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Understanding the evolution and the effect of plasticity in plant responses to environmental changes is crucial to combat global climate change. It is particularly interesting in species that survive in distinct environments, such as Eugenia uniflora, which thrives in contrasting ecosystems within the Atlantic Forest (AF). In this study, we combined transcriptome analyses of plants growing in nature (Restinga and Riparian Forest) with greenhouse experiments to unveil the DEGs within and among adaptively divergent populations of E. uniflora. We compared global gene expression among plants from two distinct ecological niches. We found many differentially expressed genes between the two populations in natural and greenhouse-cultivated environments. The changes in how genes are expressed may be related to the species’ ability to adapt to specific environmental conditions. The main difference in gene expression was observed when plants from Restinga were compared with their offspring cultivated in greenhouses, suggesting that there are distinct selection pressures underlying the local environmental and ecological factors of each Restinga and Riparian Forest ecosystem. Many of these genes engage in the stress response, such as water and nutrient transport, temperature, light intensity, and gene regulation. The stress-responsive genes we found are potential genes for selection in these populations. These findings revealed the adaptive potential of E. uniflora and contributed to our understanding of the role of gene expression reprogramming in plant evolution and niche adaptation.

Abstract Abiotic stresses such as nutritional imbalance, salt, light intensity, and high and low temperatures negatively affect plant growth and development. Through the course of evolution, plants developed multiple mechanisms to cope with environmental variations, such as physiological, morphological, and molecular adaptations. Epigenetic regulation, transcription factor activity, and post-transcriptional regulation operated by RNA molecules are mechanisms associated with gene expression regulation under stress. Epigenetic regulation, including histone and DNA covalent modifications, triggers chromatin remodeling and changes the accessibility of transcription machinery leading to alterations in gene activity and plant homeostasis responses. Soybean is a legume widely produced and whose productivity is deeply affected by abiotic stresses. Many studies explored how soybean faces stress to identify key elements and improve productivity through breeding and genetic engineering. This review summarizes recent progress in soybean gene expression regulation through epigenetic modifications and circRNAs pathways, and points out the knowledge gaps that are important to study by the scientific community. It focuses on epigenetic factors participating in soybean abiotic stress responses, and chromatin modifications in response to stressful environments and draws attention to the regulatory potential of circular RNA in post-transcriptional processing.

Abstract Myrtaceae is a large and species-rich family of woody eudicots, with prevalent distribution in the Southern Hemisphere. Classification and taxonomy of species belonging to this family is quite challenging, sometimes with difficulty in species identification and producing phylogenies with low support for species relationships. Most of the current knowledge comes from few molecular markers, such as plastid genes and intergenic regions, which can be difficult to handle and produce conflicting results. Based on plastid protein-coding sequences and nuclear markers, we present a topology for the phylogenetic relationships among Myrtaceae tribes. Our phylogenetic estimate offers a contrasting topology over previous analysis with fewer markers. Plastome phylogeny groups the tribes Syzygieae and Eucalypteae and individual chloroplast genes produce divergent topologies, especially among species within Myrteae tribe, but also in regard to the grouping of Syzygieae and Eucalypteae. Results are consistent and reproducible with both nuclear and organellar datasets. It confronts previous data about the deep nodes of Myrtaceae phylogeny.

Abstract Myrteae is the largest and most diverse tribe within Myrtaceae and represents the majority of its diversity in the Neotropics. Members of Myrteae hold ecological importance in tropical biomes for the provision of food sources for many animal species. Thus, due to its several roles, a growing interest has been addressed to this group. In this study, we report the sequencing and de novo assembly of the complete chloroplast (cp) genomes of six Myrteae species: Eugenia brasiliensis, E. pyriformis, E. nitida, Myrcianthes pungens, Plinia edulis and Psidium cattleianum. We characterized genome structure, gene content, and identified SSRs to detect variation within Neotropical Myrteae. The six newly sequenced plastomes exhibit a typical quadripartite structure, gene content and organization highly conserved among Myrtaceae species. Some differences in genome length, protein-coding genes and non-coding regions were found. Besides, IR boundaries present structural changes among species. Increased sequence diversity was observed in some intergenic regions, suggesting their suitability for investigating intraand interspecific genetic diversity in populational studies. These data also contribute to the improvement of taxa sampling in further phylogenetic investigations to understand Myrtaceae evolution.

