Certain subnetworks were extracted for further investigations. miR393b, specifically expressed at the reproductive stage, targets two F-box domain- and LRR-containing protein-coding genes, both of which are involved in flower development based on the GO annotations (Fig. 4a-1). Both target genes were highly expressed at the reproductive stage. Although the interaction miR393b–LOC_Os05g05800.1 (OsFBL21: F-box domain- and LRR-containing protein, expressed; RAP ID, Os05g0150500) is ‘not well supported’ based on our filtering criteria, evident cleavage signal could be found in the ‘reproductive’ degradome library GSM476257, strongly indicating the existence of this interaction in planta. LOC_Os02g47420 (ATROPGEF7/ROPGEF7, putative, expressed), also highly expressed at the reproductive stage, is targeted by miR5156. Interestingly, the orthologous gene of LOC_Os02g47420 in maize encodes a pollen-specific kinase (Fig. 4a-3). The target gene LOC_Os03g19480 (SET domain-containing protein, expressed) of miR2275d is implicated in reproduction, although it is not highly expressed at the reproductive stage (Fig. 4a-4). We also recognized that certain genes highly expressed at the vegetative stage were also included in the reproductive-specific miRNA-mediated subnetworks, such as miR2118c/q–LOC_Os07g04040.1 (expressed protein; RAP ID, Os07g0132500) and miR5527–LOC_Os03g32040.1 (phenazine biosynthesis protein, putative, expressed; RAP ID, Os03g0434400; Figs 4a-2, S6). Many targets within the network mediated by the reproductive-specific sRNAs are involved in reproduction-related biological processes, such as LOC_Os03g22570 (MIF4G domain-containing protein, putative, expressed) and LOC_Os03g44200 (beclin-1, putative, expressed) targeted by sRNA490 (Fig. 4b-1), LOC_Os03g57870 (RFC3: Putative clamp loader of PCNA, replication factor C subunit 3, expressed) targeted by sRNA508 (Fig. 4b-2), LOC_Os07g06970 (HEN1, putative, expressed) targeted by sRNA53 (Fig. 4b-3), and LOC_Os03g29680 (EARLY flowering protein, putative, expressed) simultaneously targeted by sRNA353, sRNA399 and sRNA430 (Fig. 4b-4). Within the network mediated by the vegetative-specific miRNAs, LOC_Os05g03040 encoding an AP2 domain-containing protein is targeted by miR172c (Fig. 4c). Both miR172c and LOC_Os05g03040 were highly expressed at the vegetative stage, and the interaction miR172c–LOC_Os05g03040 was ‘well supported’ by the degradome sequencing data. In Arabidopsis, the regulatory cascade miR172–AP2 plays an important role in flower development. And miR172 regulates AP2 genes through translational repression (Aukerman & Sakai, 2003; Chen, 2004). Intriguingly, the cleavage-based interaction between miR172c and LOC_Os05g03040 identified in rice indicates another novel role of the miR172–AP2 cascade in developmental phase maintenance or transition. This plausible conclusion needs further confirmation. Within the network mediated by the vegetative-specific sRNAs, many targets are involved in postembryonic development or leaf senescence, such as LOC_Os03g48970 (nuclear transcription factor Y subunit, putative, expressed) targeted by sRNA37 (Fig. 4d-1), LOC_Os06g12870 (leaf senescence related protein, putative, expressed) targeted by sRNA407 and sRNA46 (Fig. 4d-2), LOC_Os10g04580 (ras-related protein, putative, expressed) targeted by sRNA277 (Fig. 4d-3), LOC_Os05g48870 (auxin response factor 15, putative, expressed) targeted by sRNA395 (Fig. 4d-4), and LOC_Os02g49840 (OsMADS57: MADS-box family gene with MIKCc type-box, expressed) targeted by sRNA35 (Fig. 4d-6). For all the networks constructed in this study, we found that the interactions ‘well supported’ by degradome data tended to be the sRNA–target pairs with the sRNA regulators and the target genes sharing consistent expression patterns (Fig. 4d-6), but not the pairs with different expression patterns (Fig. 4d-5).
Figure 4. Target expression pattern- and cleavage pattern-based analysis of the subnetworks mediated by the developmental stage-specific small RNAs in rice. (a) Subnetworks mediated by the reproductive stage-specific microRNAs (miRNAs). (b) Subnetworks mediated by the reproductive stage-specific small RNAs (sRNAs). (c) Subnetworks mediated by the vegetative stage-specific miRNAs. (d) Subnetworks mediated by the vegetative stage-specific sRNAs. For all the subnetworks in (a)–(d), the miRNA/sRNA regulators are highlighted in red background color, the targets highly expressed during the vegetative stage are in blue, and the targets highly expressed during the reproductive stage are in orange. For some target genes, the annotations and the Gene Ontology (GO) terms are provided based on the information obtained from the rice genome annotation project established by the Institute for Genome Research (currently called the J. Craig Venter Institute) (TIGR rice, http://rice.plantbiology.msu.edu/expression.shtml; Yuan et al., 2003). The miRNA/sRNA–target interactions denoted by solid edges show that the stage-specific patterns of the prominent cleavage signals are highly correlated with the expression patterns of the miRNA/sRNA regulators. The miRNA/sRNA–target interactions denoted by dashed edges show that the stage-specific patterns of the prominent cleavage signals are not well correlated with the expression patterns of the miRNA/sRNA regulators. The target plots (t-plots) show the cleavage evidence for the listed miRNA/sRNA–target pairs based on the degradome sequencing data. The degradome data sets were divided into two groups: GSM434596 and GSM455938, prepared during the vegetative stage of rice (indicated by gray symbols; see detail in the first t-plot); and GSM455939 and GSM476257, prepared during the reproductive stage (black symbols). For each t-plot, the miRNA/sRNA regulator and the target transcript ID are shown at the top. The y-axis measures the intensity (in reads per million, RPM) of the cleavage signal, and the x-axis indicates the position of the cleavage signal on a specific transcript. The binding site of a miRNA/sRNA regulator on its target transcript is denoted by a blue horizontal line. The dominant cleavage signals are indicated by red arrowheads, and those that are not dominant but quite evident are indicated by green arrowheads.
Download figure to PowerPoint