Research paperInvestigating the regulatory roles of the microRNAs and the Argonaute 1-enriched small RNAs in plant metabolism
Introduction
Plant metabolism involves diverse biochemical pathways which are under accurate modulation and strict surveillance by numerous biological factors including transcription factors, enzymes, environmental cues and so on. The plant metabolic networks are becoming more and more complicated when increasing regulatory factors are being identified. Establishing highly comprehensive networks including biochemical pathways for metabolite biosynthesis and regulatory cascades for ensuring metabolic reactions taken place within specific cellular contexts or at appropriate developmental stages is an essential request for the plant biologists to perform metabolic engineering (Bocobza and Aharoni, 2014, Patra et al., 2013, Sweetlove and Fernie, 2013, Toubiana et al., 2013, Wang et al., 2012, Sweetlove and Fernie, 2005).
MicroRNAs (miRNAs) are recently identified as ~ 21-nt small RNA (sRNA) molecules playing indispensable regulatory roles in plants. After incorporating into Argonaute 1 (AGO1)-associated silencing complexes, miRNAs bind to specific target transcripts and guide the AGO1 complexes to perform target cleavages. This mode of regulation by miRNAs is implicated in various biological processes in plants, such as organ development, reproduction and stress response (Jones-Rhoades et al., 2006, Voinnet, 2009). Additionally, recent studies uncovered that some of the plant miRNAs are implicated in metabolic pathways through the regulation of specific target genes. For example, miR156 can modulate the process of anthocyanin biosynthesis by targeting SPL9 (Squamosa promoter binding Protein-Like 9) in Arabidopsis (Gou et al., 2011). miR163 and its target genes contribute to the natural variation of secondary metabolites in two Arabidopsis species and their allopolyploids (Ng et al., 2011). miR393-mediated auxin signaling could re-direct secondary metabolite biosynthesis away from camalexin and towards glucosinolates, resulting in higher biotroph resistance (Robert-Seilaniantz et al., 2011). miR395 is demonstrated to be an integral part of the regulatory network of sulfate assimilation (Matthewman et al., 2012). The above research progresses advanced our understanding of the regulatory roles of miRNAs in plant metabolism. Dozens of miRNA families have been reported in plants according to miRBase release 20 (Kozomara and Griffiths-Jones, 2014). However, whether the metabolism-related regulatory role is wide-spread among the miRNA population remains unclear. In addition to the miRNAs, the other sRNAs possess great potential of performing cleavage actions on specific targets (Baumberger and Baulcombe, 2005, Vaucheret, 2008). Notably, some evidences related to the involvement of the other sRNA species in plant metabolism are emerging. For example, SRO5 is induced during salt stress. The SRO5 transcript could pair with another stress-related, constitutively expressed transcript encoded by P5CDH. A 24-nt siRNA (small interfering RNA) is produced from the paired region of the two transcripts through DCL2-, RDR6-, SGS3- and NRPD1A-dependent pathway, which mediates the initial cleavage of the P5CDH transcript and establishes a phase for subsequent biogenesis of 21-nt siRNAs through DCL1-dependent pathway. Then, the 21-nt siRNAs could perform cis‑regulation of the P5CDH transcript through cleavages, thus forming a feedback regulatory loop implicated in salt tolerance in Arabidopsis (Borsani et al., 2005). However, except for the miRNAs, to what extent are the other sRNA species involved in plant metabolic pathways remains elusive.
In this study, we performed a comprehensive study in two model organisms Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) to investigate the biological role of miRNAs and AGO1-enriched sRNAs in metabolism. After retrieving metabolism-related transcripts from Plant Metabolic Network (PMN; http://www.plantcyc.org/), target prediction and validation were performed by using the publicly available sRNA and degradome sequencing data. As a result, 251 and 168 miRNA/sRNA-target pairs were identified in Arabidopsis and rice respectively, enabling us to establish regulatory networks potentially involved in plant metabolic processes. Interactions between the protein products of specific target genes led the networks to be more complicated. Target annotation analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa and Goto, 2000) pathway mapping revealed several functionally related subnetworks between the two plants. Summarily, our study provides a basis for further in-depth analyses of the biological functions of the sRNA molecules in plant metabolism.
Section snippets
Data sources
The genes involved in plant metabolism were obtained from Plant Metabolic Network (http://www.plantcyc.org/). The gene annotations and the cDNA sequences of the two plant species were retrieved from TAIR (The Arabidopsis Information Resource; release 10) (Huala et al., 2001) and MSU (Michigan State University) Rice Genome Annotation Project (release 7) (Kawahara et al., 2013, Yuan et al., 2003). The functional module “KEGG Mapper – Search&Color Pathway” (//www.genome.jp/kegg/tool/map_pathway2.html
Identification of the metabolism-related transcripts targeted by miRNAs and AGO1-enriched sRNAs in plants
We utilized six and three groups of sRNA HTS datasets to search for AGO1-enriched sRNAs in Arabidopsis and rice respectively (see details of the HTS datasets in Materials and methods). We adopted the following criterion to extract the AGO1-enriched sRNAs: for each group, the accumulation levels of an AGO1-enriched sRNA should be 10 RPM (reads per million) or higher in the “AGO1” dataset, and should be undetectable in the “control” set. After removing the redundant sRNAs (i.e. the identical sRNA
Conclusions
In this study, sRNAs enriched in AGO1 protein complexes were identified in Arabidopsis and rice. In addition to the miRNAs, the AGO1-associated sRNAs were sent for target site prediction on the transcripts encoding protein products related to plant metabolisms. Based on degradome validation, dozens of sRNA-target pairs were discovered, which could be valuable for further studies on the gene regulatory networks related to plant metabolisms. Notably, several sRNA-target pairs were suggested to be
Acknowledgements
We would like to thank all the publicly available datasets and the scientists behind them. This work is financially supported by the National Natural Science Foundation of China [31601062] and [31571349], Zhejiang Provincial Natural Science Foundation of China [LQ16C060003] and [LY15C060006], and Hangzhou Scientific and Technological Program [20170432B04].
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