Evaluation of antioxidant, organic acid, and volatile compounds in coffee pulp wine fermented with native yeasts isolated from coffee cherries.

In this study, coffee pulp was examined as a starting material to make alcoholic beverages (coffee pulp wine) and yeast fermentation ability. We have evaluated five yeasts, three of which were previously isolated from the coffee cherry, and the other two were commercial yeasts. The pH, °Brix, viable yeast cells, and color parameters of coffee pulp wines were measured. The antioxidant activity of coffee pulp wine were measured using the 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays. Relatively, the 2,2-diphenyl-1-picrylhydrazyl inhibition percentage of Saccharomycopsis fibuligera (strain KNU18Y4) fermented coffee pulp wine was higher than that of other yeasts. Coffee pulp wine fermented with Saccharomyces cerevisiae (strain Fermivin) had higher ferric reducing antioxidant power values. Coffee pulp wine fermented with S. fibuligera (strain KNU18Y4) produced higher total phenolic content and total flavonoid content. Coffee pulp wine fermented with S. cerevisiae (strain KNU18Y12) had lower total tannin content compared to other treatments. The citric and malic acid contents were higher in coffee pulp wine fermented with S. cerevisiae (strain Fermivin). On the other hand, high lactic and acetic acid produced, with coffee pulp wine fermented with S. fibuligera (strain KNU18Y4). Ethyl alcohol was the most abundant volatile compound found in all treatments.

Genome analysis of Hibiscus syriacus provides insights of polyploidization and indeterminate flowering in woody plants.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Plant Translation Elongation Factor 1Bß Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bß). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bß- or eEF1BÉ£-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bß interacted with eEF1A and that eEF1A and eEF1Bß interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bß play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bß deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bß are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bß is involved in the interaction with eEF1A. These results suggest that eEF1Bß could be a potential target for engineering virus-resistant plants.

Use of a secretion trap screen in pepper following Phytophthora capsici infection reveals novel functions of secreted plant proteins in modulating cell death.

In plants, the primary defense against pathogens is mostly inducible and associated with cell wall modification and defense-related gene expression, including many secreted proteins. To study the role of secreted proteins, a yeast-based signal-sequence trap screening was conducted with the RNA from Phytophthora capsici-inoculated root of Capsicum annuum 'Criollo de Morelos 334' (CM334). In total, 101 Capsicum annuum secretome (CaS) clones were isolated and identified, of which 92 were predicted to have a secretory signal sequence at their N-terminus. To identify differences in expressed CaS genes between resistant and susceptible cultivars of pepper, reverse Northern blots and real-time reverse-transcription polymerase chain reaction were performed with RNA samples isolated at different time points following P. capsici inoculation. In an attempt to assign biological functions to CaS genes, we performed in planta knock-down assays using the Tobacco rattle virus-based gene-silencing method. Silencing of eight CaS genes in pepper resulted in suppression of the cell death induced by the non-host bacterial pathogen (Pseudomonas syringae pv. tomato T1). Three CaS genes induced phenotypic abnormalities in silenced plants and one, CaS259 (PR4-l), caused both cell death suppression and perturbed phenotypes. These results provide evidence that the CaS genes may play important roles in pathogen defense as well as developmental processes.