Abstract
During early plant embryogenesis, some of the most fundamental decisions on fate and identity are taken making it a fascinating process to study. It is no surprise that higher plant embryogenesis was intensively analysed during the last century, while somatic embryogenesis is probably the most studied regeneration model. Encoded by the MIRNA, short, single-stranded, non-coding miRNAs, are commonly present in all Eukaryotic genomes and are involved in the regulation of the gene expression during the essential developmental processes such as plant morphogenesis, hormone signaling, and developmental phase transition. During the last few years dedicated to miRNAs, analytical methods and tools have been developed, which have afforded new opportunities in functional analyses of plant miRNAs, including (i) databases for in silico analysis; (ii) miRNAs detection and expression approaches; (iii) reporter and sensor lines for a spatio-temporal analysis of the miRNA-target interactions; (iv) in situ hybridisation protocols; (v) artificial miRNAs; (vi) MIM and STTM lines to inhibit miRNA activity, and (vii) the target genes resistant to miRNA. Here, we attempted to summarise the toolbox for functional analysis of miRNAs during plant embryogenesis. In addition to characterising the described tools/methods, examples of the applications have been presented.
Highlights
During early plant embryogenesis, some of the most fundamental decisions on fate and identity are taken making it a fascinating process to study
We can find more than 10,200 records for mature micro RNA (miRNA) molecules in plants from 92 species in the miRbase, while for Arabidopsis, there is information about 428 mature miRNA molecules. miRNAs are encoded by MIRNAs (MIRs), which are intergenic or intronic genes that are present in plant genomes in one hundred to as many as several hundred loci, which can be located in genomic regions that are distinct from the known transcription units and that have their own promoter and terminator sequences such as mono or polycistronic MIRs
It is worth mentioning that the biogenesis pathway is under a strict, very comprehensive regulation [15] and that one of the factors that affects the miRNA biogenesis machinery are the miRNA molecules themselves [16]. miR168 is present in all plants and acts as a regulator of AGO1 [17]; while, in Arabidopsis and Physcomitrella patens, miR162 negatively regulates the DICER-like1 RNase III endonuclease (DCL1) [18]
Summary
Artificial miRNAs (amiRNA) are extensively used in the field of plant molecular biology as a versatile tool of RNAi methods. The company Thermo Fisher Scientific introduced the mirVanaTM miRNA inhibitors, which are chemically modified, single-stranded RNA molecules that are designed to bind to and inhibit endogenous miRNA and to enable the miRNA functional analysis by downregulating the miRNA activity. That mutant became the first use of miRNA-resistant target mRNA in a miRNA functional analysis of the development of zygotic embryos in Arabidopsis [166,167] and recently during SE induction [126] Later, this approach was applied to describe the engagement of miR156/157, miR160, miR164, miR165/166, miR167, miR319, miR393, and miR396 in ZE regulation in Arabidopsis and rice [28,41,113,117,168,169,170,171,172,173]. The fact that miRNAs usually have more than one target and that the overexpression of a miRNA-resistant target may not reveal a complete picture of the miRNA functions and phenotypes corresponding to transgenic artifacts must be considered [176]
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