Genome and Transcriptome Analyses of Genes Involved in Ascorbate Biosynthesis in Pepper Indicate Key Genes Related to Fruit Development, Stresses, and Phytohormone Exposures.

Pepper (Capsicum annuum L.) is a vegetable consumed worldwide, primarily used for vitamin C uptake and condiment purposes. Ascorbate (Asc) is a multifunctional metabolite, acting as an antioxidant and enzymatic cofactor involved in multiple cellular processes. Nevertheless, there is no evidence about the contribution of biosynthesis pathways and regulatory mechanisms responsible for Asc reserves in pepper plants. Here, we present a genome- and transcriptome-wide investigation of genes responsible for Asc biosynthesis in pepper during fruit development, stresses, and phytohormone exposures. A total of 21 genes, scattered in ten of twelve pepper chromosomes were annotated. Gene expression analyses of nine transcriptomic experiments supported the primary role of the L-galactose pathway in the Asc-biosynthesizing process, given its constitutive, ubiquitous, and high expression profile observed in all studied conditions. However, genes from alternative pathways generally exhibited low expression or were unexpressed and appeared to play some secondary role under specific stress conditions and phytohormone treatments. Taken together, our findings provide a deeper spatio-temporal understanding of expression levels of genes involved in Asc biosynthesis, and they highlight GGP2, GME1 and 2, and GalLDH members from L-galactose pathway as promising candidates for future wet experimentation, addressing the attainment of increase in ascorbate content of peppers and other crops.

Transcriptome profiling of cashew apples (Anacardium occidentale) genotypes reveals specific genes linked to firmness and color during pseudofruit development.

KEY MESSAGE: We found 34 and 71 key genes potentially involved in flavonoid biosynthesis and cell wall disassembly, respectively, which could be associated with specific peel coloration and softening of each genotype. Cashew apple (Anacardium occidentale) has a great economic importance worldwide due to its high nutritional value, peculiar flavor and aroma. During ripening, the peduncle develops different peel color and becomes quickly fragile due to its oversoftening, impacting its consumers' acceptance. In view of this, the understanding about its transcriptional dynamics throughout ripening is imperative. In this study, we performed a transcriptome sequencing of two cashew apple genotypes (CCP 76 and BRS 265), presenting different firmness and color peel, in the immature and ripe stages. Comparative transcriptome analysis between immature and ripe cashew apple revealed 4374 and 3266 differentially expressed genes (DEGs) to CCP 76 and BRS 265 genotypes, respectively. These genes included 71 and 34 GDEs involved in the cell wall disassembly and flavonoid biosynthesis, respectively, which could be associated with firmness loss and anthocyanin accumulation during cashew apple development. Then, softer peduncle of CCP 76 could be justified by down-regulated EXP and up-regulation of genes involved in pectin degradation (PG, PL and PAE) and in cell wall biosynthesis. Moreover, genes related to flavonoid biosynthesis (PAL, C4H and CHS) could be associated with early high accumulation of anthocyanin in red-peel peduncle of BRS 265. Finally, expression patterns of the selected genes were tested by real-time quantitative PCR (qRT-PCR), and the qRT-PCR results were consistent with transcriptome data. The information generated in this work will provide insights into transcriptome responses to cashew apple ripening and hence, it will be helpful for cashew breeding programs aimed at developing genotypes with improved quality traits.

Genome-wide identification of ascorbate-glutathione cycle gene families in soybean (Glycine max) reveals gene duplication events and specificity of gene members linked to development and stress conditions.

