Metabolic pathways in tropical dicotyledonous albuminous seeds: Coffea arabica as a case study

Experimental site and plant material

Experiments were performed on seeds of Coffea arabica L. cv. ‘Laurina’. Developing seeds were harvested from plants grown at Grand Tampon, Reunion Island, France (21.16°S, 55.33°E, 1015 m asl) on an Andosol soil, with an average temperature of 18.5°C and a mean annual rainfall of 1300 mm. Coffee trees were planted in 2002 without shade and were in their second (2005) production cycle. Plant spacing was 2 m between rows and 1 m within rows. For each of the seven developmental stages studied (Fig. 1; Salmonaet al., 2008), three independent biological samples (pools of c. 200 seeds) were collected on 20 trees randomly selected in the experimental plot. After cross-section, the seed was separated from the pericarp and locules and immediately frozen in liquid nitrogen and stored at −80°C before RNA extraction. For chemical analysis, seed tissues were freeze-dried and ground to a fine powder using an analytical grinder (IKA A10, Staufen, Germany). Powdered seed tissue was stored over silica-gel in a hermetic plastic box at −20°C until analysis. For each developmental stage, four to five fresh fruits were dissected in order to measure separately perisperm and endosperm masses. The dry mass was measured after 1 d of drying in an oven at 103°C.

Fig. 1

Characterization of developing coffee (Coffea arabica) seeds. Sampling was carried out at seven phenological stages from aqueous perisperm to ripe endosperm. Changes in perisperm (open circles) and endosperm (closed circles) dry mass (a), relative proportion (b) and water content (c). Error bars indicate ± SD of repetitions (n = 3). Changes in seed content (d) in lipids (closed diamonds), chlorogenic acids (open triangles), sucrose (closed squares), hexoses (open squares), and cell-wall polysaccharides (open diamonds). The secondary y axis is for cell wall polysaccharides.

Real-time RT-PCR assays

Total RNA extraction and real-time RT-PCR assays were performed as previously described (Salmonaet al., 2008). Roughly, first strand cDNA synthesis was performed using the ImProm-II reverse transcription system (Promega, Madison, WI, USA) using 1 µg of total RNA (treated with RNase-free DNase I, Promega) as a template. Polymerase chain reactions were carried out in an optical 96-well plate with an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA), using SYBR Green to monitor dsDNA synthesis. Data were analyzed using the SDS 2.1 software (Applied Biosystems) to determine cycle threshold (Ct) values. PCR efficiency (E) was estimated for each gene with LinReg software, which uses absolute fluorescence data captured during the exponential phase of amplification of each reaction using the equation (1 + E) = 10^(−1/slope) (Ramakerset al., 2003). Efficiency values were taken into account in all subsequent calculations. Normalization was processed by using the mean expression (Ct value) for all genes under study as the inter-PCR run effect was considered negligible, as tested by the use of an internal reference (ubiquitin) in each plate. Relative expression rate of target gene transcripts were then calculated (Supporting Information, Table S2) using the following formula:

where

and

The specificity of the PCR products generated for each set of primers (Table S3) was tested by agarose gel electrophoresis (Fig. S1), and sequencing of PCR products.

The Coffee EST database used in this project (SOL genomics networks; http://sgn.cornell.edu/) currently contains > 15 000 unigenes (deduced from 57 000 ESTs from C. arabica and C. canephora; Lin et al., 2005). For FA and TAG biosynthesis, we performed an exhaustive search for orthologs with TBLASTN, using sequences from the Arabidopsis Lipid Gene Database as templates (Beisson et al., 2003). In the same way, for the phenylpropanoid pathway, we used the well-characterized gene repertoire for lignification in Arabidopsis thaliana (Raes et al., 2003; Kim et al., 2004; Costa et al., 2005) to find orthologs. By contrast, metabolic pathways involved in CWP biosynthesis, especially galactomannans and pectins, have not been so comprehensively described in model plants. Only a few genes were cloned and the corresponding enzymes functionally characterized. Since it is hazardous to deduce functions from weak sequence homologies, we only retained coffee sequences highly homologous to genes functionally characterized in model plants (Edwards et al., 1999; Seitz et al., 2000; Sprenger & Keller, 2000; Hegemanet al., 2001; Reid et al., 2003; Dhuggaet al., 2004; Gu & Bar-Peled, 2004; Kanter et al., 2005; Marraccini et al., 2005; Nguema-Ona et al., 2006; Sterling et al., 2006).

Lipid, sugar and chlorogenic acid determination

Total lipids were extracted from 300 mg samples of freeze-dried powder using a modified Folch method (Folchet al., 1957) as described in Laffargue et al. (2007). Fatty acid methyl esters (FAMEs) were prepared according to the ISO-5509 standard, and GC analyses of FAMEs were performed as previously described (Laffargueet al., 2007). Sugars were extracted and measured by high-performance anion exchange chromatography coupled with pulsed amperometric detection (Dionex Chromatography Co., Sunnyvale, CA, USA) as described elsewhere (Dussertet al., 2006). The seed content in CWP was estimated by measuring the defatted alcohol insoluble residue (DAIR) using the method developed by Redgwell et al. (2003). Chlorogenic acids were extracted as described in Bertrand et al. 2003 and then analyzed using the HPLC conditions employed previously (Bertrandet al., 2008). All metabolites were analyzed in triplicate (from three different extractions) using a completely random experimental design. Estimation of CWP throughout seed development allowed us to report lipids, free sugars and CGA contents on a corrected dry mass basis (cDM = DM/(100 – DAIR content) × 100, Figs 2–6) giving a better estimate of these compounds at the intracellular level.

Fig. 2

Mapping of transcripts and metabolites associated with sucrose metabolism throughout coffee (Coffea arabica) seed development. Metabolite profiles are in gray boxes and gene expression profiles are in white boxes. Dashed lines indicate multistep pathways. AGPP, ADP-glucose pyrophosphorylase; CWIN, cell wall invertase; FK, fructokinase; HK, hexokinase; INV, invertase; PFK, phosphofructokinase, PGMc and PGMp, cytosolic and plastidial phosphoglucomutase, respectively; PGI, phosphoglucose isomerase; PK, pyruvate kinase; SPP, sucrose phosphate phosphatase; SPS, sucrose phosphate synthase; SS, starch synthase; SUSY, sucrose synthase; UGPP, UDP-glucose pyrophosphorylase.

Development of the coffee seed and accumulation of reserves

Under the field conditions used in this study, fruits of C. arabica completed their maturation in approx. 260 d. Until 75 d after flowering (DAF) (stage 2), the perisperm exhibited a significant increase in dry mass (DM) (Fig. 1a), which comprised the major part of the seed until stage 3 (Fig. 1b). Afterwards, the endosperm developed rapidly and replaced the perisperm in the locule (Fig. 1a). Endosperm DM increased considerably until 225 DAF (stage 6, ripening stage when the pericarp of the cherry fruits turns yellow), then remained fairly constant during the last month of development that leads to the mature stage when the cherry fruit with the pericarp turns red and purple (Fig. 1a). At this stage, the endosperm represented > 98% of the mature seed mass (stage 7, Fig. 1b). Unlike in orthodox seeds, coffee seed development does not end with a significant drop in water content (i.e. a desiccation stage sensu stricto) (Fig. 1c). Instead, the endosperm exhibited a progressive decrease in water content throughout its development, but its final water content did not drop below 50%. Lipids and CGA started to accumulate as soon as the endosperm began to develop (from stage 3, Fig. 1d, Table S1). However, in contrast to lipids whose pattern reflected that of endosperm DM and accumulated until stage 6 (Fig.1a–d), CGA deposition ended early (i.e. by stage 4). Hexoses were the main free sugars during stages 2–3 when the perisperm predominated. Both CWP and sucrose began to accumulate by stage 4, that is, later than lipids and CGA (Fig. 1d). However, like lipids, storage of all sugar compounds ended by stage 6.

Hexose permeation pathways and starch metabolism in the transient perisperm

One of the most noticeable findings arising from sugar profiling during coffee seed development was the very high proportion of glucose, myo-inositol and fructose at early stages, when the transient perisperm predominated (16.5, 4.7 and 1.6% DM, respectively, at stage 2, Table S1). Their content dropped abruptly as soon as the endosperm started expanding (by stage 4), whereas sucrose content remained fairly constant during the perisperm-endosperm transition (7–8% DM). Assuming that sucrose is the major carbohydrate transported in the phloem of coffee trees (Geromelet al., 2006), one can undoubtedly presume that the high concentrations of glucose and fructose observed in the perisperm are caused by enhanced sucrose catabolism, which could occur either in the apoplasm, through the action of cell wall invertase, or intracellularly via invertase and/or sucrose synthase activities. The low expression of sucrose synthase genes (sus1 and sus2) in early developmental stages suggests an important role for invertases (Fig. 2). This hypothesis is strengthened by the high levels of expression of cell wall and vacuolar invertase genes (DV703215 and EE200220, respectively) observed at early stages. In order to be effective, cell wall invertase activity has to be coupled to a plasma membrane transporter for intracellular hexose uptake. We identified three putative plasma membrane hexose transporters, whose transcript abundance varied considerably over the course of seed development. Among them, only one gene (EE199490) displayed maximal expression in early developmental stages and thus represents a good candidate for functional characterization. Once present in the cytosol, fructose and glucose are further converted into their corresponding hexose phosphates for metabolic activation. Interestingly, the pattern of expression of a fructokinase gene perfectly matched that of fructose content. To a lesser extent, the drop in glucose was in line with a progressive decline in hexokinase transcript abundance. The resulting hexose phosphates can be further metabolized through glycolysis, starch synthesis and lipid synthesis. Transcripts encoding plastid enzymes involved in starch biosynthesis, such as phosphoglucomutase, glucose-1P pyrophosphorylase and starch synthase, all accumulate in early developmental stages and then progressively decline during seed development. Our data clearly revealed active starch metabolism in the transient perisperm of the coffee seed.

Sucrose permeation pathways during the storage process

In contrast to hexoses, sucrose is a nonnegligible component of storage compounds (7.5% DM in mature seeds; Table S1) and represents a source of energy that is easily mobilized during germination. There was a significant increase in sucrose content during the mid-stages of endosperm development (between stages 4 and 6) (Figs 1d, 2). Two main mechanisms may explain this increase. On the one hand, sucrose may be directly loaded intracellularly across the plasma membrane by sucrose transporters. Interestingly, the two potential sucrose/H⁺ cotransporter genes we identified both showed maximal expression at stages 3–4 when the endosperm is developing rapidly and starts to store TAG and CGA (from stage 3 on) and CWP (from stage 4 on). This hypothesis for increased sucrose import during the storage phase was corroborated by the expression profile of the sucrose synthase gene sus1, which is most likely involved in sucrose catabolism and was closely associated with the expression profiles of sucrose transporters. On the other hand, sucrose could also be synthesized de novo via the coupled enzyme activities of sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). However, the two corresponding genes were expressed at maximal levels in the transient perisperm and not at stages when sucrose content increased. Consequently, the present profiling data strongly suggest that the mid-stage increase in sucrose content is controlled at the transcriptional level through enhanced expression of sucrose transporters.

Fine transcriptional tuning during CWP synthesis: balance between genes involved in the last polymerization steps and catabolism

Hexoses resulting from sucrose cleavage are diverted to different cellular and biosynthetic functions. Among them, biosynthesis of CWP is of particular importance in coffee, since they increased dramatically when the endosperm was undergoing rapid development (between stages 3 and 6) to reach 60% DM by stage 6 (Figs 1d, 3). Key genes involved in the final polymerization steps all exhibited a transcription peak concomitant with the onset of CWP accumulation (Fig. 3), suggesting that CWP metabolism is mostly controlled at the transcriptional level. They include a cellulose synthase (CS) subunit gene and genes encoding enzymes directly involved in 1,4-β-mannan synthesis such as mannose-1P guanyltransferase (MGT) and β-1,4 mannan synthase (MS), which can be used as a signature of hemicellulosic polysaccharide biosynthesis. They also comprise genes involved in galactose-to-mannan branching responsible for galactomannan structure (a backbone of β-1,4 mannose residues with varying degrees of α-1,6 galactose substitutions), such as galactomannan galactosyltransferase (GMGT) and α-galactosidase. The transcriptional control of CWP biosynthesis in the developing coffee endosperm is also revealed by the shutdown of genes dedicated to CWP hydrolysis, such as genes encoding β-mannosidase, cellulase and, to a lesser extent, β-galactosidase, during the CWP accumulation stage.

