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Abstract Background Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Background and Aims: Allopolyploidization is a widespread phenomenon known to generate novel phenotypes by merging evolutionarily distinct parental genomes and regulatory networks in a single nucleus. The objective of this study was to investigate the transcriptional regulation associated with phenotypic novelty in coffee beans of the allotetraploid Coffea arabica., Methods: A genome-wide comparative transcriptomic analysis was performed in C. arabica and its two diploid progenitors, C. canephora and C. eugenioides. Gene expression patterns and homeologue expression were studied on seeds at five different maturation stages. The involvement of homeologue expression bias (HEB) in specific traits was addressed both by functional enrichment analyses and by the study of gene expression in the caffeine and chlorogenic acid biosynthesis pathways., Key Results: Expression-level dominance in C. arabica seed was observed for most of the genes differentially expressed between the species. Approximately a third of the genes analysed showed HEB. This proportion increased during seed maturation but the biases remained equally distributed between the sub-genomes. The relative expression levels of homeologues remained relatively constant during maturation and were correlated with those estimated in leaves of C. arabica and interspecific hybrids between C. canephora and C. eugenioides. Functional enrichment analyses performed on genes exhibiting HEB enabled the identification of processes potentially associated with physiological traits. The expression profiles of the genes involved in caffeine biosynthesis mirror the differences observed in the caffeine content of mature seeds of C. arabica and its parental species., Conclusions: Neither of the two sub-genomes is globally preferentially expressed in C. arabica seeds, and homeologues appear to be co-regulated by shared trans-regulatory mechanisms. The observed HEBs are thought to be a legacy of gene expression differences inherited from diploid progenitor species. Pre-existing functional divergences between parental species appear to play an important role in controlling the phenotype of C. arabica seeds., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Species of the genus Coffea accumulate diterpenes of the ent-kaurane family in the endosperm of their seeds, of which cafestol and kahweol are the most abundant. The diterpenes are mainly stored in esterified form with fatty acids, mostly palmitate. In contrast to the numerous studies on their effects on human health and therapeutic applications, nothing was previously known about their biological and ecological role in planta. The antifungal and anti-insect activities of cafestol and cafestol palmitate were thus investigated in this study. Cafestol significantly affected the mycelial growth of five of the six phytopathogenic fungi tested. It also greatly reduced the percentage of pupation of larvae and the pupae and adult masses of one of the two fruit flies tested. By contrast, cafestol palmitate had no significant effect against any of the fungi and insects studied. Using confocal imaging and oil body isolation and analysis, we showed that diterpenes are localized in endosperm oil bodies, suggesting that esterification with fatty acids enables the accumulation of large amounts of diterpenes in a non-toxic form. Diterpene measurements in all organs of seedlings recovered from whole seed germination or embryos isolated from the endosperm showed that diterpenes are transferred from the endosperm to the cotyledons during seedling growth and then distributed to all organs, including the hypocotyl and the root. Collectively, our findings show that coffee diterpenes are broad-spectrum defence compounds that protect not only the seed on the mother plant and in the soil, but also the seedling after germination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Background and Aims: The pacaya palm is a dioecious neotropical palm species that is exploited in Latin America for its male inflorescence, which is edible when immature. It is cultivated, in a non-intensive manner, in Guatemala, where a morphotype occurs that produces much larger, more highly branched inflorescences compared with wild palms. We sought to identify molecular factors underlying this phenotypic divergence, which is likely to be a product of domestication., Methods: We performed RNA-seq-based studies on immature pacaya palm male inflorescences in order to identify genes that might be directly or indirectly affected in their expression in relation to domestication. We also measured the accumulation of a range of soluble sugar molecules to provide information on the biochemical status of the two different types of material., Key Results: A total of 408 genes were found to display significantly different expression levels between the wild and cultivated morphotypes. Three different functional categories were found to be enriched in the gene set that was upregulated in the cultivated morphotype: redox balance; secondary metabolism; and transport. Several sugars were found to accumulate at higher levels in inflorescences of the cultivated morphotype, in particular myo-inositol, fructose and glucose., Conclusions: The observed upregulation of redox-related genes in the cultivated morphotype is corroborated by the observation of higher myo-inositol accumulation, which has been shown to be associated with enhanced scavenging of reactive oxygen species in other plants and which may affect meristem activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Detecting processes of local adaptation in forest trees and identifying environmental selective drivers are of primary importance for forest management and conservation. Transplant experiments, functional genomics and population genomics are complementary tools to efficiently characterize heritable phenotypic traits and to decipher the genetic bases of adaptive traits. Using an integrative approach combining phenotypic assessment in common garden, transcriptomics and landscape genomics, we investigated leaf adaptive traits in Coffea mauritiana, a forest tree endemic to Reunion Island. Eight populations of C. mauritiana originating from sites with contrasted environmental conditions were sampled in common garden to assess several leaf morphological traits, to analyze the leaf transcriptome and leaf cuticular wax composition. The relative alkane content of cuticular waxes was significantly correlated with major climatic gradients, paving the way for further transcriptome-based analyses. The expression pattern of cuticle biosynthetic genes was consistent with a modulation of alkane accumulation across the population studied, supporting the hypothesis that the composition of cuticular wax is involved in the local adaptation of C. mauritiana. Association tests in landscape genomics performed using RNA-seq-derived single-nucleotide polymorphisms revealed that genes associated with cell wall remodeling also likely play an adaptive role. By combining these different approaches, this study efficiently identified local adaptation processes in a non-model species. Our results provide the first evidence for local adaptation in trees endemic to Reunion Island and highlight the importance of cuticle composition for the adaptation of trees to the high evaporative demand in warm climates., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Background: Fruit abscission depends on cell separation that occurs within specialized cell layers that constitute an abscission zone (AZ). To determine the mechanisms of fleshy fruit abscission of the monocot oil palm (Elaeis guineensis Jacq.) compared with other abscission systems, we performed multi-scale comparative transcriptome analyses on fruit targeting the developing primary AZ and adjacent tissues., Results: Combining between-tissue developmental comparisons with exogenous ethylene treatments, and naturally occurring abscission in the field, RNAseq analysis revealed a robust core set of 168 genes with differentially regulated expression, spatially associated with the ripe fruit AZ, and temporally restricted to the abscission timing. The expression of a set of candidate genes was validated by qRT-PCR in the fruit AZ of a natural oil palm variant with blocked fruit abscission, which provides evidence for their functions during abscission. Our results substantiate the conservation of gene function between dicot dry fruit dehiscence and monocot fleshy fruit abscission. The study also revealed major metabolic transitions occur in the AZ during abscission, including key senescence marker genes and transcriptional regulators, in addition to genes involved in nutrient recycling and reallocation, alternative routes for energy supply and adaptation to oxidative stress., Conclusions: The study provides the first reference transcriptome of a monocot fleshy fruit abscission zone and provides insight into the mechanisms underlying abscission by identifying key genes with functional roles and processes, including metabolic transitions, cell wall modifications, signalling, stress adaptations and transcriptional regulation, that occur during ripe fruit abscission of the monocot oil palm. The transcriptome data comprises an original reference and resource useful towards understanding the evolutionary basis of this fundamental plant process.

