Three different cDNAs coding for putative plant plastid sigma 70 -type transcription initiation factors have recently been cloned and sequenced from Arabidopsis thaliana. We have analyzed the evolutionary conservation of function(s) of the N-terminal and C-terminal halves of these three sigma factors by in vitro transcription studies using heterologous transcription systems and by complementation assays using Escherichia coli thermosensitive rpoD mutants. Our results indicate differences and similarities of the three plant factors and their prokaryotic ancestors. The functions of the N-terminal parts of the plant sigma factors are considerably different from the function of the N-terminal part of the principal sigma 70 factor of E. coli. On the other hand, the C-terminal parts have kept at least two characteristics when compared with their prokaryotic ancestors: 1) they can distinguish between different promoter structures, and 2) one of them is capable of fully complementing E. coli rpoD mutants, i.e. recognizing all essential E. coli promoters that are used by the E. coli principal sigma 70 factor. This shows for the first time in vivo a strong evolutionary conservation of cis-and trans-acting elements between the prokaryotic and the plant plastid transcriptional machinery.

Caffeine is a metabolite of great economic importance, especially in coffee, where it influences the sensorial and physiological impacts of the beverage. Caffeine metabolism in the Coffea species begins with the degradation of purine nucleotides through three specific N-methyltransferases: XMT, MXMT and DXMT. A comparative analysis was performed to clarify the molecular reasons behind differences in caffeine accumulation in two Coffea species, namely Coffea arabica and Coffea canephora var. robusta. Three different genes encoding N-methyltransferase were amplified in the doubled haploid Coffea canephora: CcXMT1, CcMXMT1 and CcDXMT. Six genes were amplified in the haploid Coffea arabica: CaXMT1, CaXMT2, CaMXMT1, CaMXMT2, CaDXMT1, and CaDXMT2. A complete phylogenic analysis was performed to identify specific key amino acids defining enzymatic function for each protein identified. Furthermore, a quantitative gene-expression analysis was conducted on leaves and on maturing coffee beans, simultaneously analyzing caffeine content. In the different varieties analyzed, caffeine accumulation is higher in leaves than in the coffee bean maturation period, higher in Robusta than in Arabica. In Robusta, CcXMT1 and CcDXMT gene expressions are predominant and transcriptional activity is higher in leaves than in maturing beans, and is highly correlated to caffeine accumulation. In Arabica, the CaXMT1 expression level is high in leaves and CaDXMT2 as well to a lesser extent, while global transcriptional activity is weak during bean maturation, suggesting that the transcriptional control of caffeine-related genes differs within different organs and between Arabica and Robusta. These findings indicate that caffeine accumulation in Coffea species has been modulated by a combination of differential transcriptional regulation and genome evolution.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2170-7) contains supplementary material, which is available to authorized users.

Summary Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development – the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re‐synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered.