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

Differentially regulated induced resistance marker enzymes in soybean genotypes resistant and susceptible to Asian soybean rust

O objetivo deste trabalho foi avaliar, por meio de enzimas marcadoras, a indução de resistência à ferrugem‑asiática‑da‑soja em genótipos de soja contrastantes quanto à suscetibilidade a Phakopsora pachyrhizi. A proteína total e as atividades de cinco enzimas marcadoras da indução de resistência (lipoxigenases, peroxidases, fenilalanina amônia‑liase, quitinases e β‑1,3‑glucanases) foram avaliadas em extratos de folhas de plantas de soja dos genótipos Embrapa 48 (suscetível) e PI 561356 (resistente), submetidas à inoculação ou não com o patógeno. Foram observadas respostas de defesa discrepantes entre os dois genótipos e entre os tempos de coleta (12, 72 e 168 horas após inoculação). A resposta de indução dessas enzimas assemelha-se à defesa bifásica, para Embrapa 48, e é consistente com o observado para outros patossistemas. No entanto, o genótipo PI 561356 respondeu com diminuição da concentração de proteína total e das atividades enzimáticas, o que indica reduçãodo metabolismo geral das plantas infectadas. Há um importante mecanismo de resistência do genótipo PI 561356, ainda não relatado, embasado em vias que envolvem essas enzimas marcadoras e em mecanismos que utilizam menor concentração de proteínas, como os de via metabólica de resposta em cascata.The objective of this work was to evaluate induced resistance to Asian soybean rust by means of enzymeactivities in soybean genotypes contrasting as to their susceptibility to Phakopsora pachyrhizi. Total protein and the activities of five induced resistance marker enzymes (lipoxygenases, peroxidases, phenylalanine ammonia‑lyase, chitinases and β‑1,3‑glucanases) were evaluated in leaf extracts of soybean plants of the genotypes Embrapa 48 (susceptible) and PI 561356 (resistant), inoculated or not with the pathogen. Discrepant defense responses were obtained between the two genotypes and among the leaf harvest times (12, 72, and 168 hours after inoculation). The induction response of these enzymes resembles the biphasic defense in Embrapa 48 and is consistent with that observed in other pathological systems. However, the genotype PI 561356 responded with a decrease in total protein concentration and in enzymatic activities, indicating a general reduction in the metabolism of the infected plants. There is an important mechanism of resistance for the genotype PI 561356, not yet reported, which is grounded on the metabolic ways involving these induced resistance marker enzymes and on the mechanisms that use lower concentrations of total protein, such as the ones with metabolic pathways in response cascade

Enzimas marcadoras de indução de resistência diferencialmente reguladas em soja resistente e suscetível à ferrugem-asiática-da-soja

O objetivo deste trabalho foi avaliar, por meio de enzimas marcadoras, a indução de resistência à ferrugem-asiática-da-soja em genótipos de soja contrastantes quanto à suscetibilidade a Phakopsora pachyrhizi. Aproteína total e as atividades de cinco enzimas marcadoras da indução de resistência (lipoxigenases, peroxidases, fenilalanina amônia-liase, quitinases e β-1, 3-glucanases) foram avaliadas em extratos de folhas de plantas de soja dos genótipos Embrapa 48 (suscetível) e PI 561356 (resistente), submetidas à inoculação ou não com o patógeno. Foram observadas respostas de defesa discrepantes entre os dois genótipos e entre os tempos de coleta (12, 72 e 168 horas após inoculação). A resposta de indução dessas enzimas assemelha-se à defesa bifásica, para Embrapa 48, e é consistente com o observado para outros patossistemas. No entanto, o genótipo PI 561356 respondeu com diminuição da concentração de proteína total e das atividades enzimáticas, o que indica redução do metabolismo geral das plantas infectadas. Há um importante mecanismo de resistência do genótipo PI 561356, ainda não relatado, embasado em vias que envolvem essas enzimas marcadoras e em mecanismos que utilizam menor concentração de proteínas, como os de via metabólica de resposta em cascata.The objective of this work was to evaluate induced resistance to Asian soybean rust by means of enzyme activities in soybean genotypes contrasting as to their susceptibility to Phakopsora pachyrhizi. Total protein and the activities of five induced resistance marker enzymes (lipoxygenases, peroxidases, phenylalanine ammonia-lyase, chitinases and β-1, 3-glucanases) were evaluated in leaf extracts of soybean plants of the genotypes Embrapa 48 (susceptible) and PI 561356 (resistant), inoculated or not with the pathogen. Discrepant defense responses were obtained between the two genotypes and among the leaf harvest times (12, 72, and 168 hours after inoculation). The induction response of these enzymes resembles the biphasic defense in Embrapa 48 and is consistent with that observed in other pathological systems. However, the genotype PI 561356 responded with a decrease in total protein concentration and in enzymatic activities, indicating a general reduction in the metabolism of the infected plants. There is an important mechanism of resistance for the genotype PI 561356, not yet reported, which is grounded on the metabolic ways involving these induced resistance marker enzymes and on the mechanisms that use lower concentrations of total protein, such as the ones with metabolic pathways in response cascade