Fig. 3

Carbohydrate metabolism in the developing coffee (Coffea arabica) seed. A schematic diagram of the interactions involved in the interconversion of carbon from fructose and glucose into either sorbitol, phytic acid, galactose, raffinose, stachyose and cell wall polymers such as cellulose, pectin and mannan. Metabolite profiles are in gray boxes and gene expression profiles are in white boxes. Dashed lines indicate multistep pathways. Direct building blocks for CWP (cell wall polysaccharides) are circled. CS, cellulose synthase; GalAT, galacturonosyl transferase; α-Gal, alpha-galactosidase; GAS, galactinol synthase; GK, galactinol kinase; GMGT, galactomannan galactosyltransferase; GUT, Glc uridyltransferase; MGT, mannose-1P guanyltransferase; MIOX, myo-inositol oxygenase; MIP, myo-inositol 1-phosphate; MIPP, myo-inositol 1-phosphate phosphatase; MIPS, myo-inositol 1-phosphate synthase; MS, beta 1,4-mannan synthase; RS, raffinose synthase; SDH, sorbitol dehydrogenase; SS, stachyose synthase; UDP-Gal, UDP-galactose; UDP-GalA, UDP-galacturonic acid; UG4E, UDP-glucose 4′-epimerase; UDP-Glc, UDP-glucose; UDP-GlcA, UDP-glucuronic acid; UGA4E, UDP-glucuronic acid 4′-epimerase; UGDH, UDP-Glc dehydrogenase. #, no candidate gene found in Coffea EST databases.

Supplying CWP building blocks: evidence for transient storage forms of galactose and for a functional myo-inositol oxidation pathway

Among potential precursors of CWP, myo-inositol – a major precursor of phytic acid, glucuronic acid, and galactinol – caught our attention since it was present in substantial amounts in the transient perisperm (Fig. 3). Its decline during seed development slightly preceded the drop in myo-inositol phosphate synthase (MIPS) gene transcription that encodes the key enzyme of myo-inositol biosynthesis. By contrast, the drop in myo-inositol was closely associated with the transient expression peak of phytase, encoding an enzyme directly using myo-inositol as the substrate for phytic acid synthesis. High phytase activity during this period could explain the delay between myo-inositol drop and repression of its biosynthetic genes. However, myo-inositol could also be oxidized to glucuronic acid via the myo-inositol oxygenation pathway. The myo-inositol oxygenase (MIOX) gene, which encodes a key enzyme of this pathway, showed a strong peak of transcription by stages 4–5, corresponding perfectly to cell wall thickening during endosperm development. Alternatively, UDP-glucuronic acid may be supplied through UDP-glucose dehydrogenase (UGDH) activity (Fig. 3). In the developing coffee seed, our results suggest temporal sharing of tasks by the two pathways. Indeed, in contrast to the MIOXgene, which showed maximal expression at mid-stages, UGDHand the genes for subsequent enzymes leading to pectin synthesis (i.e. UDP-glucuronic acid 4′-epimerase and galacturonosyltransferase (GalAT)) were only expressed at early stages, suggesting they play an important role in the intense cell division activity occurring in the perisperm tissue.

Besides, myo-inositol may also be diverted towards raffinose family oligosaccharides (RFO) since, together with galactose, myo-inositol is a component of galactinol. RFO, mostly raffinose and stachyose, accumulated only transiently in the developing endosperm of coffee, reaching 0.2 and 0.8% cDM, respectively, at stages 4–5 (Table S1, Fig. 3). This transient accumulation of RFO was closely associated with several genes linked to galactose metabolism that peaked at the same stages, such as genes encoding UDP-glucose 4′-epimerase, galactokinase, α-galactosidase and a protein similar to a fungal galactose oxidase. As galactose is relatively toxic in a free form (free galactose was not detected), we hypothesize that it is transiently stored in other forms (RFO) before its incorporation in galactomannans.

