Beyond transcription factors: more regulatory layers affecting soybean gene expression under abiotic stress.

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.

Perspectives in Myrtaceae evolution from plastomes and nuclear phylogenies.

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.

Functional analysis of alternative castor bean DGAT enzymes.

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.

Transcriptional analyses of two soybean cultivars under salt stress.

Soybean is an economically important plant, and its production is affected in soils with high salinity levels. It is important to understand the adaptive mechanisms through which plants overcome this kind of stress and to identify potential genes for improving abiotic stress tolerance. RNA-Seq data of two Glycine max cultivars, a drought-sensitive (C08) and a tolerant (Conquista), subjected to different periods of salt stress were analyzed. The transcript expression profile was obtained using a transcriptogram approach, comparing both cultivars and different times of treatment. After 4 h of salt stress, Conquista cultivar had 1400 differentially expressed genes, 647 induced and 753 repressed. Comparative expression revealed that 719 genes share the same pattern of induction or repression between both cultivars. Among them, 393 genes were up- and 326 down-regulated. Salt stress also modified the expression of 54 isoforms of miRNAs in Conquista, by the maturation of 39 different pre-miRNAs. The predicted targets for 12 of those mature miRNAs also have matches with 15 differentially expressed genes from our analyses. We found genes involved in important pathways related to stress adaptation. Genes from both ABA and BR signaling pathways were modulated, with possible crosstalk between them, and with a likely post-transcriptional regulation by miRNAs. Genes related to ethylene biosynthesis, DNA repair, and plastid translation process were those that could be regulated by miRNA.

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.

Comparative analysis of the complete chloroplast genomes from six Neotropical species of Myrteae (Myrtaceae).

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.

Identification of soybean trans-factors associated with plastid RNA editing sites.

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.

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.

Identification of root transcriptional responses to shoot illumination in Arabidopsis thaliana.

KEY MESSAGE: The transcriptional profile of roots is highly affected by shoot illumination. Transcriptogram analysis allows the identification of cellular processes that are not detected by DESeq. Light is a key environmental factor regulating plant growth and development. Arabidopsis thaliana seedlings grown under light display a photomorphogenic development pattern, showing short hypocotyl and long roots. On the other hand, when grown in darkness, they display skotomorphogenic development, with long hypocotyls and short roots. Although many signals from shoots might be important for triggering root growth, the early transcriptional responses that stimulate primary root elongation are still unknown. Here, we aimed to investigate which genes are involved in the early photomorphogenic root development of dark grown roots. We found that 1616 genes 4 days after germination (days-old), and 3920 genes 7 days-old were differently expressed in roots when the shoot was exposed to light. Of these genes, 979 were up regulated in 4 days and 2784 at 7 days-old. We compared the functional categorization of differentially regulated processes by two methods: GO term enrichment and transcriptogram analysis. Expression analysis of nine selected candidate genes in roots confirmed the data observed in the RNA-seq analysis. Loss-of-function mutants of these selected differentially expressed genes suggest the involvement of these genes in root development in response to shoot illumination. Our findings are consistent with the observation that dark grown roots respond to the shoot-perceived aboveground light environment.

Transcriptome analysis of strawberry (Fragaria × ananassa) fruits under osmotic stresses and identification of genes related to ascorbic acid pathway.

Strawberry (Fragaria ananassa Duch.) is an economically important fruit with a high demand owing to its good taste and medicinal properties. However, its cultivation is affected by various biotic and abiotic stresses. Plants exhibit several intrinsic mechanisms to deal with stresses. In the case of strawberry, the mechanisms highlighting the response against these stresses remain to be elucidated, which has hampered the efforts to develop and cultivate strawberry plants with high yield and quality. Although a virtual reference genome of F. ananassa has recently been published, there is still a lack of information on the expression of genes in response to various stresses. Therefore, to provide molecular information for further studies with strawberry plants, we present the reference transcriptome dataset of F. ananassa, assembled and annotated from deep RNA-Seq data of fruits cultivated under salinity and drought stresses. We also systematically arranged a series of transcripts differentially expressed during these stresses, with an emphasis on genes related to the accumulation of ascorbic acid (AsA). Ascorbic acid is the most potent antioxidant present in these fruits and highly considered during biofortification. A comparison of the expression profile of these genes by RT-qPCR with the content of AsA in the fruits verified a tight regulation and balance between the expression of genes, from biosynthesis, degradation and recycling pathways, resulting in the reduced content of AsA in fruits under these stresses. These results provide a useful repertoire of genes for metabolic engineering, thereby improving the tolerance to stresses.

