RESUMO
Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/fisiologia , Phaseolus/genética , Phaseolus/fisiologia , Fatores de Transcrição/genética , Produtos Agrícolas/genética , Produtos Agrícolas/fisiologia , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologiaRESUMO
We recently described the activity of miR1514a in response to water deficit in Phaseolus vulgaris. Pvu-miR1514a targets a NAC transcription factor mRNA for cleavage and subsequently triggers NAC-derived phasiRNA formation. Here we show that accumulation and activity of miR1514a are also conserved in the model legume Medicago truncatula. Consistently, we identified Mtr-miR1514a and detected its increased accumulation in response to stress conditions, targeting a NAC TF mRNA for cleavage and triggering phasiRNA production. In P. vulgaris, miR1514a inhibition in transgenic hairy roots was reported to increase NAC 700 mRNA levels and to affect expression patterns of several genes, including that of a Sec 14 homolog. We report here that in adult plant roots exposed to dehydration conditions, where miR1514a levels increased and NAC 700 mRNA decreased, there was a reduction of Sec 14 homolog mRNA levels, suggesting a direct transcriptional effect. The functions of miR1514a, NAC 700 and derived phasiRNAs have just begun to be elucidated in common bean; future understanding of their activities in this and other legumes species will advance our knowledge of microRNA functions in plants.
Assuntos
MicroRNAs/metabolismo , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Medicago truncatula/genética , Medicago truncatula/metabolismo , MicroRNAs/genética , Phaseolus/genética , Phaseolus/metabolismo , Proteínas de Plantas/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Glycine max/genética , Glycine max/metabolismoRESUMO
MicroRNAs are recognized as important post-transcriptional regulators in plants. Information about the roles of miRNAs in common bean (Phaseolus vulgaris L.), an agronomically important legume, is yet scant. The objective of this work was to functionally characterize the conserved miRNA: miR398b and its target Cu/Zn Superoxide Dismutase 1 (CSD1) in common bean. We experimentally validated a novel miR398 target: the stress up-regulated Nodulin 19 (Nod19). Expression analysis of miR398b and target genes -CSD1 and Nod19- in bean roots, nodules and leaves, indicated their role in copper (Cu) homeostasis. In bean plants under Cu toxicity miR398b was decreased and Nod19 and CSD1, that participates in reactive oxygen species (ROS) detoxification, were up-regulated. The opposite regulation was observed in Cu deficient bean plants; lower levels of CSD1 would allow Cu delivery to essential Cu-containing proteins. Composite common bean plants with transgenic roots over-expressing miR398 showed ca. 20-fold higher mature miR398b and almost negligible target transcript levels as well as increased anthocyanin content and expression of Cu-stress responsive genes, when subjected to Cu deficiency. The down-regulation of miR398b with the consequent up-regulation of its targets was observed in common bean roots during the oxidative burst resulting from short-time exposure to high Cu. A similar response occurred at early stage of bean roots inoculated with Rhizobium tropici, where an increase in ROS was observed. In addition, the miR398b down-regulation and an increase in CSD1 and Nod19 were observed in bean leaves challenged with Sclerotinia scleortiorum fungal pathogen. Transient over-expression of miR398b in Nicotiana benthamiana leaves infected with S. sclerotiorum resulted in enhanced fungal lesions. We conclude that the miR398b-mediated up-regulation of CSD and Nod19 is relevant for common bean plants to cope with oxidative stress generated in abiotic and biotic stresses.