Nanoencapsulation Enhances The Post-emergence Herbicidal Activity Of Atrazine Against Mustard Plants.

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 10(12) 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

Post-Emergence Herbicidal Activity of Nanoatrazine Against Susceptible Weeds

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

Effect of Nitrogen Addition to the Soil on Atlantic Forest Tree Seedlings

The input of large amounts of fertilizers in agricultural areas may result in nitrogen (N) leakage to nearby forest fragments, which can impact the physiology and growth of trees. The current study aimed to assess the effects of soil N addition on seedlings of four tree species in the Brazilian Atlantic Forest: Croton floribundus and Astronium graveolens (pioneer), Guarea kunthiana and Aspidosperma polyneuron (non-pioneer). The experiment was carried out in a greenhouse condition with three treatments: reference (soil without addition of nutrients), N addition (N: soil with addition of ammoniacal-N), and complete (C: soil with addition of ammoniacal-N and other macro and micronutrients). Croton floribundus seedlings presented higher shoot growth with N addition, mainly in treatment C, and only this treatment increased net photosynthesis. There was great variation in the metabolic responses induced by treatments N and C, with accumulation of nitrate in the leaves and xylem sap only in seedlings in treatment N. In A. graveolens, there was a decrease in transpiration in response to treatments N and C. However, water use efficiency, leaf area, and dry mass increased only in seedlings subjected to treatment C. Regarding metabolic parameters, A. graveolens was little responsive to the treatments. In G. kunthiana seedlings, the treatments decreased net photosynthesis and increased leaf total N. Only treatment N led to decreased stem dry mass and increased nitrate contents in leaves and xylem sap. Aspidosperma polyneuron exhibited no change in growth, but there was an accumulation of N compounds in the leaves for both treatments N and C, which suggests that this species could be a good bioindicator of N addition to the soil. Although influencing different parameters, the results indicate that soil N addition affects the performance of both pioneer and non-pioneer species. Finally, the implications of these results for biomonitoring of N availability in the soil of forest fragments are discussed

Oxidative stress parameters of <i>B</i>. <i>juncea</i> plants treated with the formulations.

<p>Lipid peroxidation (A) and hydrogen peroxide content (B) were evaluated 24, 48, and 72 h after the plants were sprayed with 3.1 mL of water, empty PCL nanocapsules (NC), commercial atrazine (ATZ), or PCL nanocapsules containing atrazine (NC+ATZ). The formulations containing atrazine at 1 mg mL^-1 were used undiluted or after 10-fold dilution in water (1/10), resulting in atrazine dosages equivalent to 2000 or 200 g ha^-1, respectively. Different letters indicate significantly different values according to two-way ANOVA followed by the Tukey test (p < 0.05). Lowercase letters indicate comparison among all treatments at each time point, while capital letters indicate comparison of the same treatment along its individual time course analysis. Data are shown as means ± SE (n = 5).</p

Maximum photosystem II quantum yields of <i>B</i>. <i>juncea</i> plants treated with the formulations.

<p>Chlorophyll fluorescence parameters were evaluated 24, 48, and 72 h after the plants were sprayed with 3.1 mL of water, empty PCL nanocapsules (NC), commercial atrazine (ATZ), or PCL nanocapsules containing atrazine (NC+ATZ). The formulations containing atrazine at 1 mg mL^-1 were used undiluted or after 10-fold dilution in water (1/10), resulting in atrazine dosages equivalent to 2000 or 200 g ha^-1, respectively. Different letters indicate significantly different values according to two-way ANOVA followed by the Tukey test (p < 0.05). Lowercase letters indicate comparison among all treatments at each time point, while capital letters indicate comparison of the same treatment along its individual time course analysis. Data are shown as means ± SE (n = 5).</p