Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb

The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs. (Résumé d'auteur

Selection of reference genes for expression Study in pulp and seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality—selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality. (Résumé d'auteur

An issue of concern: unique truncated ORF8 protein variants of SARS-CoV-2

Open reading frame 8 (ORF8) shows one of the highest levels of variability among accessory proteins in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits the presentation of viral antigens by the major histocompatibility complex class I (MHC-I), which interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 in evading immunity and plays a role in SARS-CoV-2 replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein, defines the B.1.1.7 lineage of SARS-CoV-2, engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) with the Q27STOP mutations were identified among 49,055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents, which include Africa, Asia, Europe and South America. Based on various quantitative features, such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, nine possible T-ORF8 unique variants were defined. The question as to whether T-ORF8 variants function similarly to the wild type ORF8 is yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures

HVA22 from citrus: A small gene family whose some members are involved in plant response to abiotic stress

International audienceThe HVA22 gene has been isolated for the first time from the aleurone layer of barley (Hordeum vulgare). Here, we characterized the HVA22 family from citrus (C. clementina and C. sinensis). Twelve genes, 6 in each species, were identified as well as duplication events for some of them. The ORF size ranged from 235 to 804 bp and the protein molecular weight from 94 to 267 kDa. All the citrus HVA22 protein presented transmembrane location and conserved TB2/DP1/HVA22 region. Phylogenetic and gene expression analyses suggested that some citrus HVA22 play a role in flower and fruit development, and that gene expression may be regulated by hormone or environmental conditions. Other regulation levels were also predicted, such as alternative splicing and post translational modifications. The overall data indicated that citrus HVA22 may be involved in vesicular traffic in stressed cells, and that CcHVA22d could be involved in dehydration tolerance

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa

International audienceThe cupuassu tree (Theobroma grandiflorwn) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu-M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin

Cupuassu (<i>Theobroma grandiflorum</i> [Willd. ex Sprengel] Schumann) Fruit Development: Key Genes Involved in Primary Metabolism and Stress Response

Cupuassu (Theobroma grandiflorum [Willd. ex Sprengel] Schumann) seeds constitute the raw material for oil extraction and fabrication of cupulate (product similar to chocolate). However, fungal diseases such as witches’ broom caused by Moniliophthora perniciosa have interfered with the large-scale development of cupuassu plantations. Cupuassu genetic breeding programmes focus on a variety of biotechnological tools or approaches to select genes related to quality or resistance mechanisms. In this study, we used expression and interactomics analyses of preselected genes involved in fruit quality and/or resistance to better understand the molecular and physiological mechanisms associated with these plant processes. It was found that (i) resistant and susceptible cupuassu genotypes showed different pulp characteristics as well as gene expression patterns; (ii) monosaccharide and carbohydrate transport pathways were enhanced during fruit maturation; (iii) sugar accumulation participated in signal transduction associated with fruit development and stress response in maturing fruits; and (iv) maturing pulp and seeds showed increased phospholipid metabolism and translocation, as well as immune system activation. The TgSTP1, TgWRKY33, TgCZF1, and TgUBA1 genes in cupuassu and the orthologues of DIN10, CNI1, and TET8 identified by the interactomics approach may be good candidates for marker-assisted selection in breeding programmes focusing on both fruit quality and resistance/tolerance to biotic/abiotic stress