Identification of novel soybean microRNAs involved in abiotic and biotic stresses

<p>Abstract</p> <p>Background</p> <p>Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.</p> <p>Results</p> <p>The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.</p> <p>Conclusions</p> <p>Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.</p

Overall picture of expressed Heat Shock Factors in Glycine max, Lotus japonicus and Medicago truncatula

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions

Overall picture of expressed Heat Shock Factors in Glycine max, Lotus japonicus and Medicago truncatula

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions.35124725

Identification of novel soybean microRNAs involved in abiotic and biotic stresses

Background: Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and posttranscriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing. Results: The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families. Conclusions: Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses

Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR

Identification of the soybean HyPRP family and specific gene response to asian soybean rust disease

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi

Identification of the soybean HyPRP family and specific gene response to asian soybean rust disease

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi