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.

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. imbalance salt intensity development evolution variations physiological morphological adaptations posttranscriptional post transcriptional responses engineering pathways community 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 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 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.

RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.

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.

Abstract Araucaria angustifolia is endemic to southern Brazil. Known as Brazilian pine, A. angustifolia is the only native conifer species with economic and social relevance in this country. Due to massive exploitation, it has suffered a significant population decline and currently is classified as critically endangered. This 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 the inverted repeat (IR) are present as single copy, with exception of the 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 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.

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.

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.

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.

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.