BackgroundSmall 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.ResultsThe 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.ConclusionsValidation 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.

Poly(epsilon-caprolactone) (PCL) nanocapsules have been recently developed as a modified release system for atrazine, an herbicide that can have harmful effects in the environment. Here, the post-emergence herbicidal activity of PCL nanocapsules containing atrazine was evaluated using mustard (Brassica juncea) as target plant species model. Characterization of atrazine-loaded PCL nanocapsules by nanoparticle tracking analysis indicated a concentration of 7.5 x 1012 particles mL-1 and an average size distribution of 240.7 nm. The treatment of mustard plants with nanocapsules carrying atrazine at 1 mg mL-1 resulted in a decrease of net photosynthesis and PSII maximum quantum yield, and an increase of leaf lipid peroxidation, leading to shoot growth inhibition and the development of severe symptoms. Time course analysis until 72 h after treatments showed that nanoencapsulation of atrazine enhanced the herbicidal activity in comparison with a commercial atrazine formulation. In contrast to the commercial formulation, ten-fold dilution of the atrazine-containing nanocapsules did not compromise the herbicidal activity. No effects were observed when plants were treated with nanocapsules without herbicide compared to control leaves sprayed with water. Overall, these results demonstrated that atrazine-containing PCL nanocapsules provide very effective post-emergence herbicidal activity. More importantly, the use of nanoencapsulated atrazine enables the application of lower dosages of the herbicide, without any loss of efficiency, which could provide environmental benefits.

To identify differentially expressed genes in soybean grown under different drought conditions, cDNA libraries from roots of different genotypes were constructed. Genes of contrasting genotypes of soybean were found to be differentially expressed in plants exposed to drought conditions. A total of 753 no redundant clones were identified by PCR, and these were printed on microarray glass slides. Probes of total RNA were prepared from bulked roots subjected to 25 and 50 min (Bulk 1) or 75 and 100 min (Bulk 2) of drought stress. Differential expression of 145 genes, involved in metabolic pathways responsive to biotic and abiotic stresses, was observed. These genes were classified into nine functional categories, including energy, transcription factors, metabolism, stress response, protein synthesis, cell communication, cell cycle, cell transport, and unknown function. The functionality of some of these genes was confirmed by quantitative real-time PCR (qRT-PCR).

ABSTRACT. Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.

Poly(ε-caprolactone) (PCL) nanocapsules have been previously developed as a carrier system for atrazine. However, the efficacy of this nanoformulation against weeds commonly found in crop cultures has not been tested yet. Here, we evaluated the post-emergence herbicidal activity of PCL nanocapsules containing atrazine against Amaranthus viridis (slender amaranth) and Bidens pilosa (hairy beggarticks), in comparison with a commercial formulation of atrazine. For both species, treatment with atrazine-loaded nanocapsules (at 2,000 g ha −1) led to a greater decrease in the photosystem II activity (above 50% inhibition relative to the control) than the commercial atrazine formulation at the same concentration (around 40% inhibition). The growth of A. viridis plants was equally reduced by nanoatrazine and commercial formulation (above 64% for root and 75% for shoot). In the case of B. pilosa, atrazine-loaded nanocapsules decreased more effectively the root and shoot growth than the commercial formulation, leading to a loss of plant biomass. Moreover, for both species, the use of 10-fold diluted atrazine-loaded PCL nanocapsules (200 g ha −1) resulted in the same inhibitory effect in root and shoot growth as the commercial formulation at the standard atrazine dose. These results suggest that the utilization of atrazine-containing PCL nanocapsules potentiated the post-emergence control of A. viridis and B. pilosa by the herbicide. Thus, this nanoformulation emerges as an efficient alternative for weed control.