The Analysis of Partial Sequences of the Flavonone 3 Hydroxylase Gene in Lupinus mutabilis Reveals Differential Expression of Two Paralogues Potentially Related to Seed Coat Colour

Flavonone 3 hydroxylases (EC 1.14.11.9) are key enzymes in the synthesis of anthocyanins and other flavonoids. Such compounds are involved in seed coat colour and stem pigmentation. Lupinus mutabilis (tarwi) is a legume crop domesticated in the Andean region, valued for the high protein and oil content of its seeds. Tarwi accessions are being selected for cultivation in Europe under defined breeding criteria. Seed coat colour patterns are relevant breeding traits in tarwi, and these are conditioned by anthocyanin content. We identified and isolated part of the tarwi flavonone 3-hydroxylase gene (LmF3h) from two accessions with distinct seed coat colour patterns. Two partial LmF3h paralogues, with predicted 20% amino-acid changes but little predicted tertiary structure alterations, were identified in the coloured seed genotype, while only one was present in the white seed genotype. Upon selection and validation of appropriate reference genes, a RT-qPCR analysis showed that these paralogues have different levels of expression during seed development in both genotypes, although they follow the same expression patterns. DNA and transcription analyses enabled to highlight potential F3H paralogues relatable to seed coat pigmentation in tarwi and, upon biochemical and genetic confirmation, prompt marker-assisted breeding for relevant phenotypic traits associated with flavonoid synthesisinfo:eu-repo/semantics/publishedVersio

Pathological, Morphological, Cytogenomic, Biochemical and Molecular Data Support the Distinction between Colletotrichum cigarro comb. et stat. nov. and Colletotrichum kahawae

The genus Colletotrichum has witnessed tremendous variations over the years in the number of species recognized, ranging from 11 to several hundreds. Host-specific fungal species, once the rule, are now the exception, with polyphagous behavior regarded as normal in this genus. The species Colletotrichum kahawae was created to accommodate the pathogens that have the unique ability to infect green developing coffee berries causing the devastating Coffee Berry Disease in Africa, but its close phylogenetic relationship to a polyphagous group of fungi in the C. gloeosporioides species complex led some researchers to regard these pathogens as members of a wider species. In this work we combine pathological, morphological, cytogenomic, biochemical, and molecular data of a comprehensive set of phylogenetically-related isolates to show that the Coffee Berry Disease pathogen forms a separate species, C. kahawae, and also to assign the closely related fungi, previously in C. kahawae subsp. cigarro, to a new species, C. cigarro comb. et stat. nov. This taxonomic clarification provides an opportunity to link phylogeny and functional biology, and additionally enables a much-needed tool for plant pathology and agronomy, associating exclusively C. kahawae to the Coffee Berry Disease pathogen.info:eu-repo/semantics/publishedVersio

Identification of HIR, EDS1 and PAD4 genes reveals differences between Coffea species that may impact disease resistance

Coffee, a widely consumed important agricultural product, is mainly produced from two species, Coffea arabica (Arabica coffee) and C. canephora (Robusta coffee). Timor Hybrid (HDT) is a population resulting from a natural cross between C. arabica and C. canephora. HDT derivatives have a high spectrum of resistance to different races of Hemileia vastatrix (Hv), the causal agent of coffee leaf rust. A RNAseq database, obtained from HDT832/2 leaves inoculated with Hv (Host Resistance) and Uromyces vignae (Uv, Nonhost Resistance), showed the presence of genes implicated in the hypersensitive response and salicylic acid pathway. Hypersensitive Induced Reaction (HIR) gene family, Enhanced Disease Susceptibility1 gene (EDS1), and Phytoalexin Deficient 4 (PAD4) gene are involved in host and nonhost resistance. Relative expression calculated by RT-qPCR was used to confirm and expand the transcriptome analysis. HDTHIR4, HDTEDS1, and HDTPAD4 showed the highest upregulation in response to Hv and Uv inoculation, confirming a similar trend in host and nonhost resistance in HDT. HIR and EDS1/PAD4 gene families were characterized for the first time in the three available Coffea genomes. HIR genes were quite conserved between Coffea species. Surprisingly, EDS1 and PAD4 genes revealed major differences in gene structure. The PAD4 predicted protein from C. arabica does not include both conserved domains of the EDS1/PAD4 family, and the EDS1 putative protein from C. canephora includes a formin domain unusual in the same protein family. The variability shown by EDS1/PAD4 gene family may impact the disease resistance response of Coffea species, which can be surveyed for the gene sequences that will produce a more resistant phenotype.This research was co-funded by Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES ref. PTDC/ASP-PLA/29779/2017, and by FCT UNIT LEAF (UID/AGR/04129/2020)info:eu-repo/semantics/publishedVersio

Dietary antioxidants in coffee leaves: impact of botanical origin and maturity on chlorogenic acids and xanthones

Natural polyphenols are important dietary antioxidants that significantly benefit human health. Coffee and tea have been shown to largely contribute to the dietary intake of these antioxidants in several populations. More recently, the use of coffee leaves to produce tea has become a potential commercial target, therefore prompting studies on the quantification of polyphenols in coffee leaves. In this study a variety of coffee leaf species, at different development stages, were analyzed using ultra-high pressure liquid chromatography. The results demonstrate that both the botanical origin of the samples and their maturity influence significantly the concentration of the antioxidants; for total chlorogenic acids a two-fold difference was found between different species and up to a three-fold variation was observed between young and mature leaves. Furthermore, the range of concentrations of chlorogenic acids in young leaves (35.7–80.8 mg/g of dry matter) were found to be comparable to the one reported for green coffee beans. The results provide important data from which potential new commercial products can be developedinfo:eu-repo/semantics/publishedVersio

Evaluation of virus-induced gene silencing (VIGS) in coffee plants

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Exploring the role of sugars in the Kawisari coffee resistance to Hemileia vastatrix

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

First report of a genome sequence resource of Colletotrichum kahawae, the causal agent of coffee berry disease

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Assessment of carbon metabolism of Coffee Kawisari hybrid challenged by Hemileia vastatrix, the causal agent of Coffee Leaf Rust.

Plants have evolved sophisticated mechanisms to coordinate carbon metabolism during growth and development both under optimal and stress conditions. In coffee-rust biotrophic interactions, plants try to limit pathogen access to nutrients (e.g., sugars and sugar derivatives) and trigger immune responses, while Hemileia vastatrix (Hv) attempts to circumvent plant defences and control the host's primary metabolism for its own benefit. Previous proteomics data highlighted the up-regulation of proteins from photosynthesis, primary metabolism, and redox-related enzymes along the coffee resistance response. Coffee Kawisari hybrid - Hv interactions (resistant and susceptible reactions) were evaluated using a single sample fractionation method for metabolite and protein extraction. The microscopic evaluation of the Hv infection process revealed that coffee resistance was associated with early hypersensitive response and accumulation of phenolic-like compounds in host cell walls. GC-TOF-MS untargeted metabolomics allowed the identification of metabolic components, such as sugars, sugar derivatives, amino acids, phenylpropanoids, chlorogenic acids, alkaloids, and fatty acids (using the Golm Metabolome database). The overrepresentation of several caffeoylquinic acids in the resistance reaction may be linked to the accumulation of the phenolic-like compounds that were cytologically observed at the infection sites. Furthermore, sugar-related features also played a role in distinguishing between resistant and susceptible reactions, such as glucose and galactose. The cellular availability of mono and disaccharides is the result of the activity of several enzymes, e.g., sucrose synthase and invertases, that can be targeted by Hv in its strategy to manipulate plant carbon metabolism. The activity profile of these enzymes along the infection will be discussed. Proteomic analysis of the same samples (using the single sample fractionation method previously mentioned) is foreseen. The ultimate goal is to establish a connection between the metabolite and protein signatures.Financial support by the Access to Research Infrastructures, Horizon2020 Programme of the EU (EPPN2020 Grant Agreement 731013) and Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES (PTDC/ASP- PLA/29779/2017), UNIT LEAF (UID/AGR/04129/2020), UCIBIO (UIDP/04378/2020; UIDB/04378/ 2020) and i4HB (LA/P/0140/2020).N/

Integrative transcriptomic and metabolomic approaches to unravel the resistance profile of Kawisari coffee against Hemileia vastatrix

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Characterization of the transcriptional signatures associated with resistance and susceptibility to Hemileia vastatrix in the Kawisari coffee hybrid

Coffee leaf rust (CLR), a disease caused by the biotrophic fungus Hemileia vastatrix (Hv), is the main threat to the worldwide production of Arabica coffee. The gradual breakdown of resistance in coffee varieties in the last years has highlighted the need for novel sources of resistance to CLR. This work aimed to unveil the cellular and molecular resistance profile of the Kawisari hybrid (C. arabica x C. liberica), a genotype used as a resistance donor in Arabica breeding programs in India. This coffee genotype was inoculated with two Hv races that triggered either resistance or susceptibility. Progress of infection was monitored using light microscopy. Simultaneously, we conducted a time-course RNA-seq characterization of the transcriptional responses. The microscopic studies showed that the post-haustorial resistance of Kawisari was associated with the hypersensitive response, accumulation of phenolic-like compounds and haustorium encasement with callose. The transcriptomic analysis suggest the downregulation of host primary metabolism genes at the early onset of infection, followed later by activation of genes functionally associated with multiple plant defense responses, including salicylic acid and jasmonate hormonal signaling. Resistance was also accompanied by the differential regulation of genes associated with phenylpropanoid metabolism and lignin biosynthesis. Our results, further validated by qPCR, provide important new insight into the molecular mechanisms underpinning resistance against CLR in this coffee genotype.Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES PTDC/ASPPLA/ 29779/2017 and by FCT UNIT LEAF (UID/AGR/04129/2020).info:eu-repo/semantics/publishedVersio