Nathalie Chabrillange

Functional characterization of two p-coumaroyl ester 3′-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis

Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3′-hydroxylases (C3′H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3′H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3′-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step

SummaryThe standard cryopreservation process previously developed for oil palm clones using shiny white, finger-like somatic embryos could be applied in some cases to standard cultures. Its efficiency was markedly improved by completing the 7-day pregrowth period on 0.75 M sucrose by an additional dehydration period carried out either by placing the embryos in the air current of the laminar flow cabinet or in an air tight box containing silica gel. This improved process was successfully applied to 7 different clones. It will facilitate the routine uof cryopreservation for oil palm cultures.

Quantitative estimation of seed desiccation sensitivity using a quantal response model: application to nine species of the genus Coffea L.

Seed desiccation sensitivity was studied in nine species of the genus Coffea by measuring seed viability after equilibration over various saturated salt solutions. A quantal response model based on the logistic distribution was developed in order to describe the typical S-shaped patterns observed. The closeness of fit of the desiccation sensitivity model was shown, and the assumption that seed desiccation sensitivity follows a continuous distribution within species was verified. For each species, the water content at which 50% of initial viability was reached, WC 50 , and a specific parameter describing the intra-specific variability, β, were calculated using a non-linear regression. A simplified water sorption model was developed which allowed easy calculation of water activity and water potential corresponding to WC 50 ( a w50 and Ψ 50 ) for relative humidities ranging between 10 and 100%. Distribution of WC 50 and Ψ 50 (or a w50 ) in the genus Coffea was homogeneous within the following intervals: from 0.05 to 0.38 g H 2 O.g −1 dw for WC 50 and from −168 to −11 MPa for Ψ 50 . Different classifications of the coffee species studied as regards to their desiccation sensitivity were obtained depending on whether WC 50 or Ψ 50 was used for classification. The continuum for desiccation sensitivity observed within the nine species studied confirmed that coffee is an appropriate material for studying desiccation sensitivity.

Cryopreservation of seeds of four coffee species ( Coffea arabica, C. costatifructa, C. racemosa and C. sessiliflora ): importance of water content and cooling rate

In the range of water contents studied (0.1–0.4 g H 2 O g dw −1 ), Coffea arabica seeds were less sensitive to desiccation than C. costatifructa, C. racemosa and C. sessiliflora seeds. At 0.20 g H 2 O g dw −1 , 53% of C. arabica seeds germinated after direct immersion in LN (rapid cooling, 200°C min −1 ), but none of them developed into normal seedlings. By contrast, in C. costatifructa, C. racemosa and C. sessiliflora , when seeds were dehydrated to the optimal water content (0.19, 0.28 and 0.31 g H 2 O g dw −1 , respectively), the percentages of seeds which developed into normal seedlings after LN exposure were 26, 78 and 31% of the desiccation control, respectively. Normal seedlings could be recovered from cryopreserved C. arabica seeds only if they were desiccated to 0.20 g H 2 O g dw −1 and precooled slowly to −50°C prior to immersion in LN. Precooling seeds at 2°C min −1 allowed 25% of seeds to develop into normal seedlings. The thawing rate had no effect on the survival of cryopreserved C. arabica seeds. In all cryopreservation experiments, the total germination did not reflect the percentage of seeds which developed into normal seedlings. Examination of excised embryos indicated a partial explanation of this difference since only the shoot apex was destroyed in abnormal embryos, whereas the hypocotyl and radicle were normal.

Tolerance of coffee (Coffea spp.) seeds to ultra-low temperature exposure in relation to calorimetric properties of tissue water, lipid composition, and cooling procedure

The effect of exposure to ultra‐low temperature (liquid nitrogen, LN) on viability of seeds desiccated to various water contents was investigated in 9 coffee species. Three groups of species could be distinguished based on seed survival after LN exposure. In group 1 species, no seedling production could be obtained after LN exposure due to endosperm injury. In group 2 species, recovery was very low or nil after rapid cooling, and only moderate after slow cooling. In group 3 species, very high percentages of seedling development were observed after both rapid and slow cooling. A high interspecific variability for the high moisture freezing limit was observed within the species of groups 2 and 3, since it ranged from 0.14 to 0.26 g H2O g−1 dry weight. A very highly significant correlation was found for those species between the unfreezable water content, as determined from DSC analysis, and the high moisture freezing limit of their seeds. No significant correlation was found between seed lipid content, which varied from 9.8 to 34.6% dry weight, and survival after LN exposure. However, a negative relationship was found between seed unfreezable water content and lipid content. Interspecific differences in fatty acid composition of seed lipids resulted in a high variability in the percentage of unsaturated fatty acids, which ranged from 28.7 to 54.4% among the 9 species studied. For all species studied, a highly significant correlation was found between the percentage of unsaturated fatty acids and the percentage of seedling recovery after rapid or slow cooling.

Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora Pierre (Rubiaceae).

Phenylalanine ammonia lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway producing phenolics, widespread constituents of plant foods and beverages, including chlorogenic acids, polyphenols found at remarkably high levels in the coffee bean and long recognized as powerful antioxidants. To date, whereas PAL is generally encoded by a small gene family, only one gene has been characterized in Coffea canephora (CcPAL1), an economically important species of cultivated coffee. In this study, a molecular- and bioinformatic-based search for CcPAL1 paralogues resulted successfully in identifying two additional genes, CcPAL2 and CcPAL3, presenting similar genomic structures and encoding proteins with close sequences. Genetic mapping helped position each gene in three different coffee linkage groups, CcPAL2 in particular, located in a coffee genome linkage group (F) which is syntenic to a region of Tomato Chromosome 9 containing a PAL gene. These results, combined with a phylogenetic study, strongly suggest that CcPAL2 may be the ancestral gene of C. canephora. A quantitative gene expression analysis was also conducted in coffee tissues, showing that all genes are transcriptionally active, but they present distinct expression levels and patterns. We discovered that CcPAL2 transcripts appeared predominantly in flower, fruit pericarp and vegetative/lignifying tissues like roots and branches, whereas CcPAL1 and CcPAL3 were highly expressed in immature fruit. This is the first comprehensive study dedicated to PAL gene family characterization in coffee, allowing us to advance functional studies which are indispensable to learning to decipher what role this family plays in channeling the metabolism of coffee phenylpropanoids.

Variability in storage response within a coffee ( Coffea spp.) core collection under slow growth conditions

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.

Effect of various sugars and polyols on the tolerance to desiccation and freezing of oil palm polyembryonic cultures.

Au cours dun traitement de 7 jours en vue du conditionnement damas dembryons somatiques de palmier a huile (Elaeis guineenis) dans un milieu contenant 0,75 M de saccharose, les concentrations en fructose et glucose sont constantes tandis que les concentrations en saccharose et en amidon ont ete multipliees respectivement par 10 et 20. Apres conditionnement sur des milieux completes a laide de differents sucres et polyols a des osmolarites proches, la recuperation des amas embryonnaires est satisfaisante sauf pour le ribose. A la suite dune nouvelle periode de dessication, la survie etait variable selon les composes. Lorsque les embryons etaient congeles sans dessication prealable, une survie na ete notee quavec un traitement a laide du saccharose. A linverse, lorsque la congelation a ete effectuee apres la deshydratation, la survie pouvait etre observee avec differents sucres et polyols. Le taux de recouvrement de la proliferation embryonnaire etait maximum pour les amas traites avec le saccharose

Desiccation tolerance in relation to soluble sugar contents in seeds of ten coffee ( Coffea L.) species

Large differences in seed desiccation sensitivity have been observed previously among ten coffee species ( Coffea arabica, C. brevipes, C. canephora, C. eugenioides, C. humilis, C. liberica, C. pocsii, C. pseudo-zanguebariae, C. sessiliflora and C.stenophylla ). Of these species, C. liberica and C. humilis were the most sensitive to desiccation and C. pseudozanguebariae the most tolerant. A study was carried out using the same seed lots to investigate if these differences in desiccation tolerance could be correlated with differences in soluble sugar content. Soluble sugars were extracted from dry seeds and analysed using high performance liquid chromatography. The seed monosaccharide (glucose and fructose) content was very low (1.5 to 2 mg g -1 dry weight [dw]) in all species studied. The sucrose content ranged from 33 mg g -1 dw in C. liberica seeds to 89 mg g -1 dw in seeds of C. pocsii . Raffinose was detected in the seeds of only five species ( C.arabica, C.brevipes, C.humilis, C.sessiliflora, C.stenophylla ), among which only three species ( C.arabica, C.sessiliflora and C.brevipes ) also contained stachyose. Both raffinose and stachyose were present in very low quantities (0.3–1.4 mg g -1 dw and 0.1–0.7 mg g -1 dw, respectively). Verbascose was never detected. No significant relationship was found between seed desiccation sensitivity and: (i) the sugar content; (ii) the presence/absence of oligosaccharides; and (iii) the oligosaccharide:sucrose ratio.