Abstract RNA editing is a posttranscriptional process that changes nucleotide sequences, among which cytosine-to-uracil by a deamination reaction can revert non-neutral codon mutations. Pentatricopeptide repeat (PPR) proteins comprise a family of RNA-binding proteins, with members acting as editing trans-factors that recognize specific RNA cis-elements and perform the deamination reaction. PPR proteins are classified into P and PLS subfamilies. In this work, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform trans-factor specific protein isolation. Soybean cis-elements from these three different RNA probes show differences in respect to other species. Pulldown samples were submitted to mass spectrometry for protein identification. Among detected proteins, five corresponded to PPR proteins. More than one PPR protein, with distinct functional domains, was pulled down with each one of the RNA probes. Comparison of the soybean PPR proteins to Arabidopsis allowed identification of the closest homologous. Differential gene expression analysis demonstrated that the PPR locus Glyma.02G174500 doubled its expression under salt stress, which correlates with the increase of its potential rps14 editing. The present study represents the first identification of RNA editing trans-factors in soybean. Data also indicated that potential multiple trans-factors should interact with RNA cis-elements to perform the RNA editing.

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer’s fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

This article contains data of Social Transmission of Food Preference in an animal model of autism and the evaluation of a set of microRNA analyzed in autistic patients and animal model of autism. The analyses of the absolute consumption of two flavored food by male rats prenatally exposed to valproic acid (VPA) and treated with resveratrol (RSV), showed that VPA animals show a trend to eat less of the flavored food presented by a demonstrator rat. We also identified 13 microRNA with similar levels among rodents’ experimental groups, as well as 11 microRNA with no alterations between autistic and control subjects. Further evaluation of mechanisms of VPA and RSV actions on behavioral and molecular alterations can shed light in important biomarkers and etiological triggers of autistic spectrum disorders. Keywords: Autism, Social transmission of food preference, microRNA, Resveratrol, Translational research, Preclinical models, Valproate

The knowledge about plant miRNAs has increased exponentially, with thousands of miRNAs been reported in different plant taxa using high throughput sequencing technologies and bioinformatic tools. Nevertheless, several groups of plants remain unexplored, and the gap of knowledge about conifer miRNAs is considerable. There is no sequence or functional information available on miRNAs in Araucariaceae. This group is represented in Brazil by only one species, Araucaria angustifolia, an endangered species known as Brazilian pine. In the present study, Brazilian pine has its transcriptome explored with respect to small RNAs, representing the first description in a member of the Araucariaceae family. The screening for conserved miRNAs in Brazilian pine revealed 115 sequences of 30 miRNA families. A total of 106 precursors sequences were predicted. Forty one comprised conserved miRNAs from 16 families, whereas 65 were annotated as novel miRNAs. The comparison of Brazilian pine precursors with sRNA libraries of other five conifer species indicates that 9 out 65 novel miRNAs are conserved among gymnosperms, while 56 seems to be specific for Brazilian pine or restricted to Araucariaceae family. Analysis comparing novel Brazilian pine miRNAs precursors and Araucaria cunninghamii RNA-seq data identified seven orthologs between both species. Mature miRNA identified by bioinformatics predictions were validated using stem-loop RT-qPCR assays. The expression pattern of conserved and novel miRNAs was analyzed in five different tissues of 3-month-old Araucaria seedlings. The present study provides insights about the nature and composition of miRNAs in an Araucariaceae species, with valuable information on miRNAs diversity and conservation in this taxon.

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.

Abstract Plinia trunciflora is a Brazilian native fruit tree from the Myrtaceae family, also known as jaboticaba. This species has great potential by its fruit production. Due to the high content of essential oils in their leaves and of anthocyanins in the fruits, there is also an increasing interest by the pharmaceutical industry. Nevertheless, there are few studies focusing on its molecular biology and genetic characterization. We herein report the complete chloroplast (cp) genome of P. trunciflora using high-throughput sequencing and compare it to other previously sequenced Myrtaceae genomes. The cp genome of P. trunciflora is 159,512 bp in size, comprising inverted repeats of 26,414 bp and single-copy regions of 88,097 bp (LSC) and 18,587 bp (SSC). The genome contains 111 single-copy genes (77 protein-coding, 30 tRNA and four rRNA genes). Phylogenetic analysis using 57 cp protein-coding genes demonstrated that P. trunciflora, Eugenia uniflora and Acca sellowiana form a cluster with closer relationship to Syzygium cumini than with Eucalyptus. The complete cp sequence reported here can be used in evolutionary and population genetics studies, contributing to resolve the complex taxonomy of this species and fill the gap in genetic characterization.

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.

Competing endogenous RNAs (ceRNAs) are natural transcripts that can act as endogenous sponges of microRNAs (miRNAs), modulating miRNA action upon target mRNAs. Circular RNAs (circRNAs) are one among the various classes of ceRNAs. They are produced from a process called back-splicing and have been identified in many eukaryotes. In plants, their effective action as a miRNA sponge was not yet demonstrated. To address this question, public mRNAseq data from Argonaute-immunoprecipitation libraries (AGO-IP) of Arabidopsis thaliana flowers were used in association with a bioinformatics comparative multi-method to identify putative circular RNAs. A total of 27,812 circRNAs, with at least two reads at the back-splicing junction, were identified. Further analyses were used to select those circRNAs with potential miRNAs binding sites. As AGO forms a ternary complex with miRNA and target mRNA, targets count in AGO-IP and input libraries were compared, demonstrating that mRNA targets of these miRNAs are enriched in AGO-IP libraries. Through this work, five circRNAs that may function as miRNA sponges were identified and one of them were validated by PCR and sequencing. Our findings indicate that this post-transcriptional regulation can also occur in plants.

MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data.

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.

MicroRNAs are key regulators of eukaryotic gene expression whose fundamental role has already been identified in many cell pathways. The correct identification of miRNAs targets is still a major challenge in bioinformatics and has motivated the development of several computational methods to overcome inherent limitations of experimental analysis. Indeed, the best results reported so far in terms of specificity and sensitivity are associated to machine learning-based methods for microRNA-target prediction. Following this trend, in the current paper we discuss and explore a microRNA-target prediction method based on a random forest classifier, namely RFMirTarget. Despite its well-known robustness regarding general classifying tasks, to the best of our knowledge, random forest have not been deeply explored for the specific context of predicting microRNAs targets. Our framework first analyzes alignments between candidate microRNA-target pairs and extracts a set of structural, thermodynamics, alignment, seed and position-based features, upon which classification is performed. Experiments have shown that RFMirTarget outperforms several well-known classifiers with statistical significance, and that its performance is not impaired by the class imbalance problem or features correlation. Moreover, comparing it against other algorithms for microRNA target prediction using independent test data sets from TarBase and starBase, we observe a very promising performance, with higher sensitivity in relation to other methods. Finally, tests performed with RFMirTarget show the benefits of feature selection even for a classifier with embedded feature importance analysis, and the consistency between relevant features identified and important biological properties for effective microRNA-target gene alignment.

MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. Members of 59 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally 29 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. Assembled mRNA-seq contigs allowed for a search of putative new precursors and led to the identification of 13 novel miRNA families. Analysis of miRNA population between libraries reveals that several miRNAs and isomiRNAs have different abundance in developing stages compared to mature seeds. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comparative study of the miRNA transcriptome of mature and developing B. napus seeds and provides a basis for future research on individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus.

BACKGROUND: microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. RESULTS: Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. CONCLUSIONS: This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

