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Coffee is a very important crop with more than seven million tons of green beans produced every year. Moreover, after oil, coffee is ranked second on international trade exchange markets. The two main species are cultivated throughout the tropics; these are Coffea arabica and Coffea canephora, representing 70 and 30% of world coffee production, respectively. In terms of cup quality, C. arabica (Arabica) is more appreciated by consumers because it less bitter and has a better flavour compared with C. canephora (Robusta). Cup quality is a vast subject, the determinants of which are not well understood.
Sucrose is one of the major sources of the free reducing sugars participating in the Maillard reaction that occurs during the roasting of the coffee grains. This reaction generates a significant number of products, including caramel, sweet and burnt-type aromas, and dark colors, which are typically associated with coffee flavor (Russwurm, 1969; Holscher & Steinhart, 1995). Although glucose and fructose are present in the mature grain, together they only represent approx. 0.03% of the dry weight of C. arabica grains. Sucrose is by far the most important free nonreducing sugar present in mature coffee grains (Rogers W. John et al., 1999). Furthermore, the lowest-quality Arabica grains have appreciably more sucrose (7.3–11.4%) than the highest-quality Robusta grains (4–5%; Russwurm, 1969; Chahan et al., 2002). While sucrose is significantly degraded during roasting, significant amounts (0.4–2.8% dry weight; DW) still remain in the roasted grain. Hence, the sucrose content of the roasted grain adds to the sweetness of the coffee and improves cup quality. Since sucrose plays a key role in determining coffee cup quality, it is important to develop a better understanding of the factors that control sucrose metabolism and accumulation in the different coffee varieties.
In higher plants, sucrose is the principal form in which carbon is translocated between photosynthetic (source) and nonphotosynthetic (sink) cells (Ziegler, 1975). Sucrose metabolism has been extensively studied in tomato (Solanum lycopersicum), a close relative of coffee. They are both members of the asterid I class and share a large number of common genes, especially during fruit development. The key reactions regulating sucrose homeostasis in tomato fruit are as follows: (i) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy) and cytoplasmic invertase (neutral); (ii) sucrose synthesis by either SuSy or sucrose-phosphate synthase (SPS) together with sucrose phosphatase (SP); (3) sucrose hydrolysis in the vacuole and/or in the apoplast by acid vacuolar and/or cell wall-bound invertase; and (iv) the rapid synthesis and breakdown of starch in the amyloplast (Nguyen-Quoc & Foyer, 2001; Rausch & Greiner, 2004; Roitsch & Gonzalez, 2004). As in other sink organs, the pattern of sucrose unloading is not constant during tomato fruit development. In the early stages of fruit development, sucrose is unloaded directly into cells from the phloem by a symplastic pathway involving direct plasmodesmatal connections between cells, and hence sucrose is not degraded to its composite hexoses during unloading at this stage. The SuSy and acid invertase activities of the sink cells are highest at this stage and are directly correlated with sucrose unloading capacity from the phloem, contributing directly to the phenomenon called sink strength (Klann et al., 1993, 1996; Godt & Roitsch, 1997; Nguyen-Quoc & Foyer, 2001). However, the symplastic connections are severed as the fruit develops and aploplastic pathways then become the predominant means of sucrose unloading and uptake. Sucrose is rapidly hydrolyzed by the cell wall-bound acid invertases during apoplastic unloading. Therefater, the glucose and fructose products are imported into the cells by hexose transporters. Sucrose is subsequently synthesized de novo in the cytoplasm by SuSy or SPS (Miron & Schaffer, 1991; Dali et al., 1992). SPS catalyzes an essentially irreversible reaction in vivo as a result of its close association with the enzyme sucrose phosphate phosphatase (SP; Echeverria et al., 1997). Fruit SuSy activity declines in parallel with the loss of the symplastic connections and it eventually falls below detectable levels at the onset of ripening (Robinson et al., 1988; Wang et al., 1993). Thereafter, SPS and SP become the predominant enzymes of sucrose synthesis, particularly at the late stages of tomato fruit development.
Research over the past two decades has confirmed the crucial roles of the sucrose-metabolizing enzymes in organ growth and development. For example, studies on mutants with reduced SuSy activity in maize (Chourey & Nelson, 1976; Chourey et al., 1998), pea (Craig, 1996), potato (Zrenner et al., 1995) and tomato (D’Aoust et al., 1999) have demonstrated unequivocally that SuSy activity exerts a major control over tissue import capacity and it directly influences the partitioning of carbohydrates, particularly with regard to starch accumulation. The vacuolar and cell wall-bound invertases are also key proteins in the regulation of sucrose metabolism during fruit development. Red-fruited tomato species, such as the commercial Solanum lycopersicum and the wild species S. pimpinellifolium, do not store sucrose but rather accumulate hexoses (glucose and fructose). Evidence from crosses of red-fruited species with sucrose-accumulating green-fruited species (Yelle et al., 1991) has shown the crucial role of acid invertase in preventing sucrose accumulation in red-fruited tomato species. Subsequently, genetic analysis located the locus conferring high amounts of soluble solids in S. pimpinellifolium fruit. This was coincident with the known position of vacuolar invertase TIV1 (Grandillo & Tanksley, 1996; Tanksley et al., 1996). Studies involving antisense TIV1 cDNA expression in transgenic tomatoes confirmed that vacuolar invertase was responsible for high amounts of fruit-soluble solids (Klann et al., 1993, 1996). Thus, vacuolar invertase is considered to play a major role in both the regulation of tissue hexose concentrations and in the mobilization of sucrose stored from the vacuoles of mature fruit (Klann et al., 1993; Yau & Simon, 2003). The constitutive expression of cell wall-bound invertases in transgenic tomato (Dickinson et al., 1991) and tobacco (von Schaewen et al., 1990) plants led to decreased sucrose transport capacities between sink and source tissues and caused stunted growth as well as more general alterations in plant morphology. Similarly, the absence of cell wall invertase has been shown to cause dramatic effects on plant and seed development in several species. For example, transgenic carrots with reduced amounts of cell wall invertase (CWI; Tang et al., 1999) showed poor tap root formation, particularly during the early elongation phase. Similarly, mutations in the maize miniature-1 (mn1) locus, which encodes CWI-2, cause aberrant pedicel formation and a drastic reduction in endosperm size (Miller & Chourey, 1992; Cheng et al., 1996). Interestingly, global acid invertase activities are dramatically reduced in the mn1 mutant, suggesting coordinate control of both the vacuolar and cell wall-bound isoforms.
Invertases are subject to a complex network of transcriptional and posttranscriptional controls that are regulated by developmental, environmental and carbohydrate signals (Sturm, 1999). In addition, plants produce small (< 20 kDa) inhibitory proteins, called invertase inhibitors, that contribute to the posttranscriptional control of invertase activity. Sequences encoding invertase inhibitors have been characterized in tobacco NtINVINH1 (Greiner et al., 1998) and maize ZM-INVINH1 (Bate et al., 2004). The specificities of these invertase inhibitors was demonstrated in vitro by using recombinant proteins (Bate et al., 2004) and in vivo via studies on transgenic plants (Greiner et al., 1999).
In order to improve coffee bean quality by increasing the final sucrose content of ‘green’ coffee, it is necessary to have a better understanding of the factors that control sucrose metabolism and accumulation during coffee grain maturation. Over the last few years, as genomic studies in Coffea have progressed, partial and full-length sequences encoding proteins involved in sucrose metabolism have begun to appear in public databases and in the literature. For example, sequences encoding SuSy (SUS1, SUS2), vacuolar (CaVAC1, partial sequence) and cell wall invertase (CaCWI1, partial sequence) have been progressively characterized and mapped (Leroy et al., 2005). More recently, data from pulse-chase and feeding experiments have revealed that the grain perisperm and endosperm tissues have the capacity to synthesize and accumulate sucrose (Geromel et al., 2006). Moreover, these authors suggested that sucrose synthesis and accumulation resulted from high SuSy activities late in coffee grain development.
The objective of the current work was to identify potential targets (genes and enzymatic activities) that could explain the differences in sucrose accumulation in the ‘green’ coffee grain of the two major commercially exploited species, C. canephora (Robusta) and C. arabica (Arabica). The most appropriate model for sucrose metabolism during fruit development has been established in the tomato pericarp (Nguyen-Quoc & Foyer, 2001). Based on this scheme, our aim was to compare sugar contents and sucrose-metabolizing enzyme activities during development, together with the identification and analysis of the expression patterns of the genes encoding the different isoforms of the sucrose-metabolizing enzymes. The comparative analysis of sucrose metabolism was undertaken during the development of Arabica and Robusta grains that have high and low sucrose accumulation capacities, respectively, during coffee grain maturation. In this way we were able to identify genes and associated enzyme activities that can account for the differences in sucrose accumulation that are generally observed between Arabica and Robusta grains.
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- Materials and Methods
- Supporting Information
There is a strong correlation between the sucrose concentration of mature coffee grains and cup quality (Russwurm, 1969; Holscher & Steinhart, 1995). Strategies designed to increase the sucrose content of coffee grains either by marker-assisted selection, classical breeding or more direct genetic manipulation methods require a precise understanding of how sucrose metabolism is regulated during coffee grain development. The present study was designed to identify genes and associated enzyme activities that are important in determining sucrose metabolism and accumulation in Arabica and Robusta grains. Using the C. canephora (Robusta) and the C. arabica (Arabica) genomes, we first set out to isolate the full set of genes that are central to grain sucrose metabolism. The first group of sequences that we characterized encode sucrose-cleaving enzymes, including two cDNAs, CcSS1 and CcSS2. The second group is the invertase family. The three cDNAs isolated in this study, CcInv2, CcInv3 and CcInv4, encode the vacuolar, cytoplasmic and cell wall isoforms, respectively. Finally, we have isolated cDNAs encoding sucrose synthesis enzymes and characterized two different SPS sequences: CcSPS1 and CcSPS2 as well as a coffee SP cDNA, CcSP1. The identification of this new set of genes in the C. canephora or C. arabica genomes is important as it allows a more precise analysis of the gene expression patterns that underpin sucrose accumulation during grain maturation.
Sucrose homeostasis and net accumulation in sink organs involve the interaction of several ‘futile’ cycles incorporating import, degradation and re-synthesis (Nguyen-Quoc & Foyer, 2001) and encompassing reactions in the cytosol, the vacuole and the apoplast. In order to understand the relative importance of these processes in the developing coffee grain, we measured the gene expression patterns and corresponding activities of sucrose-metabolizing enzymes during the maturation of two high sucrose Arabica genotypes CCCA12 and CCCA02 and two low sucrose Robusta genotypes, FRT05 or FRT64. The Arabica genotypes accumulate approx. 30% more sucrose in the mature grain than the Robusta varieties (Table 1). The results obtained here allow us to draw the following conclusions:
• Sink strength is higher in Robusta than Arabica grains in early development. Developmental changes in SuSy activity were different in Robusta and Arabica grains. While SuSy activity was not significantly different during Arabica grain development, it showed a marked increase between the SG and LG stages in both Robusta varieties, decreasing at the yellow stage, but rising again late in development (Fig. 2a). Hence, high SuSy activity is associated with the growth phase of coffee fruit development and, more specifically, with endosperm development (De Castro & Marraccini, 2006; Geromel et al., 2006). Since SuSy activity is correlated with sink strength during the early stages of development (Sun et al., 1992; Wang et al., 1993; Zrenner et al., 1995; N'tchobo et al., 1999), our data suggest that Robusta has a greater sink strength than Arabica during grain expansion. Of the two SuSy genes characterized here, CcSS2 is highly expressed early in grain development and then decreases in all four genotypes, the decrease being most pronounced in the Robusta genotypes (Fig. 3a). By contrast, CcSS1 mRNA is present at very low levels at all stages. These results suggest that CcSS2 encodes the predominant isoform implicated in coffee endosperm development. The abundance of CcSS2 transcripts and pattern of change during development are similar in Arabica and Robusta. Variations in CcSS2 transcripts cannot therefore account alone for the significant differences in SuSy activities observed between the two species. However, SuSy activity is known to be regulated by multiple parameters, particularly substrate availability (Nguyen-Quoc & Foyer, 2001) and post-translational modification through covalent modification (Anguenot et al., 2006). The results presented here differ from those published by Geromel et al. (2006) in one aspect only; we did not observe a massive increase in SuSy activity late in Arabica grain development. This difference is explained by differences in the last maturation stage used in both studies. The last coffee cherry (RG stage) that was studied here may not be as mature as the one used by Geromel et al. (2006). Furthermore, the site location for the growth of the coffee plants was different (Brazil versus Ecuador) in the two studies. Environmental factors are important determinants of fruit maturation, and growth environment may have had an impact on sucrose metabolism.
• The expression of invertase inhibitors exerts control over acid invertase activity. Sink strength is also driven by the activity of cellular invertases, especially the vacuolar isoforms (Nguyen-Quoc & Foyer, 2001). Soluble acid invertase activity was much higher in Robusta (especially the FRT05) than in the Arabica genotype, particularly in the early stages of grain development (Fig. 2c). In the Arabica genotype CCCA12, acid invertase activity was low and relatively constant at all of the four grain developmental stages analyzed. These results show that sink strength (determined by acid invertase in association with SuSy) is higher in Robusta than in Arabica during the early stages of grain development, which rely on the symplastic sucrose import. The data presented here show that sucrose degradation is considerably higher in the Robusta than in the Arabica genotypes. The vacuolar invertase is encoded by a single gene, CcInv2, in coffee (D. Crouzillat, unpublished). Our data show that CcInv2 is a high-abundance transcript during early grain development in CCCA12 and in both Robusta varieties (Fig. 3b). Hence, differences in observed acid invertase activities between Arabica and Robusta genotypes cannot be attributed to differences in CcInv2 gene expression. Thus, we explored the possibility of post-translational controls of vacuolar invertase activity in coffee. Invertase activity has been shown to be inhibited by interaction with small proteins (< 20 kDa) (Greiner et al., 1999; Bate et al., 2004; Godt & Roitsch, 2006). Based on homologies, we isolated and characterized four cDNA-encoding potential invertase inhibitors (CcInv1–4 from the coffee databases; Table 2). CcInvI2 and CcInvI3 mRNA were more abundant in Arabica than in Robusta genotypes, with expression highly restricted to the earliest stages of grain maturation, CcInvI2 transcripts attaining the highest accumulation. The appearance of the CcInvI2 and CcInvI3 mRNAs correlates closely with the absence of detectable acid invertase activities at the earliest stages of grain maturation in CCCA12. By contrast, CcInvI2 and CcInvI3 transcripts were not detected in either Robusta genotypes at the same stages, when acid invertase activities were high and CcInv2 mRNA was most abundant. This result is interesting because it demonstrates a direct connection between the expression of specific invertase inhibitors and the inhibition invertase activity. To our knowledge, this is the first time that it has been possible to show such a relationship in the invertase inhibitor family. The cellular localization of the different invertase inhibitors could not be determined using available bioinformatic tools (TargetP, iPSORT). Further experiments are underway to establish the function and specificity of each potential invertase inhibitor, especially CcInvI2 and CcInvI3.
Neutral invertase activities, which are considered to reflect the cytoplasmic isoform, were high early in grain development and decreased in a similar manner in Arabica and Robusta grains during maturation (Fig. 2d). The changes in enzyme activities occur in parallel with changes in the abundance of CcInv3 mRNA (Fig. 3b). Neutral and vacuolar invertase activities were similar in both Arabica genotypes. However, the vacuolar isoform activity was predominant in both Robusta genotypes. The abundance of CcInv3 transcripts was much lower than that of CcInv2 transcripts (Fig. 3b), suggesting that CcInv3 gene-product was more abundant than the CcInv2 gene-product. However, we cannot rule out the possibility that there is a second gene encoding cytoplasmic invertase, as has been shown in other species.
The method used here (King et al., 1997) is effective for the extraction and assay of soluble acid and neutral invertases. The extraction of cell wall-bound invertases is much more difficult and requires very high salt concentrations (Husain et al., 2001). We therefore only assessed the expression of cell wall invertases CcInv1 and CcInv4 (Fig. 3b). CcInv1 transcripts were barely detectable at any development stage. CcInv4 transcripts were more abundant than CcInv1 transcripts early in development, and abundance was higher in Arabica than in Robusta. Studies on the expression of tomato LIN family sequences and orthologous genes in A. thaliana (Godt & Roitsch, 1997; Fridman & Zamir, 2003) have revealed that each gene is specifically expressed in vegetative and reproductive tissues with hardly any overlap. For example, LIN5 transcripts are exclusively expressed in the conductive tissues of the ovary, in the fruit placenta and in the pericarp during the early stages of cell division (Fridman et al., 2004). It may be that CcInv1 mRNA and, to a lesser extent, CcInv4 mRNA are of too low abundance to be detected in the grain studied here. It is probable that more sophisticated techniques such as in situ hybridization will be required to detect the tissue-specific expression patterns of the different cell wall invertases (Fridman et al., 2004).
Interestingly, the high sucrose-cleaving enzyme activities (SuSy, acid and neutral invertase) observed in early grain development did not result in a marked accumulation of hexoses in either Robusta genotype. Conversely, high glucose contents were detected in both Arabica genotypes at the SG stage, even though SuSy, acid and neutral invertase activities were lower than those measured in Robusta. These results suggest strongly the existence of ‘futile cycles’ of sucrose degradation and re-synthesis (Nguyen-Quoc & Foyer, 2001) in coffee grain development, as has been suggested previously (Nguyen-Quoc & Foyer, 2001; Geromel et al., 2006). Furthermore, it is possible that sucrose, glucose and fructose are translocated between different tissues in the coffee cherries (perisperm and pericarp). This would explain the observed differences between sugar contents and enzyme activities in the endosperm during early grain development.
• Higher CcSPS1 and CcSPS2 transcripts and SPS activities at the end of development account for the final sucrose content of the mature Arabica grains. High SPS activities are accompanied by sucrose accumulation during the maturation of Arabica and Robusta grains. Overall SPS activities were generally higher in Robusta than in Arabica at the earliest stages of grain development (Fig. 2b). However, both genotypes showed similar fluctuations in activity during grain development (Fig. 2b). SPS activity increased between the SG and LG stages and decreased drastically between the LG and Y stages. However, Arabica grains had significantly higher SPS activities than Robusta grains, particularly at the last developmental stages. This is associated with the higher final sucrose content of the mature Arabica grains. The changes in SPS activity at the end of grain maturation were much less marked in Robusta. CcSPS1 and CcSPS2 transcripts accumulated to the same abundance in both Robusta and Arabica grains at the early stages of grain development (Fig. 5). However, while the abundance of CcSPS1 and CcSPS2 mRNAs was constant in the Robusta genotypes, both CcSPS1 and CcSPS2 transcripts increased during the final stages of Arabica grain development, with CcSPS1 showing the greatest accumulation. CcSPS1 transcripts were predominant in Arabica and their abundance is consistent with the increase in SPS activity observed during grain development in the CCCA12 genotype.
A major aim of the coffee research program is to identify and map quantitative trait loci (QTL) involved in sucrose accumulation in the coffee grain, as well as those related to size and fruit development. It is probable that genomic locations of such QTLs will coincide with the candidate genes characterized in this study, or their related regulatory proteins. The results presented here demonstrate that C. canephora grains accumulate less sucrose than C. arabica for two reasons: higher SuSy and invertase activities early in grain development that prevent sucrose accumulation in Robusta; and lower SPS activities in Robusta than in Arabica later in grain development. While sucrose accumulation during coffee grain maturation is a complex process involving multiple genes and enzymatic activities, as shown in other species such as tomato (Fridman et al., 2004), maize (Thevenot et al., 2005), and sugarcane (Aitken et al., 2006), the data presented here have pinpointed targets for increasing the final sucrose content of ‘green’ grains and hence improving Robusta coffee quality.