The transient perisperm and the developing endosperm employ different transcriptional programs towards ER lipid biosynthesis reactions

One of the immediate findings arising from our investigations is that two paralog genes exhibiting very distinctive expression patterns were identified in most steps that occur in the ER (Figs 4, 5). Conversely, only one gene was found to be involved in the reactions that take place in the plastid. In ER reactions showing two candidate genes, one gene was assigned to lipid synthesis in the perisperm, since its transcript profile showed maximal expression at stage 1, followed by a progressive decline throughout seed development (e.g. DV697030 for DAGAT (Fig. 4)), while the other gene exhibited maximal expression at mid-stages and was consequently assigned to lipid synthesis in the endosperm (e.g. DV711950 for DAGAT (Fig. 4)). The ER-located reactions for which a perisperm form and an endosperm form could be distinguished include the enzymes involved in the sequential acylation on the sn-1, 2 and 3 positions of the glycerol backbone (i.e. GPAT, LPAAT, PAP and DAGAT), the desaturation at the Δ₁₂ position of the acyl chain, and the synthesis of long-chain fatty acids (Figs 4, 5). In contrast to reactions occurring in the ER, we identified only one candidate gene for each plastidial enzyme studied and their transcription profiles all exhibited high expression at stage 1 and stages 3–4 (Figs 4, 5), confirming the fact that the same gene was expressed in the perisperm and the endosperm at each step in the formation of the acyl chain. Another notable mode of tissue-specific expression concerned Δ₁₅ desaturase for which no paralog gene specifically expressed in the endosperm could be found (Fig. 5). Accordingly, an abrupt drop in the percentage of linolenic acid percentage occurred by stages 3–4 (Fig. 5, Table S1).

Fig. 5

Acyl CoA, diacylglycerol and phosphatidylcholine pool synthesis in the developing coffee (Coffea arabica) seed. Metabolite profiles are in gray boxes and gene expression profiles are in white boxes. The peak of transcriptional activity for fatty acid synthesis genes (stage 3) is marked by a dotted line. CPT, diacylglycerol cholinephosphotransferase; FAT, acyl-ACP thioesterase; GPAT, glycerol-phosphate acyltransferase; KAS, ketoacyl-ACP synthase; KCS, beta ketoacyl-CoA synthase; LACS, long-chain acyl-CoA synthetase; LPAAT, acylglycerol-phosphate acyltransferase; PAP, phosphatidate phosphatase; PC, phosphatidylcholine; SAD, stearoyl-ACP desaturase.

Fig. 4

Fatty acid and triacylglycerol synthesis in the developing coffee (Coffea arabica) seed. Metabolite profiles are in grAy boxes and gene expression profiles are in white boxes. The peak of transcriptional activity for fatty acid synthesis activity genes (stage 3) is marked by a dotted line. AC, acetyl-CoA carboxylase; DAG, diacylglycerol; DAGAT, diacylglycerol acyltransferase; EAR, enoyl-ACP reductase; HAD, hydroxyacyl-ACP dehydrase; KAR, ketoacyl-ACP reductase; KAS, ketoacyl-ACP synthase; LPC, lysophosphatidylcholine; LPCAT, lysophosphatidylcholine acyltransferase; MAT, malonyl-CoA ACP transacylase; PC, phosphatidylcholine; PDAT, phospholipid diacylglycerol acyltransferase; PDH, pyruvate dehydrogenase. #, no candidate gene found in Coffea expressed sequence tag (EST) databases.

