Flagellin and mannitol modulate callose biosynthesis and deposition in soybean seedlings.

Callose is a polymer deposited on the cell wall and is necessary for plant growth and development. Callose is synthesized by genes from the glucan synthase-like family (GSL) and dynamically responds to various types of stress. Callose can inhibit pathogenic infection, in the case of biotic stresses, and maintain cell turgor and stiffen the plant cell wall in abiotic stresses. Here, we report the identification of 23 GSL genes (GmGSL) in the soybean genome. We performed phylogenetic analyses, gene structure prediction, duplication patterns, and expression profiles on several RNA-Seq libraries. Our analyses show that WGD/Segmental duplication contributed to expanding this gene family in soybean. Next, we analyzed the callose responses in soybean under abiotic and biotic stresses. The data show that callose is induced by both osmotic stress and flagellin 22 (flg22) and is related to the activity of ß-1,3-glucanases. By using RT-qPCR, we evaluated the expression of GSL genes during the treatment of soybean roots with mannitol and flg22. The GmGSL23 gene was upregulated in seedlings treated with osmotic stress or flg22, showing the essential role of this gene in the soybean defense response to pathogenic organisms and osmotic stress. Our results provide an important understanding of the role of callose deposition and regulation of GSL genes in response to osmotic stress and flg22 infection in soybean seedlings.

Sugarcane Genotypes with Contrasting Biological Nitrogen Fixation Efficiencies Differentially Modulate Nitrogen Metabolism, Auxin Signaling, and Microorganism Perception Pathways.

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

Methyl-CpG binding proteins (MBD) family evolution and conservation in plants.

DNA methylation is an epigenetic mechanism that acts on cytosine residues. The methyl-CpG-binding domain proteins (MBD) are involved in the recognition of methyl-cytosines by activating a signaling cascade that induces the formation of heterochromatin or euchromatin, thereby regulating gene expression. In this study, we analyzed the evolution and conservation of MBD proteins in plants. First, we performed a genome-wide identification and analysis of the MBD family in common bean and soybean, since they have experienced one and two whole-genome duplication events, respectively. We found one pair of MBD paralogs in soybean (GmMBD2) has subfunctionalized after their recent divergence, which was corroborated with their expression profile. Phylogenetic analysis revealed that classes of MBD proteins clustered with human MBD. Interestingly, the MBD9 may have emerged after the hexaploidization event in eudicots. We found that plants and humans share a great similarity in MBDs' binding affinity in the mCpG context. MBD2 and MBD4 from different plant species have the conserved four amino acid residues -Arg (R), Asp (D), Tyr (Y) and Arg (R)- reported to be responsible for MBD-binding in the mCpG. However, MBD8, MBD9, MBD10, and MBD11 underwent substitutions in these residues, suggesting the non-interaction in the mCpG context, but a heterochromatin association as MBD5 and MBD6 from human. This study represents the first genome-wide analysis of the MBD gene family in eurosids I - soybean and common bean. The data presented here contribute towards understanding the evolution of MBDs proteins in plants and their specific binding affinity on mCpG site.

Divergence and conservation of defensins and lipid transfer proteins (LTPs) from sugarcane wild species and modern cultivar genomes.

Plant defensins and lipid transfer proteins (LTPs) constitute a large and evolutionarily diverse family of antimicrobial peptides. Defensins and LTPs are two pathogenesis-related proteins (PR proteins) whose characterization may help to uncover aspects about the sugarcane response to pathogens attack. LTPs have also been investigated for their participation in the response to different types of stress. Despite the important roles of defensins and LTPs in biotic and abiotic stresses, scarce knowledge is found about these proteins in sugarcane. By using bioinformatics approaches, we characterized defensins and LTPs in the sugarcane wild species and modern cultivar genomes. The identification of defensins and LTPs showed that all five defensins groups and eight of the nine LTPs have their respective genes loci, although some was only identified in the cultivar genome. Phylogenetic analysis showed that defensins appear to be more conserved among groups of plants than LTPs. Some defensins and LTPs showed opposite expression during pathogenic and benefic bacterial interactions. Interestingly, the expression of defensins and LTPs in shoots and roots was completely different in plants submitted to benefic bacteria or water depletion. Finally, the modeling and comparison of isoforms of LTPs and defensins in wild species and cultivars revealed a high conservation of tertiary structures, with variation of amino acids in different regions of proteins, which could impact their antimicrobial activity. Our data contributed to the characterization of defensins and LTPs in sugarcane and provided new elements for understanding the involvement of these proteins in sugarcane response to different types of stress.