Background and Aims: Oaks are the foundation and dominant tree species of most Mediterranean forests. As climate models predict dramatic changes in the Mediterranean basin, a better understanding of the ecophysiology of seed persistence and germination in oaks could help define their regeneration niches. Tunisian oaks occupy distinct geographical areas, which differ in their rainfall and temperature regimes, and are thus a valuable model to investigate relationships between seed traits and species ecological requirements., Methods: Seed morphological traits, desiccation sensitivity level, lethal freezing temperature, embryonic axis and cotyledon sugar and lipid composition, and seed and acorn germination rates at various constant temperatures were measured in Quercus canariensis, Q. coccifera, Q. ilex and Q. suber, using seeds sampled in 22 Tunisian woodlands., Key Results: Only faint differences were observed for desiccation sensitivity in the oak species studied. By contrast, the species differed significantly in sensitivity to freezing, germination rates at low temperature and base temperature. Quercus ilex and Q. canariensis, which occur at high elevations where frost events are frequent, showed the lowest freezing sensitivity. A significant correlation was found between hexose contents in the embryonic axis and freezing tolerance. Significant interspecific differences in the time for seeds to germinate and the time for the radicle to pierce the pericarp were observed. The ratio of pericarp mass to acorn mass differed significantly among the species and was negatively correlated with the acorn germination rate. Quercus coccifera, which is frequent in warm and arid environments, showed the highest acorn germination rate and synchrony., Conclusions: Seed lethal temperature, seed germination time at low temperatures, the ratio of pericarp mass to acorn mass and the embryonic axis hexose content appeared to be key functional traits that may influence the geographical ranges and ecological requirements of Mediterranean oaks in Tunisia., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Past climatic fluctuations have played a major role in shaping the current plant biodiversity. Although harbouring an exceptional biota, oceanic islands have received little attention in studies on species demographic history and past vegetation patterns. We investigated the impact of past climatic changes on the effective population size of a tree (Coffea mauritiana) that is endemic to Reunion Island, located in the south-western Indian Ocean (SWIO). Demographic changes were inferred using summary statistics calculated from genomic data. Using ecological niche modelling and the current distribution of genetic diversity, the paleodistribution of the species was also assessed. A reduction in the effective population size of C. mauritiana during the last glaciation maximum was inferred. The distribution of the species was reduced on the western side of the island, due to low rainfall. It appeared that a major reduction in rainfall and a slight temperature decrease prevailed in the SWIO. Our findings indicated that analyses on the current patterns of intraspecific genetic variations can efficiently contribute to past climatic changes characterisation in remote islands. Identifying area with higher resilience in oceanic islands could provide guidance in forest management and conservation faced to the global climate change., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Oceanic islands are commonly considered as natural laboratories for studies on evolution and speciation. The evolutionary specificities of islands associated with species biology provide unique scenarios to study the role of geography and climate in driving population divergence. However, few studies have addressed this subject in small oceanic islands with heterogeneous climates. Being widely distributed in Reunion Island forest, Coffea mauritiana represents an interesting model case for investigating patterns of within-island differentiation at small spatial scale. In this study, we examined the genetic diversity and population divergences of C. mauritiana using SNP markers obtained from 323 individuals across 34 locations in Reunion Island. Using redundancy analysis, we further evaluated the contribution of geographic and climatic factors to shaping genetic divergence among populations. Genetic diversity analyses revealed that accessions clustered according to the source population, with further grouping in regional clusters. Genetic relationships among the regional clusters underlined a recent process of expansion in the form of step-by-step colonization on both sides of the island. Divergence among source populations was mostly driven by the joint effect of geographic distance and climatic heterogeneity. The pattern of isolation-by-geography was in accordance with the dispersal characteristics of the species, while isolation-by-environment was mostly explained by the heterogeneous rainfall patterns, probably associated with an asynchronous flowering among populations. These findings advance our knowledge on the patterns of genetic diversity and factors of population differentiation of species native to Reunion Island, and will also usefully guide forest management for conservation.