Ascorbate-glutathione (AsA-GSH) cycle plays an important role in tuning beneficial ROS accumulation for intracellular signals and imparts plant tolerance to oxidative stress by detoxifying excess of ROS. Here, we present genome-wide identification of AsA-GSH cycle genes (APX, MDHAR, DHAR, and GR) in several leguminous species and expression analyses in G. max during stress, germination and tissue development. Our data revealed 24 genes in Glycine genus against the maximum of 15 in other leguminous species, which was due to 9 pars of duplicated genes mostly originated from sub/neofunctionalization. Cytosolic APX and MDHAR genes were highly expressed in different tissues and physiological conditions. Germination induced genes encoding AsA-GSH proteins from different cell compartments, whereas vegetative phase (leaves) stimulated predominantly genes related to chloroplast/mitochondria proteins. Moreover, cytosolic APX-1, 2, MDHAR-1a, 1b and GR genes were the primary genes linked to senescence and biotic stresses, while stAPX-a, b and GR (from organelles) were the most abiotic stress related genes. Biotic and abiotic stress tolerant genotypes generally showed increased MDHAR, DHAR and/or GR mRNA levels compared to susceptible genotypes. Overall, these data clarified evolutionary events in leguminous plants and point to the functional specificity of duplicated genes of the AsA-GSH cycle in G. max.

Complete Chromosome-Scale Genome Sequence Resource for Sporisorium panici-leucophaei, the Causal Agent of Sourgrass Smut Disease.

Here, we present the first complete chromosome-level genome assembly of the smut fungus strain Sporisorium panici-leucophaei SPL10A, the causal agent of the sourgrass (Digitaria insularis) smut disease. Combining Illumina paired-end and Nanopore long reads, we generated a final assembly composed of 23 chromosomes (22 nuclear and one mitochondrial) with 18,915,934 bp. Gene prediction accomplished using extrinsic evidence from the sugarcane smut fungus Sporisorium scitamineum originated a total of 6,402 protein-encoding genes. The secretome (388 proteins) and the effectorome repertoires (68 candidates) were also predicted, given their crucial roles in plant-pathogen interactions. The complete telomere-to-telomere chromosome sequences of this poorly studied fungus will provide a valuable resource for future comparative genomic studies among smuts to unravel their underlying pathogenicity mechanisms.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification and evaluation of reference genes for reliable normalization of real-time quantitative PCR data in acerola fruit, leaf, and flower.

Understanding into acerola (Malpighia emarginata) molecular and biochemical bases is still obscure, despite it is one of the most important natural source of vitamin C for humans. Recently, our research group published the first data on acerola transcriptome generating valuable information to identify reference genes for RT-qPCR in this species. Hence, this study aimed to identify the most stably expressed genes based on acerola transcriptome data, and further to evaluate the suitability of F-box, U3, Merad50-ATPase, TGD4, NOB1, PA-RNA, RCC1, RBL and PGAL candidates for accurate gene expression normalization in leaf, flower and fruit at 12, 16 and 20 days after anthesis using RT-qPCR analysis. Three algorithms, geNorm, NormFinder, and BestKeeper confirmed the expression stability of all nine candidate reference genes, whereas RefFinder consensually summarized a comprehensive gene ranking. Based on geNorm, the combination of the most stable reference genes RBL and U3 for leaf/flower group, TGD4, F-box and PGAL (fruit developmental stages or fruit/leaf), RCC1, PGAL and RBL (fruit/flower) and RCC1, RBL, TGD4 and PGAL (total samples) were required for accurate normalization. Moreover, the use of these reference genes to assess the expression profile of GMP1 and NAT3 genes confirmed the reliability of ranking and defined the best combination of genes recommended by geNorm and RefFinder. This work will benefit further RT-qPCR studies in these acerola organs by offering a foundation for accurate normalization of gene expression profiling.

Differential expression of recently duplicated PTOX genes in Glycine max during plant development and stress conditions.