In silico identification of candidate miRNA-encoded Peptides in four Fabaceae species.

MicroRNAs (miRNAs) are one of the main regulators of gene expression. Recent studies have demonstrated that primary transcripts of miRNAs (pri-miRNAs) encode regulatory peptides, called miRNA-encoded peptides (miPEPs), capable of enhancing the expression of their associated miRNAs in plants. In this work, we aimed to computationally identify miPEPs produced by small open reading frames (ORFs) in pri-miRNAs from four species of Fabaceae. Five families of miRNAs were investigated, based on their role in plant-microorganism interaction. We used the miR171 family as a training dataset centered on the information about mtr-miPEP171b and vvi-miPEP171d already described. From the sequences of the pri-miRNAs and the genomic regions where they were located, ORFs encoding putative miPEPs were predicted. The 5'-most ORFs encoding peptides on pri-miRNAs were aligned and the amino acids conservation was observed. In total, 81 sequences of potential miPEPs were identified. We found conserved miPEPs inside pri-miRNAs from soybean and between soybean, common bean, and cowpea. Besides, our results showed few conserved miPEPs among isoforms of the same miRNA and no conservation among different miRNA families, which indicate the possible specificity of miPEPs in relation to their corresponding miRNAs. Our findings contribute to the understanding of miPEPs features in plants and provide the basis for studies aiming the biotechnological use of miPEPs in leguminous species.

Cell wall formation pathways are differentially regulated in sugarcane contrasting genotypes associated with endophytic diazotrophic bacteria.

MAIN CONCLUSION: Differences in cell wall components between two BNF-contrasting sugarcane genotypes might result from genetic variations particular to the genotype and from the efficiency in diazotrophic bacteria association. Sugarcane is a plant of the grass family (Poaceae) that is highly cultivated in Brazil, as an important energy resource. Commercial sugarcane genotypes may be successfully associated with beneficial endophytic nitrogen-fixing bacteria, which can influence several plant metabolic pathways, such as cell division and growth, synthesis of hormones, and defense compounds. In this study, we investigated how diazotrophic bacteria associated with sugarcane plants could be involved in the regulation of cell wall formation pathways. A molecular and structural characterization of the cell wall was compared between two genotypes of sugarcane with contrasting rates of Biological Nitrogen Fixation (BNF): SP70-1143 (high BNF) and Chunee (low BNF). Differentially expressed transcripts were identified in transcriptomes generated from SP70-1143 and Chunee. Expression profiles of cellulose and lignin genes, which were more expressed in SP70-1134, and callose genes, which were more expressed in Chunee, were validated by RT-qPCR and microscopic analysis of cell wall components in tissue sections. A similar expression profile in both BNF-contrasting genotypes was observed in naturally colonized plants and in plants inoculated with G. diazotrophicus. Cell walls of the high BNF genotype have a greater cellulose content, which might contribute to increase biomass. In parallel, callose was concentrated in the vascular tissues of the low BNF genotype and could possibly represent a barrier for an efficient bacterial colonization and dissemination in sugarcane tissues. Our data show a correlation between the gene profiles identified in the BNF-contrasting genotypes and a successful association with endophytic diazotrophic bacteria.

Insights into the structure and role of seed-borne bacteriome during maize germination.