Pineapple (Ananas comosus (L.) Merr.) is a non-climacteric tropical fruit whose ripening could be accompanied by oxidative processes and the concurrent activation of enzymatic and non-enzymatic reactive oxygen species (ROS) scavenging systems. To better understand the variability of these processes among climatic environments or genotypes in pineapple, the temporal expression dynamics for genes encoding oxidative and antioxidative stress enzymes were analyzed by real-time RT-PCR during fruit development and ripening, among three cultivars: Queen Victoria, Flhoran 41 and MD-2 hybrid, and in two climatic areas. Pineapple development and ripening involved changes in the levels of transcripts encoding for polyphenol oxidase and transcripts involved in the first steps of the phenylpropanoid pathway and in the balance of ROS, especially those encoding for ascorbate peroxydase and metallothioneins, regardless of the cultivar. Our results confirm the same dynamic in gene expression from the two environmental crop areas, however climatic conditions influenced the level of the expression of the major transcripts studied that were linked to these oxidative and antioxidant metabolisms. MT3a and MT3b transcripts were not influenced by genetic factor. The genetic effect was not significant on the various transcripts linked to the first steps of the phenylpropanoid pathway and to phenol oxidation, except 4CL ones. In ripe pineapple, highly significant relationships were found between the contents in antioxidant metabolites, i.e., ascorbic acid and total phenolic compounds, and the transcript levels of genes involved in the enzymatic ROS-scavenging system and in the biosynthesis or regeneration of ROS-scavenging compounds, like phenylpropanoids, ascorbic acid, metallothioneins., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The 'intermediate seed' category was defined in the early 1990s using coffee (Coffea arabica) as a model. In contrast to orthodox seeds, intermediate seeds cannot survive complete drying, which is a major constraint for seed storage and has implications for both biodiversity conservation and agricultural purposes. However, intermediate seeds are considerably more tolerant to drying than recalcitrant seeds, which are highly sensitive to desiccation. To gain insight into the mechanisms governing such differences, changes in desiccation tolerance (DT), hormone contents, and the transcriptome were analysed in developing coffee seeds. Acquisition of DT coincided with a dramatic transcriptional switch characterised by the repression of primary metabolism, photosynthesis, and respiration, and the up-regulation of genes coding for late-embryogenesis abundant (LEA) proteins, heat-shock proteins (HSPs), and antioxidant enzymes. Analysis of the heat-stable proteome in mature coffee seeds confirmed the accumulation of LEA proteins identified at the transcript level. Transcriptome analysis also suggested a major role for ABA and for the transcription factors CaHSFA9, CaDREB2G, CaANAC029, CaPLATZ, and CaDOG-like in DT acquisition. The ability of CaHSFA9 and CaDREB2G to trigger HSP gene transcription was validated by Agrobacterium-mediated transformation of coffee somatic embryos.

Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regulate the expression of the uidA reporter gene in response to water deficit mimicked by polyethylene glycol (-2.0 MPa) and low relative humidity treatments. A deletion analysis of their corresponding 5'-upstream regions revealed increased specificity of β-glucuronidase activity in the polyethylene glycol and low relative humidity treatments, with high expression in leaf mesophyll and guard cells in full-length constructs. RT-qPCR assays also revealed that the HP16 haplotype (specific to clone tolerant to water deficit) had stronger and earlier activity compared with the HP15 and HP17 haplotypes. As most of the cis-regulatory elements involved in ABA-dependent and -independent networks, tissue specificity and light regulation are common to these haplotypes, we propose that their organization, as well as the nucleic acid polymorphisms present outside these boxes, may play a role in modulating activities of DREB1D promoters in guard cells., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Background and Aims: Dominant tree species in northern temperate forests, for example oak and beech, produce desiccation-sensitive seeds. Despite the potentially major influence of this functional trait on the regeneration and distribution of species under climate change, little is currently known about the ecological determinants of the persistence of desiccation-sensitive seeds in transient soil seed banks. Knowing which key climatic and microsite factors favour seed survival will help define the regeneration niche for species whose seeds display extreme sensitivity to environmental stress, Methods: Using the Mediterranean Holm oak (Quercus ilex) forest as a model system, an in situ time-course monitoring of seed water status and viability was performed during the unfavourable winter season in two years with contrasting rainfall, at an instrumented site with detailed climate records. In parallel, the characteristics of the microhabitat and their influence on the post-winter water status and viability of seeds were investigated in a regional survey of 33 woodlands representative of the French distribution of the species., Key Results: Time-course monitoring of seed water status in natural conditions confirmed that in situ desiccation is the main abiotic cause of mortality in winter. Critical water contents could be reached in a few days during drought spells. Seed dehydration rates were satisfactorily estimated using integrative climate proxies including vapour pressure deficit and potential evapotranspiration. Seed water status was therefore determined by the balance between water uptake after a rainfall event and water loss during dry periods. Structural equation modelling of microhabitat factors highlighted the major influence of canopy openness and resulting incident radiation on the ground., Conclusions: This study provides part of the knowledge required to implement species distribution models which incorporate their regeneration niche. It is an important step forward in evaluating the ecological consequences of increasing winter drought and environmental filtering due to climate change on the regeneration of the most dominant Mediterranean tree species., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin., (Copyright © 2014, American Association for the Advancement of Science.)

