Emerging roles of plant microRNAs during Colletotrichum spp. infection.

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.

Transcriptomics analysis of Psidium cattleyanum Sabine (Myrtaceae) unveil potential genes involved in fruit pigmentation.

Psidium cattleyanum Sabine is an Atlantic Forest native species that presents some populations with red fruits and others with yellow fruits. This variation in fruit pigmentation in this species is an intriguing character that could be related to species evolution but still needs to be further explored. Our goal was to provide genomic information for these morphotypes to understand the molecular mechanisms of differences in fruit colour in this species. In this study, we performed a comparative transcriptome analysis of red and yellow morphotypes of P. cattleyanum, considering two stages of fruit ripening. The transcriptomic analysis performed encompassing leaves, unripe and ripe fruits, in triplicate for each morphotype. The transcriptome consensus from each morphotype showed 301,058 and 298,310 contigs from plants with yellow and red fruits, respectively. The differential expression revealed important genes that were involved in anthocyanins biosynthesis, such as the anthocyanidin synthase (ANS) and UDP-glucose:flavonoid-o-glucosyltransferase (UFGT) that were differentially regulated during fruit ripening. This study reveals stimulating data for the understanding of the pathways and mechanisms involved in the maturation and colouring of P. cattleyanum fruits and suggests that the ANS and UFGT genes are key factors involved in the synthase and pigmentation accumulation in red fruits.

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

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.

Enzymes of glycerol-3-phosphate pathway in triacylglycerol synthesis in plants: Function, biotechnological application and evolution.

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.

Genome-wide analysis of the Glycerol-3-Phosphate Acyltransferase (GPAT) gene family reveals the evolution and diversification of plant GPATs.

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.

Genome-wide analysis of the Glycerol-3-Phosphate Acyltransferase (GPAT) gene family reveals the evolution and diversification of plant GPATs

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.

Unveiling Chloroplast RNA Editing Events Using Next Generation Small RNA Sequencing Data.

Organellar RNA editing involves the modification of nucleotide sequences to maintain conserved protein functions, mainly by reverting non-neutral codon mutations. The loss of plastid editing events, resulting from mutations in RNA editing factors or through stress interference, leads to developmental, physiological and photosynthetic alterations. Recently, next generation sequencing technology has generated the massive discovery of sRNA sequences and expanded the number of sRNA data. Here, we present a method to screen chloroplast RNA editing using public sRNA libraries from Arabidopsis, soybean and rice. We mapped the sRNAs against the nuclear, mitochondrial and plastid genomes to confirm predicted cytosine to uracil (C-to-U) editing events and identify new editing sites in plastids. Among the predicted editing sites, 40.57, 34.78, and 25.31% were confirmed using sRNAs from Arabidopsis, soybean and rice, respectively. SNP analysis revealed 58.2, 43.9, and 37.5% new C-to-U changes in the respective species and identified known and new putative adenosine to inosine (A-to-I) RNA editing in tRNAs. The present method and data reveal the potential of sRNA as a reliable source to identify new and confirm known editing sites.

Salt stress affects mRNA editing in soybean chloroplasts.

Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

Salt stress affects mRNA editing in soybean chloroplasts

Abstract Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

Diversity and evolution of plant diacylglycerol acyltransferase (DGATs) unveiled by phylogenetic, gene structure and expression analyses.

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.

Circular RNAs are miRNA sponges and can be used as a new class of biomarker.

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.

Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family.

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.

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1.

The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1).

NPK macronutrients and microRNA homeostasis.

Macronutrients are essential elements for plant growth and development. In natural, non-cultivated systems, the availability of macronutrients is not a limiting factor of growth, due to fast recycling mechanisms. However, their availability might be an issue in modern agricultural practices, since soil has been frequently over exploited. From a crop management perspective, the nitrogen (N), phosphorus (P), and potassium (K) are three important limiting factors and therefore frequently added as fertilizers. NPK are among the nutrients that have been reported to alter post-embryonic root developmental processes and consequently, impairs crop yield. To cope with nutrients scarcity, plants have evolved several mechanisms involved in metabolic, physiological, and developmental adaptations. In this scenario, microRNAs (miRNAs) have emerged as additional key regulators of nutrients uptake and assimilation. Some studies have demonstrated the intrinsic relation between miRNAs and their targets, and how they can modulate plants to deal with the NPK availability. In this review, we focus on miRNAs and their regulation of targets involved in NPK metabolism. In general, NPK starvation is related with miRNAs that are involved in root-architectural changes and uptake activity modulation. We further show that several miRNAs were discovered to be involved in plant-microbe symbiosis during N and P uptake, and in this way we present a global view of some studies that were conducted in the last years. The integration of current knowledge about miRNA-NPK signaling may help future studies to focus in good candidates genes for the development of important tools for plant nutritional breeding.

THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis.

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.

De novo assembly of Eugenia uniflora L. transcriptome and identification of genes from the terpenoid biosynthesis pathway.

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.

Metatranscriptomic analysis of small RNAs present in soybean deep sequencing libraries.

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.

Identification of novel soybean microRNAs involved in abiotic and biotic stresses.

BACKGROUND: Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing. RESULTS: The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families. CONCLUSIONS: Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.

The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean.

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.

Molecular phylogenetic analysis of Petunia Juss. (Solanaceae).

Representatives from 11 Petunia Jussieu species in south and southeast Brazil were compared with two Calibrachoa La Llave & Lex., one Bouchetia Dunal, and two Nierembergia Ruiz & Pav. taxa in relation to DNA molecular variability. A total of 4532 base pairs related to one nuclear, five plastidial and one mitochondrial systems was investigated. Petunia and Calibrachoa, although separated among themselves, clearly differentiate from the two other genera. Despite the fact that the Petunia species do not show marked molecular differences, they can be separated in two complexes, in good agreement with altitude data. Petunia + Calibrachoa should have diverged from other clades at about 25 million years before present.