Triacylglycerols (TAGs), which consist of three fatty acids bound to a glycerol backbone, are major storage lipids that accumulate in developing seeds, flower petals, pollen grains, and fruits of innumerous plant species. These storage lipids are of great nutritional and nutraceutical value and, thus, are a common source of edible oils for human consumption and industrial purposes. Two metabolic pathways for the production of TAGs have been clarified: an acyl¬ CoA-dependent pathway and an acyl-CoA-independent pathway. Lipid metabolism, specially the pathways to fatty acids and TAG biosynthesis, is relatively well understood in plants, but poorly known in algae. It is generally accepted that the basic pathways of fatty acid and TAG biosynthesis in algae are analogous to those of higher plants. However, unlike higher plants where individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, the complete pathway, from carbon dioxide fixation to TAG synthesis and sequestration, takes place within a single algal cell. Another distinguishing feature of some algae is the large amounts of very long-chain polyunsaturated fatty acids (VLC- PUFAs) as major fatty acid components. Nowadays, the focus of attention in biotechnology is the isolation of novel fatty acid metabolizing genes, especially elongases and desaturases that are responsible for PUFAs synthesis, from different species of algae, and its transfer to plants. The aim is to boost the seed oil content and to generate desirable fatty acids in oilseed crops through genetic engineering approaches. This paper presents the current knowledge of the neutral storage lipids in plants and algae from fatty acid biosynthesis to TAG accumulation.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract Araucaria angustifolia is endemic from southern Brazil. Known as Brazilian pine, A. angustifolia is the only native conifer species with economic/social relevance in this country. Due to massive exploitation, this species suffered significant population decline and currently is classified as critically endangered. It encouraged the scientific community to investigate genetic features in Brazilian pine to increase resources for management and preservation. In this work, RNA-Seq data was used to determine the complete nucleotide sequence of the A. angustifolia chloroplast genome (cpDNA). The cpDNA is 146,203 bp in length and contains 122 genes, including 80 protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes. Coding regions comprise 45.02%; 4.96% correspond to rRNAs and tRNAs, and 50.02% of the genome encompasses non-coding regions. Genes found in inverted repeat (IR) are present as single copy, with exceptions to rrn5 and trnI-CAU loci. The typical LSC, SSC, IRa and IRb organization reported in several land-plant groups is not present in A. angustifolia cpDNA. Phylogenetic analyses using Bayesian and Maximum Likelihood methods clustered A. angustifolia in the Araucariaceae family, with A. heterophylla and A. columnaris as congeneric species. The screening of A. angustifolia cpDNA reveled 100 SSRs, 14 of them corresponding to tetrapolymer loci.

Abstract The diversity of diacylglycerol acyltransferases (DGATs) indicates alternative roles for these enzymes in plant metabolism besides triacylglycerol (TAG) biosynthesis. In this work, we functionally characterized castor bean (Ricinus communis L.) DGATs assessing their subcellular localization, expression in seeds, capacity to restore triacylglycerol (TAG) biosynthesis in mutant yeast and evaluating whether they provide tolerance over free fatty acids (FFA) in sensitive yeast. RcDGAT3 displayed a distinct subcellular localization, located in vesicles outside the endoplasmic reticulum (ER) in most leaf epidermal cells. This enzyme was unable to restore TAG biosynthesis in mutant yeast; however, it was able to outperform other DGATs providing higher tolerance over FFA. RcDAcTA subcellular localization was associated with the ER membranes, resembling RcDGAT1 and RcDGAT2, but it failed to rescue the long-chain TAG biosynthesis in mutant yeast, even with fatty acid supplementation. Besides TAG biosynthesis, our results suggest that RcDGAT3 might have alternative functions and roles in lipid metabolism.

Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice.

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.

As microRNAs (miRNAs) are important regulators of many biological processes, a series of small RNAomes from plants have been produced in the last decade. However, miRNA data from several groups of plants are still lacking, including some economically important crops. Here microRNAs from Coffea canephora leaves were profiled and 58 unique sequences belonging to 33 families were found, including two novel microRNAs that have never been described before in plants. Some of the microRNA sequences were also identified in Coffea arabica that, together with C. canephora, correspond to the two major sources of coffee production in the world. The targets of almost all miRNAs were also predicted on coffee expressed sequences. This is the first report of novel miRNAs in the genus Coffea, and also the first in the plant order Gentianales. The data obtained establishes the basis for the understanding of the complex miRNA-target network on those two important crops.

Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.

Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five "genetic clusters" (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three "genetic clusters". These results are in agreement with the pattern of geographic distribution of the studied populations.

AtGRP3 is a glycine-rich protein (GRP) from Arabidopsis thaliana shown to interact with the receptor-like kinase AtWAK1 in yeast, in vitro and in planta. In this work, phenotypic analyses using transgenic plants were performed in order to better characterize this GRP. Plants of two independent knockout alleles of AtGRP3 develop longer roots suggesting its involvement in root size determination. Confocal microscopy analysis showed an abnormal cell division and elongation in grp3-1 knockout mutants. Moreover, we also show that grp3-1 exhibits an enhanced Aluminum (Al) tolerance, a feature also described in AtWAK1 overexpressing plants. Together, these results implicate AtGRP3 function root size determination during development and in Al stress.

MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants ranging from root to flower and fruit development. Members of the MADS-box gene family share a highly conserved sequence of approximately 180 nucleotides that encodes a DNA-binding domain. We used bioinformatics tools to investigate the information generated by the Eucalyptus Expressed Sequence Tag (FORESTs) genome project in order to identify and annotate MADS-box genes. The comparative phylogenetic analysis of the Eucalyptus MADS-box genes with Arabidopsis homologues allowed us to group them into one of the well-known subfamilies. Trends in gene expression of these putative Eucalyptus MADS-box genes were investigated by hierarchical clustering analysis. Among 24 MADS-box genes identified by our analysis, 12 are expressed in vegetative organs. Out of these, five are expressed predominately in wood. Understanding of the molecular mechanisms performed by MADS-box proteins underlying Eucalyptus growth, development and stress reactions would provide important insights into tree development and could reveal means by which tree characteristics could be modified for the improvement of industrial properties.

The occurrence of quasi-repetitive glycine-rich peptides has been reported in different organisms. Glycine-rich regions are proposed to be involved in protein-protein interactions in some mammalian protein families. In plants, a set of glycine-rich proteins (GRPs) was characterized several years ago, and since then a wealth of new GRPs have been identified. GRPs may have very diverse sub-cellular localization and functions. The only common feature among all different GRPs is the presence of glycine-rich repeat domains. The expression of genes encoding GRPs is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. In addition to the highly modulated expression, several GRPs also show tissue-specific localization. GRPs specifically expressed in xylem, phloem, epidermis, anther tapetum and roots have been described. In this paper, the structural and functional features of these proteins in Eucalyptus are summarized. Since this is the first description of GRPs in this species, particular emphasis has been given to the expression pattern of these genes by analyzing their abundance and prevalence in the different cDNA-libraries of the Eucalyptus Genome Sequencing Project Consortium (ForEST). The comparison of GRPs from Eucalyptus and other species is also discussed.

The expression of AtchitIV gene was analysed in Arabidopsis plants submitted to abiotic stresses. Transcript accumulation was detected in leaves in response to UV light exposure, exogenous salicylic acid administration and wounding. Transgenic Arabidopsis plants carrying AtchitIV promoter::gus fusion also showed differential expression of the reporter gene in response to these treatments. The AtchitIV expression was also analysed during Arabidopsis embryo development. GUS assay demonstrated AtchitIV promoter activation in zygotic embryos from torpedo stage up to full maturation. Promoter deletion analysis indicated that all the 5' cis-acting elements responsible for the specific tissue expression are located in a region of 1083 bp, adjacent to the start of transcription. A negative regulatory region located between portions -1083 and -600 was also observed.

Bacteria are highly diverse and ubiquitous organisms that play a key role as drivers for ecosystem processes. The application of NGS (next-generation sequencing technologies) for 16S analysis has been broadly used for understanding bacterioplankton composition and structure. Most of studies conducted on aquatic ecosystems with 16S NGS have been in seawater and lakes. A few studies using NGS have been conducted in river environments and have suggested the presence of a bacterial seed-bank. We performed 16S highly variable V4 region high-throughput analysis in the Sinos River, which is located in one of most important Brazilian industrial centers. This region has several contrasts in its environmental characteristics, presenting a longitudinal gradient of eutrophication and making it a remarkable study site for observing the dynamics of bacterioplankton. We demonstrated consistent evidence for the existence of a bacterial seed-bank and its longitudinal persistence. Seasonal shifts reinforce the importance of the source of the river in maintaining the bacterial seed-bank that spreads throughout the river. Therefore, the preservation of the source of the river is important not only for hydrologic reasons but also to maintain the microbial composition and the ecological integrity of the river.

Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST) data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB) como pertencentes à sub-sub-classe E.C.3.4.22, foram usadas na busca de clusters no banco de dados do SUCEST (SUgarCane EST project), utilizando-s o programa T-BLAST-n. Homologia de seqüências foram encontradas com 76 clusters que correspondem a prováveis proteinases cisteínicas. O alinhamento destas seqüências com a de outras proteases cisteínicas, de diversas origens, forneceu informação quanto à classificação e possível função das proteinases de cana-de-açúcar. Além disso, o padrão de expressão de cada gene foi postulado a partir da correlação direta com as bibliotecas de cDNA do SUCEST dos quais os clusters foram derivados. Uma vez que nenhum gene de protease cisteínica foi anteriormente evidenciado em cana-de-açúcar, este estudo representa uma etapa inicial para o estudo de novos aspectos bioquímicos, fisiológicos e biotecnológicos destas enzimas.

The cystatins are tightly binding, but reversible, inhibitors of cysteine proteinases, which constitute a superfamily of evolutionary related proteins. They have been subdivided into three families: the cystatin family which contain two disulfide bonds, the stefin family which lack disulfide bonds, and the kininogen family composed of large glycoproteins containing three repeats similar to those found in the cystatin family. Members of the cystatin superfamily occurring in plants are currently known as phytocystatins, defined as proteins lacking disulfide bonds but possessing a conserved N-terminal amino acid sequence (L-A-R-[FY]-A-[VI]-X(3)-N). We have used the protein sequence deduced from seven phytocystatins (from the Arabidopsis thaliana genome project) and from the SUgarCane EST project (SUCEST) database to identify 25 possible sugarcane phytocystatins. Phylogenetic analysis has allowed us to cluster these phytocystatins into four distinct groups: (i) those with a characteristic N-terminal consensus, (ii) those with the same consensus plus a long C-terminal extension; (iii) those that lack the consensus but contain the highly conserved QxVxG motif found in all members of the superfamily and (iv) those that lack both the consensus and the QxVxG motif.As cistatinas são inibidores competitivos e reversíveis de proteinases cisteínicas, compondo uma superfamília de proteínas evolutivamente relacionadas. Esta superfamília está dividida em três famílias: a primeira, das estefinas, é composta de proteínas destituídas de pontes de enxofre; a segunda, das cistatinas, agrupa proteínas que possuem pontes de sulfeto; a terceira família, dos kininogênios, se caracteriza por ser formada por glicoproteínas de alto peso molecular com três domínios repetidos similares aos da família das cistatinas. As fitocistatinas constituem uma família de cistatinas que possui representantes exclusivamente em plantas. Elas são proteínas que não possuem pontes de enxofre e contém uma seqüência conservada L-A-R-[FY]-A-[VI]-X3-N na região N-terminal. Neste trabalho nos usamos seqüências derivadas de sete fitocistatinas, identificadas no projeto genoma de Arabidopsis, para identificar possíveis membros da superfamília das cistatinas presentes no banco de dados de cana-de-açúcar do SUCEST. Foram identificadas vinte e cinco possíveis fitocistatinas de cana-de-açúcar. Uma análise filogenética permitiu agrupar as fitocistatinas em quatro grupos: (i) contendo o consenso N-terminal ; (ii) contendo o consenso N-terminal e mais uma extensão C-terminal; (iii) destituído do consenso N-terminal e (iv) destituído do consenso N-terminal e do motivo conservado QxVxG encontrado em todos os membros da superfamília.

O melhoramento vegetal vem sendo praticado pelo homem há milhares de anos. Entretanto, esse processo de domesticação tornou os vegetais mais susceptíveis a pragas e doenças. O melhoramento clássico permitiu, por cruzamento, a manipulação genética dos vegetais com conseqüente aumento na produtividade agrícola. Recentemente, a tecnologia do DNA recombinante ampliou as possibilidades de integração de genes exógenos ao genoma vegetal, resultando na produção das plantas transgênicas. Apesar das grandes discussões em torno do assunto, essas plantas representam hoje um caminho promissor para o melhoramento vegetal. Inúmeros exemplos de estratégias de transferência de genes conferiram, com sucesso, resistência a herbicidas, vírus, fungos, bactérias e insetos ou produziram um aumento na qualidade dos alimentos. Além das aplicações biotecnológicas, as plantas transgênicas têm contribuído significativamente para o estudo do funcionamento dos genes, tais como a análise da regulação da expressão gênica e o estudo das funções das proteínas codificadas pelos diferentes genes da planta.