Abstract
In plants, microRNAs have been indicated to control various developmental processes, including somatic embryogenesis (SE), which is triggered in the in vitro cultured somatic cells of plants. Although a transcriptomic analysis has indicated that numerous MIRNAs are differentially expressed in the SE of different plants, the role of specific miRNAs in the embryogenic reprogramming of the somatic cell transcriptome is still poorly understood. In this study, we focused on performing a functional analysis of miR396 in SE given that the transcripts of MIR396 genes and the mature molecules of miR396 were found to be increased during an SE culture of Arabidopsis. In terms of miR396 in embryogenic induction, we observed the SE-associated expression pattern of MIR396b in explants of the β-glucuronidase (GUS) reporter line. In order to gain insight into the miR396-controlled mechanism that is involved in SE induction, the embryogenic response of mir396 mutants and the 35S:MIR396b overexpressor line to media with different 2,4-Dichlorophenoxyacetic acid (2,4-D) concentrations was evaluated. The results suggested that miR396 might contribute to SE induction by controlling the sensitivity of tissues to auxin treatment. Within the targets of miR396 that are associated with SE induction, we identified genes encoding the GROWTH-REGULATING FACTOR (GRF) transcription factors, including GRF1, GRF4, GRF7, GRF8, and GRF9. Moreover, the study suggested a regulatory relationship between miR396, GRF, and the PLETHORA (PLT1 and PLT2) genes during SE induction. A complex regulatory relationship within the miR396–GRF1/4/8/9–PLT1/2 module that involves the negative and positive control of GRFs and PLT (respectively) by miR396 might be assumed.
Highlights
Somatic embryogenesis (SE), a unique plant-specific developmental process, results in the formation of somatic embryos from in vitro cultured somatic cells/tissue
Given that Indolilic-3-acetic acid (IAA) contributed to the pool of indolic compounds, these results suggest a negative impact of the miR396 expression on the IAA accumulation in the SE-induced explants, and we assume that miR396 might control the auxin biosynthesis pathway during SE induction
To confirm that the miR396-regulated GROWTH-REGULATING FACTOR (GRF) contributed to SE induction via an auxin-related mechanism, we evaluated the embryogenic response of the 35S:GRF1 transgenic line and the grf4 mutant in media with different concentrations of 2,4-D
Summary
Somatic embryogenesis (SE), a unique plant-specific developmental process, results in the formation of somatic embryos from in vitro cultured somatic cells/tissue. In concert with TFs, microRNAs, which seem to preferentially target TF genes to control plant development [10,11], have been suggested as regulating SE induction. In line with this assumption, the differential expression of numerous miRNAs has been indicated in an embryogenic culture of Arabidopsis, and the function of the candidate microRNAs was attributed mainly to hormone and stress responses [12]. In order to gain insight into the function of miR396 in SE induction, we studied the embryogenic response that is induced in in vitro cultured Arabidopsis explants in relation to the miR396 expression level and auxin treatment. The regulatory relationship between the miR396–GRF module and the PLETHORA genes, including PLT2, was investigated
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