Laurence Mondolot

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.

Ant‐plants and fungi: a new threeway symbiosis

Symbioses between plants and fungi, fungi and ants, and ants and plants all play important roles in ecosystems. Symbioses involving all three partners appear to be rare. Here, we describe a novel tripartite symbiosis in which ants and a fungus inhabit domatia of an ant-plant, and present evidence that such interactions are widespread. We investigated 139 individuals of the African ant-plant Leonardoxa africana for occurrence of fungus. Behaviour of mutualist ants toward the fungus within domatia was observed using a video camera fitted with an endoscope. Fungi were identified by sequencing a fragment of their ribosomal DNA. Fungi were always present in domatia occupied by mutualist ants but never in domatia occupied by opportunistic or parasitic ants. Ants appear to favour the propagation, removal and maintenance of the fungus. Similar fungi were associated with other ant-plants in Cameroon. All belong to the ascomycete order Chaetothyriales; those from L. africana formed a monophyletic clade. These new plant-ant-fungus associations seem to be specific, as demonstrated within Leonardoxa and as suggested by fungal phyletic identities. Such tripartite associations are widespread in African ant-plants but have long been overlooked. Taking fungal partners into account will greatly enhance our understanding of symbiotic ant-plant mutualisms.

Plant-ants use symbiotic fungi as a food source: new insight into the nutritional ecology of ant–plant interactions

Usually studied as pairwise interactions, mutualisms often involve networks of interacting species. Numerous tropical arboreal ants are specialist inhabitants of myrmecophytes (plants bearing domatia, i.e. hollow structures specialized to host ants) and are thought to rely almost exclusively on resources derived from the host plant. Recent studies, following up on century-old reports, have shown that fungi of the ascomycete order Chaetothyriales live in symbiosis with plant-ants within domatia. We tested the hypothesis that ants use domatia-inhabiting fungi as food in three ant–plant symbioses: Petalomyrmex phylax/Leonardoxa africana, Tetraponera aethiops/Barteria fistulosa and Pseudomyrmex penetrator/Tachigali sp. Labelling domatia fungal patches in the field with either a fluorescent dye or 15N showed that larvae ingested domatia fungi. Furthermore, when the natural fungal patch was replaced with a piece of a 15N-labelled pure culture of either of two Chaetothyriales strains isolated from T. aethiops colonies, these fungi were also consumed. These two fungi often co-occur in the same ant colony. Interestingly, T. aethiops workers and larvae ingested preferentially one of the two strains. Our results add a new piece in the puzzle of the nutritional ecology of plant-ants.

Polyphenol investigation of Argania spinosa (Sapotaceae) endemic tree from Morocco.

Abstract The leaves of Argania spinosa from Morocco were investigated for flavonoids and condensed tannins. Four flavonol glycosides were identified by 1H NMR as myricitrin, quercitrin, hyperoside and myricetin 3-O-galactoside. UV spectrophotometric and histochemical methods were carried out to quantify and locate flavonoids and condensed tannins from leaves, stems and thorns of A. spinosa. Both high content and cell localisation of total polyphenols could explain the Argan tree adaptation to aridity.

A survey of mangiferin and hydroxycinnamic acid ester accumulation in coffee (Coffea) leaves: biological implications and uses

BACKGROUND AND AIMS The phenolic composition of Coffea leaves has barely been studied, and therefore this study conducts the first detailed survey, focusing on mangiferin and hydroxycinnamic acid esters (HCEs). METHODS Using HPLC, including a new technique allowing quantification of feruloylquinic acid together with mangiferin, and histochemical methods, mangiferin content and tissue localization were compared in leaves and fruits of C. pseudozanguebariae, C. arabica and C. canephora. The HCE and mangiferin content of leaves was evaluated for 23 species native to Africa or Madagascar. Using various statistical methods, data were assessed in relation to distribution, ecology, phylogeny and use. KEY RESULTS Seven of the 23 species accumulated mangiferin in their leaves. Mangiferin leaf-accumulating species also contain mangiferin in the fruits, but only in the outer (sporophytic) parts. In both leaves and fruit, mangiferin accumulation decreases with ageing. A relationship between mangiferin accumulation and UV levels is posited, owing to localization with photosynthetic tissues, and systematic distribution in high altitude clades and species with high altitude representatives. Analyses of mangiferin and HCE content showed that there are significant differences between species, and that samples can be grouped into species, with few exceptions. These data also provide independent support for various Coffea lineages, as proposed by molecular phylogenetic analyses. Sampling of the hybrids C. arabica and C. heterocalyx cf. indicates that mangiferin and HCE accumulation may be under independent parental influence. CONCLUSIONS This survey of the phenolic composition in Coffea leaves shows that mangiferin and HCE accumulation corresponds to lineage recognition and species delimitation, respectively. Knowledge of the spectrum of phenolic accumulation within species and populations could be of considerable significance for adaptation to specific environments. The potential health benefits of coffee-leaf tea, and beverages and masticatory products made from the fleshy parts of Coffea fruits, are supported by our phenolic quantification.