Seed germination events modulate microbial community composition, which ultimately influences seed-to-seedling growth performance. Here, we evaluate the germinated maize (variety SHS 5050) root bacterial community of disinfected seed (DS) and non-disinfected seed (NDS). Using a gnotobiotic system, sodium hypochlorite (1.25%; 30 min)-treated seeds showed a reduction of bacterial population size and an apparent increase of bacterial community diversity associated with a significant selective reduction of Burkholderia-related sequences. The shift in the bacterial community composition in DS negatively affects germination speed, seedling growth and reserve mobilization rates compared with NDS. A synthetic bacterial community (syncom) formed by 12 isolates (9 Burkholderia spp., 2 Bacillus spp., and 1 Staphylococcus sp.) obtained from natural microbiota maize seeds herein was capable of recovering germination and seedling growth when reintroduced in DS. Overall, results showed that changes in bacterial community composition and selective reduction of Burkholderia-related members' dominance interfere with germination events and the initial growth of the maize. By cultivation-dependent and -independent approaches, we deciphered seed-maize microbiome structure, bacterial niches location and bacterial taxa with relevant roles in seedling growth performance. A causal relationship between seed microbial community succession and germination performance opens opportunities in seed technologies to build-up microbial communities to boost plant growth and health.

Genome-Wide Analysis of the COBRA-Like Gene Family Supports Gene Expansion through Whole-Genome Duplication in Soybean (Glycine max).

The COBRA-like (COBL) gene family has been associated with the regulation of cell wall expansion and cellulose deposition. COBL mutants result in reduced levels and disorganized deposition of cellulose causing defects in the cell wall and inhibiting plant development. In this study, we report the identification of 24 COBL genes (GmCOBL) in the soybean genome. Phylogenetic analysis revealed that the COBL proteins are divided into two groups, which differ by about 170 amino acids in the N-terminal region. The GmCOBL genes were heterogeneously distributed in 14 of the 20 soybean chromosomes. This study showed that segmental duplication has contributed significantly to the expansion of the COBL family in soybean during all Glycine-specific whole-genome duplication events. The expression profile revealed that the expression of the paralogous genes is highly variable between organs and tissues of the plant. Only 20% of the paralogous gene pairs showed similar expression patterns. The high expression levels of some GmCOBLs suggest they are likely essential for regulating cell expansion during the whole soybean life cycle. Our comprehensive overview of the COBL gene family in soybean provides useful information for further understanding the evolution and diversification of COBL genes in soybean.

Altered bacteria community dominance reduces tolerance to resident fungus and seed to seedling growth performance in maize (Zea mays L. var. DKB 177).

Seeds are reservoirs of beneficial and harmful microorganism that modulates plant growth and health. Here, we access seed to seedling bacteriome assembly modified by seed-disinfection and the underlined effect over maize germination performance and root-seedlings microbial colonization. Seed-disinfection was performed with sodium hypochlorite (1.25 %, 30 min), resulting in a reduction of the cultivable-dependent fraction of seed-borne bacteria population, but not significantly detected by real-time PCR, microscopy, and biochemical analysis of the roots on germinated seeds. 16S rRNA sequencing revealed that bacteriome of non-germinated seeds and roots of 5-d germinated seeds exhibited similar diversity and did not differ in the structure concerning seed-disinfection. On the other hand, the relative abundance reduction of the genera f_Enterobacteriaceae_922761 (unassigned genus), Azospirillum, and Acinetobacter in disinfected-seed prior germination seems to display changes in prominence of several new taxa in the roots of germinated seeds. Interestingly, this bacteriome community rebuilt negatively affected the germination speed and growth of maize plantlets. Additionally, bacteriome re-shape increased the maize var. DKB 177 susceptible to the seed-borne plant pathogen Penicillium sp. Such changes in the natural seed-borne composition removed the natural barrier, increasing susceptibility to pathogens, impairing disinfected seeds to germinate, and develop. We conclude that bacteria borne in seeds modulate the relative abundance of taxa colonizing emerged roots, promote germination, seedling growth, and protect the maize against fungal pathogens.

Systematic analysis of 1298 RNA-Seq samples and construction of a comprehensive soybean (Glycine max) expression atlas.