PC-DAG interconversion as the major route for TAG enrichment in linoleic acid

In the classical Kennedy pathway of TAG formation, the third acylation is catalyzed by DAGAT using an acyl-CoA from the ER pool (Fig. 4). Since acyl-CoAs resulting from FA synthesis are saturated or monounsaturated, high 18:2 percentages in TAG, such as in the mature coffee endosperm, imply the involvement of a complementary pathway. Diacylglycerols (DAG) formed through the Kennedy pathway may be converted to PC through CPT activity (Fig. 5). Oleate esterified to the resulting PC could then be desaturated to 18:2 by the Δ₁₂ desaturase. The first possible route for the incorporation of 18:2 in TAG consists in the transfer of linoleoyl from PC to DAG by the PDAT enzyme to form lyso-PC and TAG. A PDAT gene (DV707770), highly homologous to the Arabidopsis PDAT gene At5g13640, was highly expressed at stage 1, and was then progressively shut down during coffee seed development (Fig. 5). This route could therefore contribute significantly to the enrichment of TAG in 18:2 in the perisperm but not in the endosperm. A second route involves the reversibility of the reaction catalyzed by CPT. Linoleate-containing PC can therefore be converted into DAG, which, in turn, enrich TAG in PUFA through the Kennedy pathway (Figs 4, 5). A significant increase in CPT transcription was observed at mid-stages, concomitantly with the other genes involved in TAG storage (Figs 4, 5). Therefore, PC-DAG interconversion appears to be the major route for TAG enrichment in linoleic acid in the developing coffee endosperm.

Acyl chain and DAG synthesis precedes formation of TAG and oil bodies in the developing endosperm

Even if lipid storage appeared to be a continuous process during the first 120 d of endosperm development (from stage 3 to stage 6, Fig. 1d), detailed analysis of the several pathways that contribute to TAG accumulation revealed two tightly coordinated sequential stages of transcription (Figs 4, 5). Plastidial genes involved in formation of acetyl-CoA, sequential addition of 2-carbon units, desaturation at the Δ₉ position and, finally, FA release in the form of acyl-CoA all showed high levels of transcription at stage 3. Similarly, ER genes contributing to acylation on positions sn-1 and sn-2 of the glycerol (EL610524, DV681807, DV703295) were highly expressed from the onset of endosperm development. By contrast, genes for enzymes governing desaturation of 18:1–18:2 (Δ₁₂ desaturase, EE196983), PC-DAG interconversion (CPT), elongation of very long acyl chains (KCS, FD528594) and acylation on sn-3 position of glycerol (DAGAT, DV711950), exhibited maximal gene transcription at stages 4–5, concomitantly with peak synthesis of the oil body proteins (oleosin, caleosin and steroleosin). There was therefore a striking delay of c. 30 d between the burst of gene expression related to the specific last steps of TAG assembly and storage and that related to FA and DAG biosynthesis.

The singular fatty acid composition of the coffee endosperm could rely on high FATb transcription during lipid synthesis

The endosperm of the mature coffee seed exhibits a rather unusual seed fatty acid composition since it is rich in both palmitic (16:0) and linoleic (18:2) acids (i.e. c. 40%, Table S1). 16:0-ACP represents the first major branch point in FA biosynthesis since it is the substrate for two major activities, KASII, which initiates the biosynthesis of C18 FA, and FATb, which in turn triggers the entry of 16:0 in the ER acyl-CoA pool and, consequently, its incorporation in TAG (Fig. 5). High palmitate and linoleate content implies that neither FATb nor any of the genes encoding enzymes acting from 16:0-ACP to linoleoyl-PC are down-regulated during endosperm development. We observed that FATb as well as KASII, SAD, FaTA and Δ₁₂ desaturase-encoding genes exhibited a pronounced and coordinated increase in transcription during the stage with the highest lipid synthesis rate (Figs 1d, 4). Consequently, one may hypothesize that this coordinated transcription of genes involved in the two branches starting from 16:0-ACP results in a balanced proportion of their respective end-products.

Chlorogenic acids accumulate massively at the beginning of endosperm development only

Chlorogenic acids already represented 5% DM of the seed at stage 2, suggesting that these compounds are synthesized early in the perisperm tissue and are then loaded, probably via the apoplast, in the developing endosperm. Moreover, for the first time, we showed that CGA accumulate within a short period, at the beginning of endosperm development (between stages 3 and 4) (Fig. 6). This increase corresponded to a fourfold rise and total CGA reached almost 20% of DM at stage 4 (7% of DM in the mature seed) and was mostly explained by an abrupt rise in 5-caffeoyl quinate (5-CQA), which represented c. 82% of total CGA at stage 4 (69% in mature seeds) (Table S1). A rapid overview of expression profiles of phenylpropanoid-related genes suggests that the tight coordination of gene expression observed in the biosynthesis of CWP and lipids is not a characteristic shared by CGA metabolism. However, a more in-depth analysis of this complex pathway network enabled identification of various potential transcriptional control points for CGA synthesis in coffee. First, genes encoding PAL1, PAL2, C4H, two 4CL (AM117807 and EH662404) and three CCoAOMT (EL645414, DV682513 and DV703981) showed high expression levels at stages 3 or 4, when the endosperm was undergoing rapid development (Fig. 6). Expression of these genes therefore coincided with maximal CGA biosynthetic activity (Figs 1d, 6), suggesting they play a key role in their biosynthesis. Other genes encoding putative upstream enzymes such as 4CL (DV689729) and C3H (DQ269126) could also be related to CGA biosynthesis activity, as their expression at stage 3 was close to their maximal expression. Similarly, the bimodal expression pattern of HCT1 suggests a dual role for this enzyme that could be directly involved in caffeoyl quinate anabolism. However, another key phenylpropanoid gene, HQT, did not show an expression profile that matches the CGA accumulation pattern, suggesting that this transcript is restricted to the perisperm and not directly involved in CGA accumulation in the endosperm.

Fig. 6

Changing patterns of gene expression (white boxes) associated with chlorogenic acids and with monolignols biosynthetic pathway in the developing coffee (Coffea arabica) seed. Caffeoyl quinic and feruloyl quinic acids (gray boxes) are intermediate metabolites leading to G and S monolignols. Dashed lines indicate multistep pathways or uncharacterized enzymes. C3H, p-coumarate 3-hydroxylase; C4H, trans-cinnamate 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; CHS, chalcone synthase; CHI, chalcone isomerase; 4CL, 4-coumarate:CoA ligase; CCR, cinnamoyl-CoA reductase; COMT, caffeic acid O-methyltransferase; CQAs, caffeoyl quinic acids; F5H, ferulate 5-hydroxylase; FQAs, feruloyl quinic acids; HCT, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase; HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase; PAL, phenylalanine ammonia lyase; POD, secretory peroxidase. *, p-coumaroyl quinate or p-coumaroyl shikimate.

The phenylpropanoid transcriptional program switches from CGA to lignin biosynthesis during endosperm development

Chlorogenic acid biosynthetic activity ceased very early during endosperm development and was even followed by a drop in CGA relative content at stage 5 (Table S1 and Fig. 6). This drop was the result of the dilution of ACG in the growing endosperm as the absolute CGA content expressed on a seed basis remained stable (Fig. 1d). However, a slight decrease in absolute CGA content was still noticeable at stage 6 (Fig. 1d) and could be explained by the metabolic re-routing of these precursors towards lignin biosynthesis (Fig. 6). Searches were therefore conducted to identify genes potentially involved in CGA remobilization at late stages. Three CCR genes (DV689388, EH662414 and DV703229), two F5H genes (DV689190 and EH662405), CCoAOMT1, HCT1 and one CAD gene (EE198746) exhibited maximal transcription levels at late stages. These genes encode enzymes that act downstream of caffeoyl quinate, supporting the hypothesis that the CGA pool serves as an intermediate in lignin biosynthesis during endosperm development. The high expression level of HCT1 during late stages suggests the involvement of this enzyme in caffeoyl quinate catabolism. Moreover, a cationic peroxidase (POD) gene, encoding an enzyme which is directly involved in monolignol polymerization, displayed high induction and maximal expression at stage 5 during the process of endosperm hardening. Besides, one can hypothesize that genes corresponding to DV689190, DV703229 and EE198746 encode the F5H, CCR and CAD isoforms that are directly involved in the last steps of the monolignol biosynthesis (peak of expression at stage 5). It is also worth noting that none of the genes encoding enzymes involved in the first three steps of the phenylpropanoid pathway, namely PAL, C4H and 4CL, displayed an expression profile that matched the time window of the lignification process associated with endosperm hardening. This reinforces the hypothesis that the phenylpropanoid pathway is dissociated into two independent transcriptional events during seed development: first, CGA biosynthesis during the perisperm-endosperm transition; and second, CGA remobilization for monolignol biosynthesis during endosperm hardening.