Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis

* 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.     

Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.     

Genome evolution in diploid and tetraploid Coffea species as revealed by comparative analysis of orthologous genome segments

Sequence comparison of orthologous regions enables estimation of the divergence between genomes, analysis of their evolution and detection of particular features of the genomes, such as sequence rearrangements and transposable elements. Despite the economic importance of Coffea species, little genomic information is currently available. Coffea is a relatively young genus that includes more than one hundred diploid species and a single tetraploid species. Three Coffea orthologous regions of 470-900 kb were analyzed and compared: both subgenomes of allotetraploid Coffea arabica (contributed by the diploid species Coffea eugenioides and Coffea canephora) and the genome of diploid C. canephora. Sequence divergence was calculated on global alignments or on coding and non-coding sequences separately. A search for transposable elements detected 43 retrotransposons and 198 transposons in the sequences analyzed. Comparative insertion analysis made it possible to locate 165 TE insertions in the phylogenetic tree of the three genomes/subgenomes. In the tetraploid C. arabica, a homoeologous non-reciprocal transposition (HNRT) was detected and characterized: a 50 kb region of the C. eugenioides derived subgenome replaced the C. canephora derived counterpart. Comparative sequence analysis on three Coffea genomes/subgenomes revealed almost perfect gene synteny, low sequence divergence and a high number of shared transposable elements. Compared to the results of similar analysis in other genera (Aegilops/Triticum and Oryza), Coffea genomes/subgenomes appeared to be dramatically less diverged, which is consistent with the relatively recent radiation of the Coffea genus. Based on nucleotide substitution frequency, the HNRT was dated at 10,000-50,000 years BP, which is also the most recent estimation of the origin of C. arabica.     

Coffee seeds contain 11S storage proteins

A legumin-like seed protein was purified from the endosperm of coffee ( Coffea arabica L. cv. Colombia). In contrast to legumes, where efficient storage globulin extraction requires buffered saline solutions well above the acidic pK_I of the globulins, coffee legumin is readily extracted with acidic aqueous buffers. The coffee legumin migrates like other 11S storage globulins in sucrose gradients. Subunits of coffee legumin have an apparent molecular mass of about 55 kDa after one-dimensional SDS-polyacrylamide gel electrophoresis in the absence of a reducing agent. In the presence of 2-mercaptoethanol, two polypeptides appear that have apparent molecular masses of 33 and 24 kDa. Two full-length cDNAs were generated from mRNA of developing seeds that were more than 98% homologous. They had open reading frames of 1 458 and 1 467 bp. Each encoded legumin precursors of 486 and 489 amino acids, respectively (M_r=54 136 and 54 818). Examination of a 5' promoter region from a coffee legumin gene revealed a putative legumin-box. Genomic DNA from C . arabica was digested with six different restriction endonucleases. After separation of the fragments by electrophoresis, single discrete fragments on DNA blots hybridized strongly to a cDNA probe for the acidic chain. Other fragments that hybridized weakly with this probe were visible after hybridization at very low stringency. DNA from other species and commercially important cultivars that comprise the genus Coffea produced similar results. Immunocytochemical studies revealed that some legumin was detected in the cytoplasm in mature coffee seeds, but that the majority of it was in large storage vacuoles that accounted for most of the cell volume.     

Characterization and expression of a novel member (JBURE-II) of the urease gene family from jackbean [Canavalia ensiformis (L.) DC

Canavalia ensiformis (jackbean) seeds contain the proteins urease and canatoxin, a variant form of the jackbean urease. Here we have cloned a cDNA encoding another isoform of urease, called JBURE-II. This cDNA was obtained by RT-PCR using as template total RNA extracted from C. ensiformis tissues. Nucleotide sequence analysis showed that JBURE-II clones share 86% similarity with known jackbean urease. The presence in C. ensiformis of a family of urease-related genes with at least three members was demonstrated by Southern blot analysis. In order to understand the pattern of expression of the JBURE-II gene, we collected tissue samples from different stages of flower and embryo development. The results of RT-PCR show that JBURE-II is expressed from flower buds throughout seed maturation. Semi-quantitative RT-PCR indicates that expression of urease and JBURE-II genes is induced in seedlings and in leaves treated with abscisic acid, a phytohormone involved in seed maturation and wound response. This work constitutes the first report on the presence of a family of urease genes in jackbean, and provides characterization of a cDNA encoding a new member of this gene family.     

Homeologous gene expression in response to growing temperature in a recent Allopolyploid (Coffea arabica L.)

Allopolyploidy is considered as a major factor contributing to speciation, diversification, and plant ecological adaptation. In particular, the expression of duplicate genes (homeologs) can be altered leading to functional plasticity and to phenotypic novelty. This study investigated the influence of growing temperatures on homeologous gene expression in Coffea arabica L., a recent allopolyploid involving 2 closely related diploid parental species. The relative expression of homeologs of 13 genes all located in the same genomic region was analyzed using an SNP ratio quantification method based on dideoxy-terminated sequences of cDNA amplicons. The relative expression of homeologous genes varied depending on the gene, the organ, and the growing condition. Nevertheless, expression of both homeologs was always detected (i.e., no silencing). Although the growing conditions were suitable for one or other of the parental species, neither subgenome appeared preferentially expressed. Furthermore, relative homeologous expression showed moderate variations across organs and conditions and appeared uncorrelated between adjacent genes. These results indicate the absence of signs of subfunctionalization suggesting C. arabica has not undergone noticeable diploidization. Furthermore, these results suggest that the expression of homeologous genes in C. arabica is regulated by a shared trans-regulation mechanism acting similarly on the 2 subgenomes and that the observed biases in the relative homeolog expression may result from cis fine-scale factors.