Soybean (Glycine max [L.] Merr.) is a major crop in animal feed and human nutrition, mainly for its rich protein and oil contents. The remarkable rise in soybean transcriptome studies over the past 5 years generated an enormous amount of RNA-seq data, encompassing various tissues, developmental conditions and genotypes. In this study, we have collected data from 1298 publicly available soybean transcriptome samples, processed the raw sequencing reads and mapped them to the soybean reference genome in a systematic fashion. We found that 94% of the annotated genes (52 737/56 044) had detectable expression in at least one sample. Unsupervised clustering revealed three major groups, comprising samples from aerial, underground and seed/seed-related parts. We found 452 genes with uniform and constant expression levels, supporting their roles as housekeeping genes. On the other hand, 1349 genes showed heavily biased expression patterns towards particular tissues. A transcript-level analysis revealed that 95% (70 963 of 74 490) of the assembled transcripts have intron chains exactly matching those from known transcripts, whereas 3256 assembled transcripts represent potentially novel splicing isoforms. The dataset compiled here constitute a new resource for the community, which can be downloaded or accessed through a user-friendly web interface at http://venanciogroup.uenf.br/resources/. This comprehensive transcriptome atlas will likely accelerate research on soybean genetics and genomics.

Programmed cell death in soybean seed coats.

Seed coat is the tissue which establishes an interface between the seed inner tissues and external environment. Our group has shown that cowpea seed coat undergoes coordinated events of programmed cell death (PCD) during development. In relation to germinating seed coats, little is known on PCD events. The goal here was to investigate the biochemical aspects of germinating soybean seed coat, focusing on proteolytic activities related to PCD. In gel and in solution activity profiles of quiescent and germinating seed coat extracts revealed a complex pattern of caspase- and metacaspase-like cysteine protease activities. Trypsin inhibitor and reserve proteins were revealed as potential substrates for these proteases. A pancaspase inhibitor (z-VAD-CHO) affected the radicle length of seeds germinated under its presence. Ultrastructural analysis showed the absence of cell organelles in all seed coat layers after imbibition, while oligonucleosome fragments peaked at 72 h after imbibition (HAI). Altogether, the data suggest the presence of biochemical PCD hallmarks in germinating soybean seed coat and point to the involvement of the detected protease activities in processes such as reserve protein mobilization and weakening of seed coat.

Cell wall dynamics and gene expression on soybean embryonic axes during germination.

MAIN CONCLUSION: Identification of the structural changes and cell wall-related genes likely involved in cell wall extension, cellular water balance and cell wall biosynthesis on embryonic axes during germination of soybean seeds. Cell wall is a highly organized and dynamic structure that provides mechanical support for the cell. During seed germination, the cell wall is critical for cell growth and seedling establishment. Although seed germination has been widely studied in several species, key aspects regarding the regulation of cell wall dynamics in germinating embryonic axes remain obscure. Here, we characterize the gene expression patterns of cell wall pathways and investigate their impact on the cell wall dynamics of embryonic axes of germinating soybean seeds. We found 2143 genes involved in cell wall biosynthesis and assembly in the soybean genome. Key cell wall genes were highly expressed at specific germination stages, such as expansins, UDP-Glc epimerases, GT family, cellulose synthases, peroxidases, arabinogalactans, and xyloglucans-related genes. Further, we found that embryonic axes grow through modulation of these specific cell wall genes with no increment in biomass. Cell wall structural analysis revealed a defined pattern of cell expansion and an increase in cellulose content during germination. In addition, we found a clear correlation between these structural changes and expression patterns of cell wall genes during germination. Taken together, our results provide a better understanding of the complex transcriptional regulation of cell wall genes that drive embryonic axes growth and expansion during soybean germination.

A miniature inverted-repeat transposable element, AddIn-MITE, located inside a WD40 gene is conserved in Andropogoneae grasses.

