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MAIN CONCLUSION: This review provides valuable insights into plant molecular regulatory mechanisms during fungus attacks, highlighting potential miRNA candidates for future disease management. Plant defense responses to biotic stress involve intricate regulatory mechanisms, including post-transcriptional regulation of genes mediated by microRNAs (miRNAs). These small RNAs play a vital role in the plant's innate immune system, defending against viral, bacterial, and fungal attacks. Among the plant pathogenic fungi, Colletotrichum spp. are notorious for causing anthracnose, a devastating disease affecting economically important crops worldwide. Understanding the molecular machinery underlying the plant immune response to Colletotrichum spp. is crucial for developing tools to reduce production losses. In this comprehensive review, we examine the current understanding of miRNAs associated with plant defense against Colletotrichum spp. We summarize the modulation patterns of miRNAs and their respective target genes. Depending on the function of their targets, miRNAs can either contribute to host resistance or susceptibility. We explore the multifaceted roles of miRNAs during Colletotrichum infection, including their involvement in R-gene-dependent immune system responses, hormone-dependent defense mechanisms, secondary metabolic pathways, methylation regulation, and biosynthesis of other classes of small RNAs. Furthermore, we employ an integrative approach to correlate the identified miRNAs with various strategies and distinct phases of fungal infection. This study provides valuable insights into the current understanding of plant miRNAs and their regulatory mechanisms during fungus attacks.

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Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic factors is very relevant to help in understanding the processes of local adaptation. 1-Pyrroline-5-carboxylate synthetase (P5CS) is one interesting gene to study in this species since it encodes a key enzyme of proline biosynthesis, which is an osmoprotectant during abiotic stress. Applying in silico analysis, we identified one P5CS gene sequence of E. uniflora (EuniP5CS). Phylogenetic analysis, as well as, gene and protein structure investigation, revealed that EuniP5CS is a member of P5CS gene family. Plants of E. uniflora from two distinct environments (restinga and riparian forest) presented differences in the proline accumulation and P5CS expression levels under growth-controlled conditions. Both proline accumulation and gene expression level of EuniP5CS were higher in the genotypes from riparian forest than those from restinga. When these plants were submitted to drought stress, EuniP5CS gene was up-regulated in the plants from restinga, but not in those from riparian forest. These results demonstrated that EuniP5CS is involved in proline biosynthesis in this species and suggest that P5CS gene may be an interesting candidate gene in future studies to understand the processes of local adaptation in E. uniflora.

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Triacylglycerols (TAG) are the major form of energy storage in plants. TAG are primarily stored in seeds and fruits, but vegetative tissues also possess a high capacity for their synthesis and storage. These storage lipids are essential to plant development, being used in seedling growth during germination, pollen development, and sexual reproduction, for example. TAG are also an important source of edible oils for animal and human consumption, and are used for fuel and industrial feedstocks. The canonical pathway leading to TAG synthesis is the glycerol-3-phosphate, or Kennedy, pathway, which is an evolutionarily conserved process in most living organisms. The enzymatic machinery for synthesizing TAG is well known in several plant species, and the genes encoding these enzymes have been the focus of many studies. Here, we review recent progress on the understanding of evolutionary, functional and biotechnological aspects of the glycerol-3-phosphate pathway enzymes that produce TAG. We discuss current knowledge about their functional aspects, and summarize valuable insights into genetically engineered plants for enhancing TAG accumulation. Also, we highlight the evolutionary history of these genes and present a meta-analysis linking positive selection to gene family and plant diversification, and also to the domestication processes in oilseed crops.

