surrounding layers are removed in one husking step. The chemical composition of green coffee beans and the corresponding quality of the beverage produced by these two processes differ signifi cantly ( Bytof et al. 2005 , Knopp et al. 2006 ). Recently, it has been shown that the variation in the drying procedure in the course of wet processing strongly affects the abundance of various sugars, representing impor- tant aroma precursors ( Kleinwächter and Selmar 2010 ). Although sun drying is considered to yield the best coffee quali- ties, in this study the direct infl uence of sunlight could be excluded. Kleinwächter and Selmar (2010) noted that the main difference between continuous machine drying and diurnal sun drying is the lack of pauses during the night. Differences in relevant aroma compounds in differentially processed coffees can be attributed to variations in the extent and time courses of metabolic processes, especially those related to germination that occur within the seeds during processing ( Selmar et al. 2006 , Bytof et al. 2007 ). Additionally, drought stress-induced reactions also appear to be important. The occurrence of corresponding stress-induced processes was deduced from the fi nding that the typical stress metabolite γ -aminobutyric acid (GABA) is accumulated in drying coffee beans ( Bytof et al. 2005 ). This non-protein amino acid is thought to be accumulated in response to several stress conditions ( Satya Narayan and Nair 1990 , Bown and Shelp 1997 ). It is obvious that the strong decrease in water potential during the drying of coffee beans corresponds to classical drought stress. Based on these observations, GABA accumulation should rep- resent a characteristic drought stress response ( Bytof et al. 2005 ), but because GABA is also accumulated in a number of plants in the course of normal germination ( Inatomi and Slaughter 1971 , Kuo et al. 2004 ), the question arose as to whether the GABA accumulation that occurs in the course of Stress Metabolism in Green Coffee Beans ( Coffea arabica L.): Expression of Dehydrins and Accumulation of GABA during Drying Daniela Kramer , Björn Breitenstein , Maik Kleinwächter and Dirk Selmar ∗ Institut für Pfl anzenbiologie, Technische Universität Braunschweig, Mendelssohnstr. 4, D-38106 Braunschweig, Germany ∗ Corresponding author: E-mail, d.selmar@tu-bs.de Fax, + 49-531-391-8180 . (Received January 8, 2010 Accepted February 16, 2010) Plant Cell Physiol. 51(4): 546–553 (2010) doi:10.1093/pcp/pcq019, available online at www.pcp.oxfordjournals.org © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org 546 Plant Cell Physiol. 51(4): 546–553 (2010) doi:10.1093/pcp/pcq019 © The Author 2010. Regular Paper at Nara Institute of Science and Technology on May 16, 2010 http://pcp.oxfordjournals.org Downloaded from

green coffee processing is a stress response or due to metabolic steps involved in the ongoing germination of the coffee beans. In this context, dehydrins provide additional markers for drought stress. Apart from their abundance in late embryogen- esis, an increase in gene expression of dehydrins is strongly cor- related with several types of stress conditions ( Close 1997 , Allagulova et al. 2003 , Bouché and Fromm 2004 ). Due to their great hydrophilicity and thermostability, it is assumed that dehydrins are structure stabilizers with detergent- and chaperone-like properties ( Borowski et al. 2002 ). Dehydrins are often associated with drought stress and dehydration ( Nylander et al. 2001 , Caruso et al. 2002 , Rodriguez et al. 2005 , Samarah et al. 2006 ). In this work, we considered gene expres- sion of dehydrins as a suitable tool to monitor metabolic responses to putative drought stress. Additionally, gene expres- sion of isocitrate lyase (ICL) was analyzed to describe germina- tion processes. The alignment of these various metabolic events should contribute to the general understanding of the complex metabolism of coffee seeds during processing, thereby estab- lishing the basis for deliberately modulating coffee quality by modifying drying conditions. Results and Discussion When fresh coffee seeds were dried continuously at 30 ° C and the residual water content declined to less than 43 % a signifi cant increase in the expression of dehydrins occurred ( Fig. 2 , left). Notably, when the drying was interrupted by pauses of 8 h, the enhancement of dehydrin mRNA was detectable earlier, although the drying process was delayed. A similar situation was also found for GABA accumulation ( Fig. 2 , left section). If we assume that drying of the seeds induces drought stress, which generally leads to typical metabolic stress responses such as GABA accumulation and dehydrin synthesis, these fi ndings seem contradictory. Dehydration is much faster in seeds that are dried continuously compared with seeds resulting from an interrupted drying pattern. Consequently, drought stress- related responses should occur earlier in continuously dried seeds than in those dried with 8 h pauses. However, when seeds are dried at 40 ° C, gene expression of dehydrins, as well as the accumulation of GABA, appear nearly simultaneously in both drying regimes ( Fig. 2 , right section). Surprisingly, the pattern of GABA accumulation is bimodal. The occurrence of two maxima suggests that two distinct events are responsible for stress induction. The metabolic steps responsible for stress induction must be more complex than previously expected. In order to get a clearer picture of these processes, an enhanced sampling frequency was required. Accordingly, drying under laboratory conditions was per- formed. The resulting data confi rmed the complex pattern of GABA accumulation and also revealed that the gene expression of dehydrins possesses two maxima ( Fig. 3 ). Although we earlier assumed that GABA accumulation is primarily related to drought stress, the results of more diligent seeds surrounded by the parchment (endocarp) seed surrounded by testa parchment cut embryo 5 mm endosperm endosperm fruit flesh (exo- and mesocarp) (a) (c) (b) Fig. 1 Seed and fruit morphology of Coffea arabica. (a) Sectional image of a mature coffee fruit. Generally, in each coffee cherry, two seeds that are surrounded by the endocarpal parchment are embedded in the fruit fl esh. (b) Sectional image of parchment coffee. After removing the entire fruit fl esh by depulping and fermentation, the coffee seeds are still covered by the endocarp. When this corneous layer is removed, the testa surrounding the seeds becomes visible. During wet processing, both layers—the parchment and the seed coat—are removed by hulling. (c) External appearance of coffee seeds. On the left side, an endocarpal, exterior view of a coffee seed is shown. The hazy silhouette of the embryo can be recognized vaguely in the opaque endosperm. On the right side, a corresponding cross-section is displayed. 547 Stress responses in drying coffee beans Plant Cell Physiol. 51(4): 546–553 (2010) doi:10.1093/pcp/pcq019 © The Author 2010. at Nara Institute of Science and Technology on May 16, 2010 http://pcp.oxfordjournals.org Downloaded from