For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes. Klebsiella ...pneumoniae is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on K. pneumoniae. In this study, the expression profile of eleven candidate reference genes was assessed in K. pneumoniae cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta C
and RefFinder. The statistical analyses ranked recA, rho, proC and rpoD as the most suitable reference genes for accurate RT-qPCR data normalization in K. pneumoniae. The reliability of the proposed reference genes was validated by normalizing the relative expression of iron-regulated genes in K. pneumoniae cells submitted to iron-replete and iron-limited conditions. This work emphasizes that the stable expression of any potential reference candidate gene must be validated in each physiological condition or experimental treatment under study.
Key message
The manuscript by Alves et al. entitled “
Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants
” describes the identification and characterization ...of tRNAderived sRNA fragments in plants. By combining bioinformatic analysis and genetic and molecular approaches, we show that tRF biogenesis does not rely on canonical microRNA/siRNA processing machinery (i.e., independent of DICER-LIKE proteins). Moreover, we provide evidences that the Arabidopsis S-like Ribonuclease 1 (RNS1) might be involved in the biogenesis of tRFs. Detailed analyses showed that plant tRFs are sorted into different types of ARGONAUTE proteins and that they have potential target candidate genes. Our work advances the understanding of the tRF biology in plants by providing evidences that plant and animal tRFs shared common features and raising the hypothesis that an interplay between tRFs and other sRNAs might be important to fine-tune gene expression and protein biosynthesis in plant cells.
Small RNA (sRNA) fragments derived from tRNAs (3′-loop, 5′-loop, anti-codon loop), named tRFs, have been reported in several organisms, including humans and plants. Although they may interfere with gene expression, their biogenesis and biological functions in plants remain poorly understood. Here, we capitalized on small RNA sequencing data from distinct species such as
Arabidopsis thaliana, Oryza sativa
, and
Physcomitrella patens
to examine the diversity of plant tRFs and provide insight into their properties.
In silico
analyzes of 19 to 25-nt tRFs derived from 5′ (tRF-5s) and 3′CCA (tRF-3s) tRNA loops in these three evolutionary distant species showed that they are conserved and their abundance did not correlate with the number of genomic copies of the parental tRNAs. Moreover, tRF-5 is the most abundant variant in all three species.
In silico
and in vivo expression analyses unraveled differential accumulation of tRFs in
Arabidopsis
tissues/organs, suggesting that they are not byproducts of tRNA degradation. We also verified that the biogenesis of most
Arabidopsis
19–25 nt tRF-5s and tRF-3s is not primarily dependent on DICER-LIKE proteins, though they seem to be associated with ARGONAUTE proteins and have few potential targets. Finally, we provide evidence that
Arabidopsis
ribonuclease RNS1 might be involved in the processing and/or degradation of tRFs. Our data support the notion that an interplay between tRFs and other sRNAs might be important to fine tune gene expression and protein biosynthesis in plant cells.
Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are regulators of many central developmental and physiological ...processes including photomorphogenesis, leaf and seed formation, energy homeostasis, and abiotic and biotic stress responses. Here we performed a comprehensive phylogenetic analysis of bZIP genes from algae, mosses, ferns, gymnosperms and angiosperms.
We identified 13 groups of bZIP homologues in angiosperms, three more than known before, that represent 34 Possible Groups of Orthologues (PoGOs). The 34 PoGOs may correspond to the complete set of ancestral angiosperm bZIP genes that participated in the diversification of flowering plants. Homologous genes dedicated to seed-related processes and ABA-mediated stress responses originated in the common ancestor of seed plants, and three groups of homologues emerged in the angiosperm lineage, of which one group plays a role in optimizing the use of energy.
Our data suggest that the ancestor of green plants possessed four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments.
Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage ...on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.
Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of ...expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.
Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric CO₂. ...The effects of increased CO₂ on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (~370 ppm) and elevated (~720 ppm) CO₂ during 50 weeks in open-top chambers. The plants grown under elevated CO₂ showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient CO₂. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO₂. The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher CO₂, and that this might be related, as previously observed for maize and sorghum, to transient drought stress.
Sugarcane is an important crop and a major source of sugar and alcohol. In this study, we performed de novo assembly and transcriptome annotation for six sugarcane genotypes involved in bi-parental ...crosses. The de novo assembly of the sugarcane transcriptome was performed using short reads generated using the Illumina RNA-Seq platform. We produced more than 400 million reads, which were assembled into 72,269 unigenes. Based on a similarity search, the unigenes showed significant similarity to more than 28,788 sorghum proteins, including a set of 5,272 unigenes that are not present in the public sugarcane EST databases; many of these unigenes are likely putative undescribed sugarcane genes. From this collection of unigenes, a large number of molecular markers were identified, including 5,106 simple sequence repeats (SSRs) and 708,125 single-nucleotide polymorphisms (SNPs). This new dataset will be a useful resource for future genetic and genomic studies in this species.
Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic ...biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.
Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to ...and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.
Flowering is of utmost relevance for the agricultural productivity of the sugarcane bioeconomy, but data and knowledge of the genetic mechanisms underlying its photoperiodic induction are still ...scarce. An understanding of the molecular mechanisms that regulate the transition from vegetative to reproductive growth in sugarcane could provide better control of flowering for breeding. This study aimed to investigate the transcriptome of +1 mature leaves of a sugarcane cultivar subjected to florally inductive and non-inductive photoperiodic treatments to identify gene expression patterns and molecular regulatory modules. We identified 7,083 differentially expressed (DE) genes, of which 5,623 showed significant identity to other plant genes. Functional group analysis showed differential regulation of important metabolic pathways involved in plant development, such as plant hormones (i.e., cytokinin, gibberellin, and abscisic acid), light reactions, and photorespiration. Gene ontology enrichment analysis revealed evidence of upregulated processes and functions related to the response to abiotic stress, photoprotection, photosynthesis, light harvesting, and pigment biosynthesis, whereas important categories related to growth and vegetative development of plants, such as plant organ morphogenesis, shoot system development, macromolecule metabolic process, and lignin biosynthesis, were downregulated. Also, out of 76 sugarcane transcripts considered putative orthologs to flowering genes from other plants (such as
Arabidopsis thaliana
,
Oryza sativa
, and
Sorghum bicolor
), 21 transcripts were DE. Nine DE genes related to flowering and response to photoperiod were analyzed either at mature or spindle leaves at two development stages corresponding to the early stage of induction and inflorescence primordia formation. Finally, we report a set of flowering-induced long non-coding RNAs and describe their level of conservation to other crops, many of which showed expression patterns correlated against those in the functionally grouped gene network.
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