Miniature inverted-repeat transposable elements (MITEs) have been associated with genic regions in plant genomes and may play important roles in the regulation of nearby genes via recruitment of small RNAs (sRNA) to the MITEs loci. We identified eight families of MITEs in the sugarcane genome assembly with MITE-Hunter pipeline. These sequences were found to be upstream, downstream or inserted into 67 genic regions in the genome. The position of the most abundant MITE (Stowaway-like) in genic regions, which we call AddIn-MITE, was confirmed in a WD40 gene. The analysis of four monocot species showed conservation of the AddIn-MITE sequence, with a large number of copies in their genomes. We also investigated the conservation of the AddIn-MITE' position in the WD40 genes from sorghum, maize and, in sugarcane cultivars and wild Saccharum species. In all analyzed plants, AddIn-MITE has located in WD40 intronic region. Furthermore, the role of AddIn-MITE-related sRNA in WD40 genic region was investigated. We found sRNAs preferentially mapped to the AddIn-MITE than to other regions in the WD40 gene in sugarcane. In addition, the analysis of the small RNA distribution patterns in the WD40 gene and the structure of AddIn-MITE, suggests that the MITE region is a proto-miRNA locus in sugarcane. Together, these data provide insights into the AddIn-MITE role in Andropogoneae grasses.

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots.

Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress.

Analysis of Three Sugarcane Homo/Homeologous Regions Suggests Independent Polyploidization Events of Saccharum officinarum and Saccharum spontaneum.

Whole genome duplication has played an important role in plant evolution and diversification. Sugarcane is an important crop with a complex hybrid polyploid genome, for which the process of adaptation to polyploidy is still poorly understood. In order to improve our knowledge about sugarcane genome evolution and the homo/homeologous gene expression balance, we sequenced and analyzed 27 BACs (Bacterial Artificial Chromosome) of sugarcane R570 cultivar, containing the putative single-copy genes LFY (seven haplotypes), PHYC (four haplotypes), and TOR (seven haplotypes). Comparative genomic approaches showed that these sugarcane loci presented a high degree of conservation of gene content and collinearity (synteny) with sorghum and rice orthologous regions, but were invaded by transposable elements (TE). All the homo/homeologous haplotypes of LFY, PHYC, and TOR are likely to be functional, because they are all under purifying selection (dN/dS ≪ 1). However, they were found to participate in a nonequivalently manner to the overall expression of the corresponding gene. SNPs, indels, and amino acid substitutions allowed inferring the S. officinarum or S. spontaneum origin of the TOR haplotypes, which further led to the estimation that these two sugarcane ancestral species diverged between 2.5 and 3.5 Ma. In addition, analysis of shared TE insertions in TOR haplotypes suggested that two autopolyploidization may have occurred in the lineage that gave rise to S. officinarum, after its divergence from S. spontaneum.

PlantRNA_Sniffer: A SVM-Based Workflow to Predict Long Intergenic Non-Coding RNAs in Plants.

Non-coding RNAs (ncRNAs) constitute an important set of transcripts produced in the cells of organisms. Among them, there is a large amount of a particular class of long ncRNAs that are difficult to predict, the so-called long intergenic ncRNAs (lincRNAs), which might play essential roles in gene regulation and other cellular processes. Despite the importance of these lincRNAs, there is still a lack of biological knowledge and, currently, the few computational methods considered are so specific that they cannot be successfully applied to other species different from those that they have been originally designed to. Prediction of lncRNAs have been performed with machine learning techniques. Particularly, for lincRNA prediction, supervised learning methods have been explored in recent literature. As far as we know, there are no methods nor workflows specially designed to predict lincRNAs in plants. In this context, this work proposes a workflow to predict lincRNAs on plants, considering a workflow that includes known bioinformatics tools together with machine learning techniques, here a support vector machine (SVM). We discuss two case studies that allowed to identify novel lincRNAs, in sugarcane (Saccharum spp.) and in maize (Zea mays). From the results, we also could identify differentially-expressed lincRNAs in sugarcane and maize plants submitted to pathogenic and beneficial microorganisms.

Roles of Non-Coding RNA in Sugarcane-Microbe Interaction.

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408-a copper-microRNA-was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5'RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

Sugarcane transcriptome analysis in response to infection caused by Acidovorax avenae subsp. avenae.