The seed of Coffea arabica accumulates large amounts of cell wall storage polysaccharides (CWSPs) of the mannan family in the cell walls of the endosperm. The variability induced by the growing environment and extensive pairwise correlation analysis with stringent significance thresholds was used to investigate transcript-transcript and transcript-metabolite relationships among 26 sugar-related genes, and the amount of CWSPs and seven soluble low molecular weight carbohydrates in the developing coffee endosperm. A dense module of nine quantitatively co-expressed genes was detected at the mid-developmental stage when CWSPs accumulate. This module included the five genes of the core galactomannan synthetic machinery, namely genes coding for the enzymes needed to assemble the mannan backbone (mannan synthase, ManS), and genes that introduce the galactosyl side chains (galactosyltransferase, GMGT), modulate the post-depositional degree of galactose substitution (α-galactosidase), and produce the nucleotide sugar building blocks GDP-mannose and UDP-galactose (mannose-1P guanyltransferase and UDP-glucose 4'-epimerase, respectively). The amount of CWSPs stored in the endosperm at the onset of their accumulation was primarily and quantitatively modulated at the transcriptional level (i.e. positively correlated with the expression level of these key galactomannan biosynthetic genes). This analysis also suggests a role for sorbitol and raffinose family oligosaccharides as transient auxiliary sources of building blocks for galactomannan synthesis. Finally, a microarray-based analysis of the developing seed transcriptome revealed that all genes of the core galactomannan synthesis machinery grouped in a single cluster of 209 co-expressed genes. Analysis of the gene composition of this cluster revealed remarkable functional coherence and identified transcription factors that putatively control galactomannan biosynthesis in coffee.

In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Oil palm (Elaeis guineensis) produces two oils of major economic importance, commonly referred to as palm oil and palm kernel oil, extracted from the mesocarp and the endosperm, respectively. While lauric acid predominates in endosperm oil, the major fatty acids (FAs) of mesocarp oil are palmitic and oleic acids. The oil palm embryo also stores oil, which contains a significant proportion of linoleic acid. In addition, the three tissues display high variation for oil content at maturity. To gain insight into the mechanisms that govern such differences in oil content and FA composition, tissue transcriptome and lipid composition were compared during development. The contribution of the cytosolic and plastidial glycolytic routes differed markedly between the mesocarp and seed tissues, but transcriptional patterns of genes involved in the conversion of sucrose to pyruvate were not related to variations for oil content. Accumulation of lauric acid relied on the dramatic up-regulation of a specialized acyl-acyl carrier protein thioesterase paralog and the concerted recruitment of specific isoforms of triacylglycerol assembly enzymes. Three paralogs of the WRINKLED1 (WRI1) transcription factor were identified, of which EgWRI1-1 and EgWRI1-2 were massively transcribed during oil deposition in the mesocarp and the endosperm, respectively. None of the three WRI1 paralogs were detected in the embryo. The transcription level of FA synthesis genes correlated with the amount of WRI1 transcripts and oil content. Changes in triacylglycerol content and FA composition of Nicotiana benthamiana leaves infiltrated with various combinations of WRI1 and FatB paralogs from oil palm validated functions inferred from transcriptome analysis.

Background and Aims: Several widespread tree species of temperate forests, such as species of the genus Quercus, produce recalcitrant (desiccation-sensitive) seeds. However, the ecological significance of seed desiccation sensitivity in temperate regions is largely unknown. Do seeds of such species suffer from drying during the period when they remain on the soil, between shedding in autumn and the return of conditions required for germination in spring?, Methods: To test this hypothesis, the Mediterranean holm oak (Quercus ilex) forest was used as a model system. The relationships between the climate in winter, the characteristics of microhabitats, acorn morphological traits, and the water status and viability of seeds after winter were then investigated in 42 woodlands sampled over the entire French distribution of the species., Key Results: The percentages of germination and normal seedling development were tightly linked to the water content of seeds after the winter period, revealing that in situ desiccation is a major cause of mortality. The homogeneity of seed response to drying suggests that neither intraspecific genetic variation nor environmental conditions had a significant impact on the level of desiccation sensitivity of seeds. In contrast, the water and viability status of seeds at the time of collection were dramatically influenced by cumulative rainfall and maximum temperatures during winter. A significant effect of shade and of the type of soil cover was also evidenced., Conclusions: The findings establish that seed desiccation sensitivity is a key functional trait which may influence the success of recruitment in temperate recalcitrant seed species. Considering that most models of climate change predict changes in rainfall and temperature in the Mediterranean basin, the present work could help foresee changes in the distribution of Q. ilex and other oak species, and hence plant community alterations.

