Sylvie Doulbeau

Metabolic pathways in tropical dicotyledonous albuminous seeds: Coffea arabica as a case study

The genomic era facilitates the understanding of how transcriptional networks are interconnected to program seed development and filling. However, to date, little information is available regarding dicot seeds with a transient perisperm and a persistent, copious endosperm. Coffea arabica is the subject of increasing genomic research and is a model for nonorthodox albuminous dicot seeds of tropical origin. The aim of this study was to reconstruct the metabolic pathways involved in the biosynthesis of the main coffee seed storage compounds, namely cell wall polysaccharides, triacylglycerols, sucrose, and chlorogenic acids. For this purpose, we integrated transcriptomic and metabolite analyses, combining real-time RT-PCR performed on 137 selected genes (of which 79 were uncharacterized in Coffea) and metabolite profiling. Our map-drawing approach derived from model plants enabled us to propose a rationale for the peculiar traits of the coffee endosperm, such as its unusual fatty acid composition, remarkable accumulation of chlorogenic acid and cell wall polysaccharides. Comparison with the developmental features of exalbuminous seeds described in the literature revealed that the two seed types share important regulatory mechanisms for reserve biosynthesis, independent of the origin and ploidy level of the storage tissue.

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.

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages.

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.

The Greater Phenotypic Homeostasis of the Allopolyploid Coffea arabica Improved the Transcriptional Homeostasis Over that of Both Diploid Parents

Polyploidy impacts the diversity of plant species, giving rise to novel phenotypes and leading to ecological diversification. In order to observe adaptive and evolutionary capacities of polyploids, we compared the growth, primary metabolism and transcriptomic expression level in the leaves of the newly formed allotetraploid Coffea arabica species compared with its two diploid parental species (Coffea eugenioides and Coffea canephora), exposed to four thermal regimes (TRs; 18–14, 23–19, 28–24 and 33–29°C). The growth rate of the allopolyploid C. arabica was similar to that of C. canephora under the hottest TR and that of C. eugenioides under the coldest TR. For metabolite contents measured at the hottest TR, the allopolyploid showed similar behavior to C. canephora, the parent which tolerates higher growth temperatures in the natural environment. However, at the coldest TR, the allopolyploid displayed higher sucrose, raffinose and ABA contents than those of its two parents and similar linolenic acid leaf composition and Chl content to those of C. eugenioides. At the gene expression level, few differences between the allopolyploid and its parents were observed for studied genes linked to photosynthesis, respiration and the circadian clock, whereas genes linked to redox activity showed a greater capacity of the allopolyploid for homeostasis. Finally, we found that the overall transcriptional response to TRs of the allopolyploid was more homeostatic compared with its parents. This better transcriptional homeostasis of the allopolyploid C. arabica afforded a greater phenotypic homeostasis when faced with environments that are unsuited to the diploid parental species.

The embryo and the endosperm contribute equally to argan seed oil yield but confer distinct lipid features to argan oil

In the perspective of studying lipid biosynthesis in the argan seed, the anatomy, ploidy level and lipid composition of mature seed tissues were investigated using an experimental design including two locations in Algeria and four years of study. Using flow cytometry, we determined that mature argan seeds consist of two well-developed tissues, the embryo and the endosperm. The lipid content of the embryo was higher than that of the endosperm, but the dry weight of the endosperm was higher. Consequently, both tissues contribute equally to seed oil yield. Considerable differences in fatty acid composition were observed between the two tissues. In particular, the endosperm 18:2 percentage was twofold higher than that of the embryo. The tocopherol content of the endosperm was also markedly higher than that of the embryo. In contrast, the endosperm and the embryo had similar sterol and triterpene alcohol contents and compositions.

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.

Influence of Environmental Conditions and Genetic Background of Arabica Coffee (C. arabica L) on Leaf Rust (Hemileia vastatrix) Pathogenesis

Global warming is a major threat to agriculture worldwide. Between 2008 and 2013, some coffee producing countries in South and Central America suffered from severe epidemics of coffee leaf rust (CLR), resulting in high economic losses with social implications for coffee growers. The climatic events not only favored the development of the pathogen but also affected the physiological status of the coffee plant. The main objectives of the study were to evaluate how the physiological status of the coffee plant modified by different environmental conditions impact on the pathogenesis of CLR and to identify indicators of the physiological status able to predict rust incidence. Three rust susceptible genotypes (one inbred line and two hybrids) were grown in controlled conditions with a combination of thermal regime (TR), nitrogen and light intensity close to the field situation before being inoculated with the rust fungus Hemileia vastatrix. It has been demonstrated that a TR of 27-22°C resulted in 2000 times higher sporulation than with a TR of 23–18°C. It has been also shown that high light intensity combined with low nitrogen fertilization modified the CLR pathogenesis resulting in huge sporulation. CLR sporulation was significantly lower in the F1 hybrids than in the inbred line. The hybrid vigor may have reduced disease incidence. Among the many parameters studied, parameters related to photosystem II and photosynthetic electron transport chain components appeared as indicators of the physiological status of the coffee plant able to predict rust sporulation intensity. Taken together, these results show that CLR sporulation not only depends on the TR but also on the physiological status of the coffee plant, which itself depends on agronomic conditions. Our work suggests that vigorous varieties combined with a shaded system and appropriate nitrogen fertilization should be part of an agro-ecological approach to disease control.