Plastid terminal oxidase (PTOX) is a chloroplast enzyme that catalyzes oxidation of plastoquinol (PQH2) and reduction of molecular oxygen to water. Its function has been associated with carotenoid biosynthesis, chlororespiration and environmental stress responses in plants. In the majority of plant species, a single gene encodes the protein and little is known about events of PTOX gene duplication and their implication to plant metabolism. Previously, two putative PTOX (PTOX1 and 2) genes were identified in Glycine max, but the evolutionary origin and the specific function of each gene was not explored. Phylogenetic analyses revealed that this gene duplication occurred apparently during speciation involving the Glycine genus ancestor, an event absent in all other available plant leguminous genomes. Gene expression evaluated by RT-qPCR and RNA-seq data revealed that both PTOX genes are ubiquitously expressed in G. max tissues, but their mRNA levels varied during development and stress conditions. In development, PTOX1 was predominant in young tissues, while PTOX2 was more expressed in aged tissues. Under stress conditions, the PTOX transcripts varied according to stress severity, i.e., PTOX1 mRNA was prevalent under mild or moderate stresses while PTOX2 was predominant in drastic stresses. Despite the high identity between proteins (97%), molecular docking revealed that PTOX1 has higher affinity to substrate plastoquinol than PTOX2. Overall, our results indicate a functional relevance of this gene duplication in G. max metabolism, whereas PTOX1 could be associated with chloroplast effectiveness and PTOX2 to senescence and/or apoptosis.

Transcriptome analysis of acerola fruit ripening: insights into ascorbate, ethylene, respiration, and softening metabolisms.

KEY MESSAGE: The first transcriptome coupled to metabolite analyses reveals major trends during acerola fruit ripening and shed lights on ascorbate, ethylene signalling, cellular respiration, sugar accumulation, and softening key regulatory genes. Acerola is a fast growing and ripening fruit that exhibits high amounts of ascorbate. During ripening, the fruit experience high respiratory rates leading to ascorbate depletion and a quickly fragile and perishable state. Despite its growing economic importance, understanding of its developmental metabolism remains obscure due to the absence of genomic and transcriptomic data. We performed an acerola transcriptome sequencing that generated over 600 million reads, 40,830 contigs, and provided the annotation of 25,298 unique transcripts. Overall, this study revealed the main metabolic changes that occur in the acerola ripening. This transcriptional profile linked to metabolite measurements, allowed us to focus on ascorbate, ethylene, respiration, sugar, and firmness, the major metabolism indicators for acerola quality. Our results suggest a cooperative role of several genes involved in AsA biosynthesis (PMM, GMP1 and 3, GME1 and 2, GGP1 and 2), translocation (NAT3, 4, 6 and 6-like) and recycling (MDHAR2 and DHAR1) pathways for AsA accumulation in unripe fruits. Moreover, the association of metabolites with transcript profiles provided a comprehensive understanding of ethylene signalling, respiration, sugar accumulation and softening of acerola, shedding light on promising key regulatory genes. Overall, this study provides a foundation for further examination of the functional significance of these genes to improve fruit quality traits.

Polymorphisms in plastoquinol oxidase (PTOX) from Arabidopsis accessions indicate SNP-induced structural variants associated with altitude and rainfall.

Plant plastoquinol oxidase (PTOX) is a chloroplast oxidoreductase involved in carotenoid biosynthesis, chlororespiration, and response to environmental stresses. The present study aimed to gain insight of the potential role of nucleotide/amino acid changes linked to environmental adaptation in PTOX gene/protein from Arabidopsis thaliana accessions. SNPs in the single-copy PTOX gene were identified in 1190 accessions of Arabidopsis using the Columbia-0 PTOX as a reference. The identified SNPs were correlated with geographical distribution of the accessions according to altitude, climate, and rainfall. Among the 32 identified SNPs in the coding region of the PTOX gene, 16 of these were characterized as non-synonymous SNPs (in which an AA is altered). A higher incidence of AA changes occurred in the mature protein at positions 78 (31%), 81 (31.4%), and 323 (49.9%). Three-dimensional structure prediction indicated that the AA change at position 323 (D323N) leads to a PTOX structure with the most favorable interaction with the substrate plastoquinol, when compared with the reference PTOX structure (Columbia-0). Molecular docking analysis suggested that the most favorable D323N PTOX-plastoquinol interaction is due to a better enzyme-substrate binding affinity. The molecular dynamics revealed that plastoquinol should be more stable in complex with D323N PTOX, likely due a restraint mechanism in this structure that stabilize plastoquinol inside of the reaction center. The integrated analysis made from accession geographical distribution and PTOX SNPs indicated that AA changes in PTOX are related to altitude and rainfall, potentially due to an adaptive positive environmental selection.

Procedures of Mitochondria Purification and Gene Expression to Study Alternative Respiratory and Uncoupling Pathways in Fruits.

We describe detailed procedures to get intact and well-coupled mitochondria from a variety of fruit species such as papaya (Carica papaya), guava (Psidium guajava), tomato (Solanum lycopersicum), and strawberry (Fragaria x ananassa) as well as the protocols to assess the capacities of AOX and UCP pathways in intact fruit mitochondria. The procedures presented here were tested for the species mentioned above; their use with other types of fruits must be tested for yield of intact and active mitochondria. This is possible from individual adjustments. Strict care during extraction, including the use of osmotic protectants (i.e., mannitol/sucrose) and antioxidants (i.e., cysteine, ascorbate) at defined concentrations, are critical factors to ensure mitochondrial integrity and to obtain higher yields. The mitochondria purified using the discontinuous Percoll gradients described here can be used for the analysis of the capacity of alternative respiration and uncoupling pathways in fruits. In addition, protocols for quantitative and semiquantitative PCR applicable for the analysis of AOX and UCP gene expression in fruits are shown. Microarray and RNA-seq data from public databases are also valuable for the analysis of AOX and UCP genes. In both cases having the sequences of genes or cDNAs to be used in primer design or probe identification is necessary.

A Step-by-Step Protocol for Classifying AOX Proteins in Flowering Plants.

The potential of alternative oxidase (AOX) genes to develop functional markers for plant breeding programs has been emphasized. In this sense, it is essential to have a reliable classification system, which could aid in the selection of candidate AOX genes from different species. In the case of angiosperms AOX, a robust classification system is required because this enzyme is encoded by variable gene numbers (1-6 genes) with variable AOX subfamilies and subtypes. Thus, in this protocol, we present a detailed guideline to application of a classification scheme of AOX based on specific amino acids and phylogeny. We believe that this classification protocol provides an easier and practical way of classifying new angiosperm AOX genes besides that it can help to standardize AOX gene names used in AOX research community.

Phylogenetic analysis and differential expression of EF1α genes in soybean during development, stress and phytohormone treatments.

The EF1α is a multifunctional protein with additional unrelated activities to its primary function in translation. This protein is encoded by a multigene family and few studies are still available in plants. Expression of six EF1α genes in Glycine max was performed using RT-qPCR and RNA-seq data to advance in the function of each gene during plant development, stress conditions and phytohormone treatments. A phylogenetic classification in Phaseoleae tribe was used to identify the G. max EF1α genes (EF1α 1a1, 1a2, 1b, 2a, 2b and 3). Three EF1α types (1-3) were found in Phaseoleae revealing duplications in G. max types 1 and 2. EF1α genes were expressed in all studied tissues, however, specific amount of each transcript was detected. In plant development, all EF1α transcripts were generally more expressed in younger tissues, however, in unifoliolate leaves and cotyledons a higher expression occurred in older tissues. Five EF1α genes (except 2a) were up-regulated under stress in a response tissue/stress/cultivar-dependent. EF1α 3 was the most stress-induced gene linked to cultivar stress tolerance mainly in aerial tissues. Auxin, salicylate and ethylene induced differentially the EF1α expression. Overall, this study provides a consistent EF1α classification in Phaseoleae tribe to better understand their functional evolution. The RT-qPCR and RNA-seq EF1α expression profiles were consistent, both exhibiting expression diversification of each gene (spatio-temporal, stress and phytohormone stimuli). Our results point out the EF1α genes, especially EF1α 3, as candidate for developing a useful tool for future G. max breeding.