Fruit provide essential nutrients and vitamins for the human diet. Not only is the lipid-rich fleshy mesocarp tissue of the oil palm (Elaeis guineensis) fruit the main source of edible oil for the world, but it is also the richest dietary source of provitamin A. This study examines the transcriptional basis of these two outstanding metabolic characters in the oil palm mesocarp. Morphological, cellular, biochemical, and hormonal features defined key phases of mesocarp development. A 454 pyrosequencing-derived transcriptome was then assembled for the developmental phases preceding and during maturation and ripening, when high rates of lipid and carotenoid biosynthesis occur. A total of 2,629 contigs with differential representation revealed coordination of metabolic and regulatory components. Further analysis focused on the fatty acid and triacylglycerol assembly pathways and during carotenogenesis. Notably, a contig similar to the Arabidopsis (Arabidopsis thaliana) seed oil transcription factor WRINKLED1 was identified with a transcript profile coordinated with those of several fatty acid biosynthetic genes and the high rates of lipid accumulation, suggesting some common regulatory features between seeds and fruits. We also focused on transcriptional regulatory networks of the fruit, in particular those related to ethylene transcriptional and GLOBOSA/PISTILLATA-like proteins in the mesocarp and a central role for ethylene-coordinated transcriptional regulation of type VII ethylene response factors during ripening. Our results suggest that divergence has occurred in the regulatory components in this monocot fruit compared with those identified in the dicot tomato (Solanum lycopersicum) fleshy fruit model.

Background: Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics., Results: The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study., Conclusion: We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Developing Coffea arabica seeds accumulate large amounts of chlorogenic acids (CGAs) as a storage form of phenylpropanoid derivatives, making coffee a valuable model to investigate the metabolism of these widespread plant phenolics. However, developmental and environmental regulations of CGA metabolism are poorly understood. In the present work, the expression of selected phenylpropanoid genes, together with CGA isomer profiles, was monitored throughout seed development across a wide set of contrasted natural environments. Although CGA metabolism was controlled by major developmental factors, the mean temperature during seed development had a direct impact on the time-window of CGA biosynthesis, as well as on final CGA isomer composition through subtle transcriptional regulations. We provide evidence that the variability induced by the environment is a useful tool to test whether CGA accumulation is quantitatively modulated at the transcriptional level, hence enabling detection of rate-limiting transcriptional steps [quantitative trait transcripts (QTTs)] for CGA biosynthesis. Variations induced by the environment also enabled a better description of the phenylpropanoid gene transcriptional network throughout seed development, as well as the detection of three temporally distinct modules of quantitatively co-expressed genes. Finally, analysis of metabolite-to-metabolite relationships revealed new biochemical characteristics of the isomerization steps that remain uncharacterized at the gene level.

* 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.

The chemotaxonomic relationships between Coffea (subgenus Coffea) species have been poorly studied to date and the compounds tested so far - chlorogenic acids, diterpenoids and purine alkaloids - did not enable the establishment of phylogenetic relationships analogous to those revealed by chloroplast and nuclear DNA studies. In the present study, the relationships between African Coffea species were assessed on the basis of their seed lipid composition. Fatty acids and sterols were determined in 59 genotypes belonging to 17 distinct Coffea species/origins. Principal Component Analysis of fatty acid and sterol data enabled easy identification of the few species for which one or several compounds could serve as a quantitative signature. Hierarchical Clustering classified the Coffea species in seven groups with both fatty acids and sterols. However, while groupings based on seed fatty acid composition showed remarkable ecological and geographical coherence, no phylogeographic explanation was found for the clusters retrieved from sterol data. When compared with previous phylogenetic studies, the groups deduced from seed fatty acid composition were remarkably congruent with the clades inferred from nuclear and plastid DNA sequences.