First report on mangiferin (C-glucosyl-xanthone) isolated from leaves of a wild coffee plant, Coffea pseudozanguebariae (Rubiaceae)

Abstract Bean biochemical composition has been extensively analyzed in Coffea species because of its impact on coffee beverage quality. C. pseudozanguebariae appeared as an interesting wild species because of its low caffeine and chlorogenic acid content, compounds involved in coffee bitterness. Extending the study to its leaves revealed the presence of two unknown phenolic compounds. Isolation, then identification by MS and NMR analysis proved that these compounds are two C-glucosyl-xanthones: mangiferin and isomangiferin. HPLC analyses indicated very high mangiferin content in young leaves. This class of compounds is described for the first time not only in coffee plants but also in the Rubiaceae family. The interest of mangiferin identification in the Coffea genus is discussed.

Differential compartmentation of o-diphenols and peroxidase activity in the inner sapwood of the Juglans nigra tree

Abstract Black walnut ( Juglans nigra L.) heartwood is characterised by the appearance of a dark-brown colour in the inner part of the trunk. At the cellular level, phenolic compounds and peroxidases (PODs, EC 1.11.1.7) are most likely involved in the generation of this colour. Ortho -diphenols are revealed by Arnow’s reagent and condensed tannins by vanillin-HCl or p -dimethylaminocinnamaldehyde. These reagents reveal that only a small percentage of the living sapwood parenchyma cells accumulate phenolic compounds in the vacuole. POD activity is revealed either with guaiacol or 3,3’-diaminobenzidine. The simultaneous disclosure of o -diphenols and POD in the same cross-section clearly indicates that POD activity and its phenolic substrates are localised in different ray parenchyma cells of walnut sapwood. None of the cells exhibiting POD activity contain o -diphenols and vice versa. This compartmentation of active PODs and of their potential substrates in different cells within the living woody tissue of walnut is discussed.

New Applications for an Old Lignified Element Staining Reagent

The use is reported of Mirandes reagent in epifluorescence microscopy which permits a clear distinction between cellulosic and lignified tissues. Homogeneous Prespermatophytae and gymnosperm xylem appeared entirely green with Mirandes reagent under ultraviolet excitation, whereas heteroxyled angiosperm wood showed a mixed pink and blue–green colour. This coloration was due to the fluorescence of cellulose, since certain elements in dicotyledonous wood (parenchyma, fibres, xylem rays) are not entirely lignified. Monocotyledonous (Poaceae) lignin showed an intense blue fluorescence due to hydroxycinnamic acids bound to the cell wall.The method showed that lignification occurs first in the middle lamella, and later in the secondary wall of xylem cells. In addition, this staining technique proved useful in the study of lignin and suberin deposition in response to various stress factors.

Spot inoculation of Medicago laciniata root by Sinorhizobium meliloti C1-4 or Glomus sp. S043 induces local accumulation of flavonoids

Abstract Medicago laciniata roots were in vitro inoculated with Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus sp. S043. Inocula were applied on well-defined parts of the root system. We studied the chemical response of root cells in presence of the bacteria or the fungus, using visible and fluorescent microscopy. The inoculation spot induced localised accumulation of flavonoids in the root tissue for both Sinorhizobium meliloti and Glomus sp. S043. There was no flavonoid accumulation in other areas of the inoculated root. No tannin accumulation was observed in the roots of the control as well as in the inoculated tissues. The role of these flavonoids is discussed.

Juvenile Coffee Leaves Acclimated to Low Light Are Unable to Cope with a Moderate Light Increase

The understorey origin of coffee trees and the strong plasticity of Coffea arabica leaves in relation to contrasting light environments have been largely shown. The adaptability of coffee leaves to changes in light was tested under controlled conditions by increasing the illumination rate on C. arabica var. Naryelis seedlings acclimated to low light conditions and observing leaf responses at three different developmental stages (juvenile, growing and mature). Only mature leaves proved capable of adapting to new light conditions. In these leaves, different major mechanisms were found to contribute to maintaining a good photosynthetic level. With increased illumination, a high photosynthetic response was conserved thanks to fast nitrogen remobilization, as indicated by SPAD values and the photorespiration rate. Efficient photoprotection was accompanied by a great ability to export sucrose, which prevented excessive inhibition of the Calvin cycle by hexose accumulation. In contrast, in younger leaves, increased illumination caused photodamage, observable even after 9 days of treatment. One major finding was that young coffee leaves rely on the accumulation of chlorogenic acids, powerful antioxidant phenolic compounds, to deal with the accumulation of reactive oxygen species rather than on antioxidant enzymes. Due to a lack of efficient photoprotection, a poor ability to export sucrose and inadequate antioxidant protection, younger leaves seemed to be unable to cope with increased illumination. In these leaves, an absence of induced antioxidant enzyme activity was accompanied, in growing leaves, by an absence of antioxidant synthesis or, in juvenile leaves, inefficient synthesis of flavonoids because located in some epidermis cells. These observations showed that coffee leaves, at the beginning of their development, are not equipped to withstand quick switches to higher light levels. Our results confirm that coffee trees, even selected for full sunlight conditions, remain shade plants possessing leaves able to adapt to higher light levels only when mature.