The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

Sucrose metabolism and the role of sucrose synthase were investigated in the fruit tissues (pericarp, perisperm, and endosperm) of Coffea arabica during development. Acid invertase, sucrose phosphate synthase, and sucrose synthase activities were monitored and compared with the levels of sucrose and reducing sugars. Among these enzymes, sucrose synthase showed the highest activities during the last stage of endosperm and pericarp development and this activity paralleled closely the accumulation of sucrose in these tissues at this stage. Carbon partitioning in fruits was studied by pulse-chase experiments with (14)C-sugars and revealed high rates of sucrose turnover in perisperm and endosperm tissues. Additional feeding experiments with (14)CO(2) showed that leaf photosynthesis contributed more to seed development than the pericarp in terms of photosynthate supply to the endosperm. Sugar analysis, feeding experiments, and histological studies indicated that the perisperm plays an important role in this downloading process. It was observed that the perisperm presents a transient accumulation of starch which is degraded as the seed develops. Two full-length cDNAs (CaSUS1 and CaSUS2) and the complete gene sequence of the latter were also isolated. They encode sucrose synthase isoforms that are phylogenetically distinct, indicating their involvement in different physiological functions during cherry development. Contrasting expression patterns were observed for CaSUS1 and CaSUS2 in perisperm, endosperm, and pericarp tissues: CaSUS1 mRNAs accumulated mainly during the early development of perisperm and endosperm, as well as during pericarp growing phases, whereas those of CaSUS2 paralleled sucrose synthase activity in the last weeks of pericarp and endosperm development. Taken together, these results indicate that sucrose synthase plays an important role in sugar metabolism during sucrose accumulation in the coffee fruit.

Coffee fruits grown in shade are characterized by larger bean size than those grown under full-sun conditions. The present study assessed the effects of shade on bean characteristics and sugar metabolism by analyzing tissue development, sugar contents, activities of sucrose metabolizing enzymes and expression of sucrose synthase-encoding genes in fruits of coffee (Coffea arabica L.) plants submitted to full-sun (FS) and shade (SH) conditions. Evolution of tissue fresh weights measured in fruits collected regularly from flowering to maturation indicated that this increase is due to greater development of the perisperm tissue in the shade. The effects of light regime on sucrose and reducing sugar (glucose and fructose) contents were studied in fresh and dry coffee beans. Shade led to a significant reduction in sucrose content and to an increase in reducing sugars. In pericarp and perisperm tissues, higher activities of sucrose synthase (EC 2.4.1.13) and sucrose-phosphate synthase (SPS: EC 2.4.1.14) were detected at maturation in the shade compared with full sun. These two enzymes also had higher peaks of activities in developing endosperm under shade than in full sun. It was also noted that shade modified the expression of SUS-encoding genes in coffee beans; CaSUS2 gene transcripts levels were higher in SH than in FS. As no sucrose increase accompanied these changes, this suggests that sucrose metabolism was redirected to other metabolic pathways that need to be identified.