Exploring the Hospital Microbiome by High-Resolution 16S rRNA Profiling.

The aim of this work was to analyze and compare the bacterial communities of 663 samples from a Brazilian hospital by using high-throughput sequencing of the 16S rRNA gene. To increase taxonomic profiling and specificity of 16S-based identification, a strict sequence quality filtering process was applied for the accurate identification of clinically relevant bacterial taxa. Our results indicate that the hospital environment is predominantly inhabited by closely related species. A massive dominance of a few taxa in all taxonomic levels down to the genera was observed, where the ten most abundant genera in each facility represented 64.4% of all observed taxa, with a major predominance of Acinetobacter and Pseudomonas. The presence of several nosocomial pathogens was revealed. Co-occurrence analysis indicated that the present hospital microbial network had low connectedness, forming a clustered topology, but not structured among groups of nodes (i.e., modules). Furthermore, we were able to detect ecologically relevant relationships between specific microbial taxa, in particular, potential competition between pathogens and non-pathogens. Overall, these results provide new insight into different aspects of a hospital microbiome and indicate that 16S rRNA sequencing may serve as a robust one-step tool for microbiological identification and characterization of a wide range of clinically relevant bacterial taxa in hospital settings with a high resolution.

Araucaria angustifolia chloroplast genome sequence and its relation to other Araucariaceae.

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.

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.

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.

Complete sequence and comparative analysis of the chloroplast genome of Plinia trunciflora.

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.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

Exploring developmental gene toolkit and associated pathways in a potential new model crustacean using transcriptomic analysis.

The crustaceans are one of the largest, most diverse, and most successful groups of invertebrates. The diversity among the crustaceans is also reflected in embryonic development models. However, the molecular genetics that regulates embryonic development is not known in those crustaceans that have a short germ-band development with superficial cleavage, such as Macrobrachium olfersi. This species is a freshwater decapod and has great potential to become a model for developmental biology, as well as for evolutionary and environmental studies. To obtain sequence data of M. olfersi from an embryonic developmental perspective, we performed de novo assembly and annotation of the embryonic transcriptome. Using a pooling strategy of total RNA, paired-end Illumina sequencing, and assembly with multiple k-mers, a total of 25,636,097 pair reads were generated. In total, 99,751 unigenes were identified, and 20,893 of these returned a Blastx hit. KEGG pathway analysis mapped a total of 6866 unigenes related to 129 metabolic pathways. In general, 21,845 unigenes were assigned to gene ontology (GO) categories: molecular function (19,604), cellular components (10,254), and biological processes (13,841). Of these, 2142 unigenes were assigned to the developmental process category. More specifically, a total of 35 homologs of embryonic development toolkit genes were identified, which included maternal effect (one gene), gap (six), pair-rule (six), segment polarity (seven), Hox (four), Wnt (eight), and dorsoventral patterning genes (three). In addition, genes of developmental pathways were found, including TGF-ß, Wnt, Notch, MAPK, Hedgehog, Jak-STAT, VEGF, and ecdysteroid-inducible nuclear receptors. RT-PCR analysis of eight genes related to embryonic development from gastrulation to late morphogenesis/organogenesis confirmed the applicability of the transcriptome analysis.

Rice ASR1 and ASR5 are complementary transcription factors regulating aluminium responsive genes.

Rice is the most tolerant staple crop to aluminium (Al) toxicity, which is a limiting stress for grain production worldwide. This Al tolerance is the result of combined mechanisms that are triggered in part by the transcription factor ASR5. ASRs are dual target proteins that participate as chaperones in the cytoplasm and as transcription factors in the nucleus. Moreover, these proteins respond to biotic and abiotic stresses, including salt, drought and Al. Rice plants with silenced ASR genes are highly sensitive to Al. ASR5, a well-characterized protein, binds to specific cis elements in Al responsive genes and regulates their expression. Because the Al sensitive phenotype found in silenced rice plants could be due to the mutual silencing of ASR1 and ASR5, we investigated the effect of the specific silencing of ASR5. Plants with artificial microRNA silencing of ASR5 present a non-transformed phenotype in response to Al because of the induction of ASR1. ASR1 has the same subcellular localization as ASR5, binds to ASR5 cis-regulatory elements, regulates ASR5 regulated genes in a non-preferential manner and might replace ASR5 under certain conditions. Our results indicate that ASR1 and ASR5 act in concert and complementarily to regulate gene expression in response to Al.

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.