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sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is an important enzyme that catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 or sn-2 position of sn-glycerol-3-phosphate (G3P) to generate lysophosphatidic acids (LPAs). The functional studies of GPAT in plants demonstrated its importance in controlling storage and membrane lipid. Identifying genes encoding GPAT in a variety of plant species is crucial to understand their involvement in different metabolic pathways and physiological functions. Here, we performed genome-wide and evolutionary analyses of GPATs in plants. GPAT genes were identified in all algae and plants studied. The phylogenetic analysis showed that these genes group into three main clades. While clades I (GPAT9) and II (soluble GPAT) include GPATs from algae and plants, clade III (GPAT1-8) includes GPATs specific from plants that are involved in the biosynthesis of cutin or suberin. Gene organization and the expression pattern of GPATs in plants corroborate with clade formation in the phylogeny, suggesting that the evolutionary patterns is reflected in their functionality. Overall, our results provide important insights into the evolution of the plant GPATs and allowed us to explore the evolutionary mechanism underlying the functional diversification among these genes.

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Abstract sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is an important enzyme that catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 or sn-2 position of sn-glycerol-3-phosphate (G3P) to generate lysophosphatidic acids (LPAs). The functional studies of GPAT in plants demonstrated its importance in controlling storage and membrane lipid. Identifying genes encoding GPAT in a variety of plant species is crucial to understand their involvement in different metabolic pathways and physiological functions. Here, we performed genome-wide and evolutionary analyses of GPATs in plants. GPAT genes were identified in all algae and plants studied. The phylogenetic analysis showed that these genes group into three main clades. While clades I (GPAT9) and II (soluble GPAT) include GPATs from algae and plants, clade III (GPAT1-8) includes GPATs specific from plants that are involved in the biosynthesis of cutin or suberin. Gene organization and the expression pattern of GPATs in plants corroborate with clade formation in the phylogeny, suggesting that the evolutionary patterns is reflected in their functionality. Overall, our results provide important insights into the evolution of the plant GPATs and allowed us to explore the evolutionary mechanism underlying the functional diversification among these genes.

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Since the first diacylglycerol acyltransferase (DGAT) gene was characterized in plants, a number of studies have focused on understanding the role of DGAT activity in plant triacylglycerol (TAG) biosynthesis. DGAT enzyme is essential in controlling TAGs synthesis and is encoded by different genes. DGAT1 and DGAT2 are the two major types of DGATs and have been well characterized in many plants. On the other hand, the DGAT3 and WS/DGAT have received less attention. In this study, we present the first general view of the presence of putative DGAT3 and WS/DGAT in several plant species and report on the diversity and evolution of these genes and its relationships with the two main DGAT genes (DGAT1 and DGAT2). According to our analyses DGAT1, DGAT2, DGAT3 and WS/DGAT are very divergent genes and may have distinct origin in plants. They also present divergent expression patterns in different organs and tissues. The maintenance of several types of genes encoding DGAT enzymes in plants demonstrates the importance of DGAT activity for TAG biosynthesis. Evolutionary history studies of DGATs coupled with their expression patterns help us to decipher their functional role in plants, helping to drive future biotechnological studies.

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Circular RNAs (circRNAs) are a class of non-coding RNAs (ncRNAs) that are involved in transcriptional and posttranscriptional gene expression regulation. The development of deep sequencing of ribosomal RNA (rRNA)-depleted RNA libraries, associated with improved computational tools, has provided the identification of several new circRNAs in all sorts of organisms, from protists, plants and fungi to animals. Recently, it was discovered that endogenous circRNAs can work as microRNA (miRNA) sponges. This means that the circRNAs bind to miRNAs and consequently repress their function, providing a new model of action for this class of ncRNA, as well as indicating another mechanism that regulates miRNA activity. As miRNAs control a large set of biological processes, circRNA sponge activity will also affect these pathways. Several studies have associated miRNA sponges with human diseases, including osteoarthritis, diabetes, neurodegenerative pathologies and several types of cancer. Additionally, high stability, abundance and tissue-specific expression patterns make circRNA sponges very attractive for clinical research. Herein, we review the biogenesis, properties and function of endogenous circRNA sponges, with a special focus on those related to human cancer. A list of web tools available for the study of circRNAs is also given. Additionally, we discuss the possibility of using circRNAs as molecular markers for the diagnosis of diseases.

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Lysophosphatidic acid acyltransferases (LPAATs) perform an essential cellular function by controlling the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane, signaling and storage lipids. Although LPAATs have been extensively explored by functional and biotechnological studies, little is known about their molecular evolution and diversification. We performed a genome-wide analysis using data from several plants and animals, as well as other eukaryotic and prokaryotic species, to identify LPAAT genes and analyze their evolutionary history. We used phylogenetic and molecular evolution analysis to test the hypothesis of distinct origins for these genes. The reconstructed phylogeny supported the ancient origin of some isoforms (plant LPAAT1 and LPAATB; animal AGPAAT1/2), while others emerged more recently (plant LPAAT2/3/4/5; AGPAAT3/4/5/8). Additionally, the hypothesis of endosymbiotic origin of the plastidic isoform LPAAT1 was confirmed. LPAAT genes from plants and animals mainly experienced strong purifying selection pressures with limited functional divergence after the species-specific duplications. Gene expression analyses of LPAAT isoforms in model plants demonstrated distinct LPAAT expression patterns in these organisms. The results showed that distinct origins followed by diversification of the LPAAT genes shaped the evolution of TAG biosynthesis. The expression pattern of individual genes may be responsible for adaptation into multiple ecological niches.

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The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.

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Pitanga (Eugenia uniflora L.) is a member of the Myrtaceae family and is of particular interest due to its medicinal properties that are attributed to specialized metabolites with known biological activities. Among these molecules, terpenoids are the most abundant in essential oils that are found in the leaves and represent compounds with potential pharmacological benefits. The terpene diversity observed in Myrtaceae is determined by the activity of different members of the terpene synthase and oxidosqualene cyclase families. Therefore, the aim of this study was to perform a de novo assembly of transcripts from E. uniflora leaves and to annotation to identify the genes potentially involved in the terpenoid biosynthesis pathway and terpene diversity. In total, 72,742 unigenes with a mean length of 1048bp were identified. Of these, 43,631 and 36,289 were annotated with the NCBI non-redundant protein and Swiss-Prot databases, respectively. The gene ontology categorized the sequences into 53 functional groups. A metabolic pathway analysis with KEGG revealed 8,625 unigenes assigned to 141 metabolic pathways and 40 unigenes predicted to be associated with the biosynthesis of terpenoids. Furthermore, we identified four putative full-length terpene synthase genes involved in sesquiterpenes and monoterpenes biosynthesis, and three putative full-length oxidosqualene cyclase genes involved in the triterpenes biosynthesis. The expression of these genes was validated in different E. uniflora tissues.

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A large number of small RNAs unrelated to the soybean genome were identified after deep sequencing of soybean small RNA libraries. A metatranscriptomic analysis was carried out to identify the origin of these sequences. Comparative analyses of small interference RNAs (siRNAs) present in samples collected in open areas corresponding to soybean field plantations and samples from soybean cultivated in greenhouses under a controlled environment were made. Different pathogenic, symbiotic and free-living organisms were identified from samples of both growth systems. They included viruses, bacteria and different groups of fungi. This approach can be useful not only to identify potentially unknown pathogens and pests, but also to understand the relations that soybean plants establish with microorganisms that may affect, directly or indirectly, plant health and crop production.

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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a robust and widely applied technique used to investigate gene expression. However, for correct analysis and interpretation of results, the choice of a suitable gene to use as an internal control is a crucial factor. These genes, such as housekeeping genes, should have a constant expression level in different tissues and across different conditions. The advances in genome sequencing have provided high-throughput gene expression analysis and have contributed to the identification of new genes, including microRNAs (miRNAs). The miRNAs are fundamental regulatory genes of eukaryotic genomes, acting on several biological functions. In this study, miRNA expression stability was investigated in different soybean tissues and genotypes as well as after abiotic or biotic stress treatments. The present study represents the first investigation into the suitability of miRNAs as housekeeping genes in plants. The transcript stability of 10 miRNAs was compared to those of six previously reported housekeeping genes for the soybean. In this study, we provide evidence that the expression stabilities of miR156b and miR1520d were the highest across the soybean experiments. Furthermore, these miRNAs genes were more stable than the most commonly protein-coding genes used in soybean gene expression studies involving RT-qPCR.

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