Due to its economic importance, Coffea arabica is becoming the subject of increasing genomic research and, in particular, the genes involved in the final chemical composition of the bean and the sensorial quality of the coffee beverage. The aim of the present study was to decipher the transcriptional networks that govern the development of the C. arabica seed, a model for non-orthodox albuminous seeds of tropical origin. For this purpose, we developed a transcriptomic approach combining two techniques: targeted cDNA arrays, containing 266 selected candidate gene sequences, and real-time RT-PCR on a large subset of 111 genes. The combination of the two techniques allowed us to limit detection of false positives and to reveal the advantages of using large real-time RT-PCR screening. Multivariate analysis was conducted on both datasets and results were broadly convergent. First, principle component analysis (PCA) revealed a dramatic re-programming of the transcriptional machinery between early cell division and elongation, storage and maturation phases. Second, hierarchical clustering analysis (HCA) led to the identification of 11 distinct patterns of gene expression during seed development as well as to the detection of genes expressed at specific developmental stages that can be used as functional markers of phenological changes. In addition, this study led to the description of gene expression profiles for quality-related genes, most of them formerly uncharacterised in Coffea. Their involvement in storage compound synthesis and accumulation during endosperm development and final metabolic re-adjustments during maturation is discussed.

Plasmodium vivax is the second most common species of malaria parasite and causes up to 80 million episodes of infection each year. New drug targets are urgently needed because of emerging resistance to current treatments. To study new potential targets, we have functionally characterized two natural variants of the hexose transporter of P. vivax (PvHT) after heterologous expression in Xenopus oocytes. We show that PvHT transports both glucose and fructose. Differences in the affinity for fructose between the two variants of PvHT establishes that sequence variation is associated with phenotypic plasticity. Mutation of a single glutamine residue, Gln(167), predicted to lie in transmembrane helix 5, abolishes fructose transport by PvHT, although glucose uptake is preserved. In contrast, the exofacial site located between predicted helices 5 and 6 of PvHT is not an important determinant of substrate specificity, despite exhibiting sequence polymorphisms between hexose transporters of different Plasmodium spp. Indeed, replacement of twelve residues located within this region of PvHT by those found in the orthologous Plasmodium falciparum sequence (PfHT) is functionally silent with respect to affinity for hexoses. All PvHT variants are inhibited by compound 3361, a long-chain O-3 derivative of D-glucose effective against PfHT. Furthermore, compound 3361 kills short term cultures of P. vivax isolated from patients. These data provide unique insights into the function of hexose transporters of Plasmodium spp. as well as further evidence that they could be targeted by drugs.

Despite substantial efforts at control over several decades, malaria is still a major global health problem as chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of these parasites are wholly dependent upon host glucose for energy and malarial proteins involved in hexose permeation are therefore attractive new drug targets. For Plasmodium falciparum, the causative agent of severe malaria, a facilitative hexose transporter (PfHT), encoded by a single-copy gene mediates glucose uptake. We first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. This review describes the value of using Xenopus oocytes in heterologous studies of P. falciparum-encoded proteins and summarises the properties of PfHT. Comparisons between Gluts (mammalian facilitative hexose transporters) and PfHT using this expression system have highlighted important mechanistic and structural differences between parasite and host proteins. Certain O-methyl derivatives of glucose proved particularly useful discriminators between mammalian transporters and PfHT. We exploited this selectivity and synthesised a long-chain O-3-hexose derivative (compound 3361) that potently inhibits PfHT expressed in oocytes and also kills P. falciparum when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the Plasmodium vivax orthologue of PfHT, and demonstrate inhibition of glucose uptake by compound 3361. These findings validate malarial hexose transporters as a novel target. We now aim to design a new class of antimalarials by the discovery of highly specific inhibitors which could act with a broad spectrum of action on different Plasmodium spp. infections.

Chemotherapy of apicomplexan parasites is limited by emerging drug resistance or lack of novel targets. PfHT1, the Plasmodium falciparum hexose transporter 1, is a promising new drug target because asexual-stage malarial parasites depend wholly on glucose for energy. We have performed a comparative functional characterization of PfHT1 and hexose transporters of the simian malarial parasite P. knowlesi (PkHT1), the rodent parasite P. yoelii (PyHT1) and the human apicomplexan parasite Toxoplasma gondii ( T. gondii glucose transporter 1, TgGT1). PkHT1 and PyHT1 share >70% amino acid identity with PfHT1, while TgGT1 is more divergent (37.2% identity). All transporters mediate uptake of D-glucose and D-fructose. PyHT1 has an affinity for glucose ( K (m) approximately 0.12 mM) that is higher than that for PkHT1 ( K (m) approximately 0.67 mM) or PfHT1 ( K (m) approximately 1 mM). TgGT1 is highly temperature dependent (the Q (10) value, the fold change in activity for a 10 degrees C change in temperature, was >7) compared with Plasmodium transporters ( Q (10), 1.5-2.5), and overall has the highest affinity for glucose ( K (m) approximately 30 microM). Using active analogues in competition for glucose uptake, experiments show that hydroxyl groups at the C-3, C-4 and C-6 positions are important in interacting with PkHT1, PyHT1 and TgGT1. This study defines models useful to study the biology of apicomplexan hexose permeation pathways, as well as contributing to drug development.

Plasmodium falciparum infection induces alterations in the transport properties of infected erythrocytes that have recently been defined using electrophysiological techniques. Mechanisms responsible for transport of substrates into intraerythrocytic parasites have also been clarified by studies of three substrate-specific (hexose, nucleoside and aquaglyceroporin) parasite plasma membrane transporters. These have been characterised functionally using the Xenopus laevis oocyte heterologous expression system. The same expression system is currently being used to define the function of parasite 'P' type ATPases responsible for intraparasitic [Ca(2+)] homeostasis. We review studies on these transport processes and examine their potential as novel drug targets.

Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.

Cyclic electron flow around photosystem (PS) I has been widely described in vitro in chloroplasts or thylakoids isolated from C(3) plant leaves, but its occurrence in vivo is still a matter of debate. Photoacoustic spectroscopy and kinetic spectrophotometry were used to analyze cyclic PS I activity in tobacco (Nicotiana tabacum cv Petit Havana) leaf discs illuminated with far-red light. Only a very weak activity was measured in air with both techniques. When leaf discs were placed in anaerobiosis, a high and rapid cyclic PS I activity was measured. The maximal energy storage in far-red light increased to 30% to 50%, and the half-time of the P(700) re-reduction in the dark decreased to around 400 ms; these values are comparable with those measured in cyanobacteria and C(4) plant leaves in aerobiosis. The stimulatory effect of anaerobiosis was mimicked by infiltrating leaves with inhibitors of mitochondrial respiration or of the chlororespiratory oxidase, therefore, showing that changes in the redox state of intersystem electron carriers tightly control the rate of PS I-driven cyclic electron flow in vivo. Measurements of energy storage at different modulation frequencies of far-red light showed that anaerobiosis-induced cyclic PS I activity in leaves of a tobacco mutant deficient in the plastid Ndh complex was kinetically different from that of the wild type, the cycle being slower in the former leaves. We conclude that the Ndh complex is required for rapid electron cycling around PS I.

Besides electron transfer reactions involved in the 'Z' scheme of photosynthesis, alternative electron transfer pathways have been characterized in chloroplasts. These include cyclic electron flow around photosystem I (PS I) or a respiratory chain called chlororespiration. Recent work has supplied new information concerning the molecular nature of the electron carriers involved in the non-photochemical reduction of the plastoquinone (PQ) pool. However, until now little is known concerning the nature of the electron carriers involved in PQ oxidation. By using mass spectrometric measurement of oxygen exchange performed in the presence of 18O-enriched O2 and Chlamydomonas mutants deficient in PS I, we show that electrons can be directed to a quinol oxidase sensitive to propyl gallate but insensitive to salicyl hydroxamic acid. This oxidase has immunological and pharmacological similarities with a plastid protein involved in carotenoid biosynthesis.