Sugarcane is an important tropical crop mainly cultivated to produce ethanol and sugar. Crop productivity is negatively affected by Acidovorax avenae subsp avenae (Aaa), which causes the red stripe disease. Little is known about the molecular mechanisms triggered in response to the infection. We have investigated the molecular mechanism activated in sugarcane using a RNA-seq approach. We have produced a de novo transcriptome assembly (TR7) from sugarcane RNA-seq libraries submitted to drought and infection with Aaa. Together, these libraries present 247 million of raw reads and resulted in 168,767 reference transcripts. Mapping in TR7 of reads obtained from infected libraries, revealed 798 differentially expressed transcripts, of which 723 were annotated, corresponding to 467 genes. GO and KEGG enrichment analysis showed that several metabolic pathways, such as code for proteins response to stress, metabolism of carbohydrates, processes of transcription and translation of proteins, amino acid metabolism and biosynthesis of secondary metabolites were significantly regulated in sugarcane. Differential analysis revealed that genes in the biosynthetic pathways of ET and JA PRRs, oxidative burst genes, NBS-LRR genes, cell wall fortification genes, SAR induced genes and pathogenesis-related genes (PR) were upregulated. In addition, 20 genes were validated by RT-qPCR. Together, these data contribute to a better understanding of the molecular mechanisms triggered by the Aaa in sugarcane and opens the opportunity for the development of molecular markers associated with disease tolerance in breeding programs.

Computational identification and comparative analysis of miRNA precursors in three palm species.

MAIN CONCLUSION: In the present study, miRNA precursors in the genomes of three palm species were identified. Analyzes of sequence conservation and biological function of their putative targets contribute to understand the roles of miRNA in palm biology. MicroRNAs are small RNAs of 20-25 nucleotides in length, with important functions in the regulation of gene expression. Recent genome sequencing of the palm species Elaeis guineensis, Elaeis oleifera and Phoenix dactylifera have enabled the discovery of miRNA genes, which can be used as biotechnological tools in palm trees breeding. The goal of this study is the identification of miRNA precursors in the genomes of these species and their possible biological roles suggested by the mature miRNA-based regulation of target genes. Mature miRNA sequences from Arabidopsis thaliana, Oryza sativa, and Zea mays available at the miRBase were used to predict microRNA precursors in the palm genomes. Three hundred and thirty-eight precursors, ranging from 76 to 220 nucleotide (nt) in size and distributed in 33 families were identified. Moreover, we also identified 266 miRNA precursors of Musa acuminata, which are phylogenetically close to palms species. To understand the biological function of palm miRNAs, 374 putative miRNA targets were identified. An enrichment analysis of target-gene function was carried out using the agriGO tool. The results showed that the targets are involved in plant developmental processes, mainly regulating root development. Our findings contribute to increase the knowledge on microRNA roles in palm biology and could help breeding programs of palm trees.

Genome-wide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize.

BACKGROUND: Small RNA (sRNA) has been described as a regulator of gene expression. In order to understand the role of maize sRNA (Zea mays-hybrid UENF 506-8) during association with endophytic nitrogen-fixing bacteria, we analyzed the sRNA regulated by its association with two diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. RESULTS: Deep sequencing analysis was done with RNA extracted from plants inoculated with H. seropedicae, allowing the identification of miRNA and siRNA. A total of 25 conserved miRNA families and 15 novel miRNAs were identified. A dynamic regulation in response to inoculation was also observed. A hypothetical model involving copper-miRNA is proposed, emphasizing the fact that the up-regulation of miR397, miR398, miR408 and miR528, which is followed by inhibition of their targets, can facilitate association with diazotrophic bacteria. Similar expression patterns were observed in samples inoculated with A. brasilense. Moreover, novel miRNA and siRNA were classified in the Transposable Elements (TE) database, and an enrichment of siRNA aligned with TE was observed in the inoculated samples. In addition, an increase in 24-nt siRNA mapping to genes was observed, which was correlated with an increase in methylation of the coding regions and a subsequent reduction in transcription. CONCLUSION: Our results show that maize has RNA-based silencing mechanisms that can trigger specific responses when plants interact with beneficial endophytic diazotrophic bacteria. Our findings suggest important roles for sRNA regulation in maize, and probably in other plants, during association with diazotrophic bacteria, emphasizing the up-regulation of Cu-miRNA.