An important feature of RNA silencing in plants is that it can be amplified, notably in a selective manner: in some cases, RNAs targeted by sRNAs, in parallel to their degradation, become templates for RNA-DEPENDENT RNA POLYMERASES (RDRs), resulting in de novo synthesis of dsRNA. This dsRNA is in turn processed by DCLs into so-called ‘secondary siRNAs’, which amplify the original sRNA signal and allow silencing of RNA related in sequence to the primarily targeted RNA. Trans-acting siRNAs (ta-siRNAs) from a highly conserved class of plant sRNA that characterizes this type of amplification mechanism. MiR390-mediated cleavage of TAS3 ncRNAs triggers the recruitment of SUPPRESSOR OF GENE SILENCING 3 (SGS3) and RDR6, resulting in the production, by DCL4, of 21-nt ta-siRNAs acting to silence complementary mRNA in trans. The resulting conserved ta-siRNAs are known as ‘ta-siARF’ as it targets AUXIN RESPONSE FACTOR (ARF) gene (Lin et al. 2015).
To date, there are two kinds of TAS3 genes described in plants; they both contain two target sites for miR390, but TAS3-short (TAS3S) can be cleaved at both sites, producing a single, centrally-located ta-siARF. While in TAS3-long (TAS3L), only the 3’ target site is cleavable and sets the phase of ta-siRNA production, generating two ta-siARFs. 5’
miR390 target site of TAS3L is usually non-cleavable because of the presence of a central mismatch (Xia et al. 2015). In our study, when examining the PARE data, we validate the target of Ssp-miR390 is truly SsTAS3. Similar to TAS3 in other species, SsTAS3 have two miR390 target sites. Intriguingly, the 3’ site show a sharp and clear cleaved signal, whereas none of the reads were found to truncated at the 5’ target site. When aligning the sequences of Ssp-miR390 and SsTAS3, we found a central mismatch at the 10th and 11th nt from the 5’ end of miRNA in the complementary region of the target transcript. Thus,
we confirmed the cleavage event of Ssp-miR390 on SsTAS33’ site. In addition, the central mismatch at 5’ target site allow us to classify our SsTAS3 to the TAS3L type. The absent of cleavage signal on TAS3S is identical to the findings in Mimulus corolla, suggesting that the TAS3S gene is not expressed in the flower (Ding et al., 2018). Next, to examine the production of ta-siARFs, the reads from sRNA-Seq are mapped against the SsTAS3.
We found two abudant 21-nt mapped reads that were in phase with the 3’ site; These two siRNAs show high sequence similarity to each other, and also identical to the ta-siARFs of Arabidopsis ta-ta-siARFs. These results validate the production of ta-siARF from SsTAS3, and the 3’ site is the main trigger site of ta-siARF generation. The above evidence proved the existence of conserved miR390/TAS3/ta-siARF in the Gloxinia petal.
Regarding the dorsoventrally expression pattern of Ssp-miR390, we hypothesized that the differential morphogenesis of dorsal and ventral petal may mediated through the tasiARF-ARF pathway. Because Ssp-miR390 expresses higher in the ventral petal, our hypothesis makes two clear predictions: (i) The cleavage event on SsARFs should be more dramatic in the ventral petal. (ii) The abundance of SsARFs transcripts should be lower in the ventral petal compared to that in the dorsal petals. To test the first prediction, we examined the PARE data, intriguing, the cleavage signal of ta-siARF on SsARF3 could only be found in ventral petal. The results confirmed the first prediction. To test the second prediction, qRT-PCR measurement in Gloxinia showed that SsARF3 appeared more depleted in the ventral petal, supporting our second prediction.
After confirmation of miR390/ARF3 being dorsoventrally expressed in Gloxinia, we are curious about the biological role it plays in the establishment of the zygomorphic flower. MiR390/ARF3 has been reported to participate in distinct aspect of plant development. This includes leaf polarity, lateral root growth, somatic embryo development and the formation of corolla tube (Marin et al., 2010, Rubio-Somoza and
Weigel, 2011, Lin et al., 2015). Among them, the roles in the pathway of miR390/TAS3/AGO7/ARF3 in leaf polarity determination is well-studied and clear. In most plants, the two surfaces of the leaf are specialized, with the photosynthesizing light harvesting cells concentrated at the upper side and gas-exchanging cells at the lower side.
The up-down polarity of a leaf is formally known as adaxial-abaxial polarity. The whole set of the miR390/ta-siARF pathway are highly conserved in plants; however, the detailed mechanism varies among species. In rice, the mature miR390 and OsAGO7 localizes to the adaxial side of the incipient primordium, where it triggers the production of ta-siARF small RNAs. These ta-siARFs then travel toward the abaxial side, providing a mobile component in ad-ab patterning (Fig. 12). However, in Arabidopsis, miR390 is ubiquitously present in the meristem and leaf primordia. The polarity of ta-siARF is built because of the restricted expression of AtAGO7 and AtTAS3. Thus, as in rice, the ta-siARF small RNAs gradient of expression rely on spatially-restriction of both the Osa-miR390 and OsAGO7. This gradient results in a precisely defined abaxial expression of OsARF3/4 (Fig. 12). Whereas Arabidopsis leaf builds the gradient by the spatially-restricted AGO7 and TAS3, the spatially expression of miR390 seems an inessential factor.
Based on the study in leaf polarity, now we discovered the whole set of miR390/TAS3/ta-siARF pathway are also present in the Gloxinia flower. Furthermore, the polarity appears to build across the dorsal and ventral petal. The findings raise an intriguing question regarding whether the miR390/TAS3/ARF3 evolved a co-option of leaf abaxial-adaxial polarity into floral dorsi-ventral zygomorphy. To further examine the possibility of this interesting hypothesis, we cloned the SsTAS3 and SsAGO7, and then performed a qRT-PCR examination. Interestingly, the preliminary data indicated that SsAGO7 showed dorsoventrally differentially expression (data not shown). The SsAGO7 displayed abundantly in the ventral petal. The result concurs with our previous finding
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that SsARF3cleavage event specifically detected in the ventral petal. Beside the abaxial determinant, we also discover Ssp-miR166, an adaxial determinant in the leaf polarity, expressed more intensively in the dorsal petal. In other word, the key players participated in the determination of leaf polarity now evolve to show dorsoventrally pattern in the gloxinia zygomorphic flower. However, to further validate the hypothesis, more tasks should be done, including the expression profile of the SsTAS3 and HD-ZIPIII mRNAs.
Also, to correlate the molecular mechanism to the flower symmetry, we planned to recruit the actinomorphic flower of Gloxinia, if the miR390/TAS3/ta-siARF pathway indeed the crucial determinant in deciding the zygomorphic symmetry, we expect to find an evenly distributed of miR390, AGO7 and ARF3, instead of a dorsoventrally pattern.
(Siobhan A. Braybrook and CrisKuhlemeier, 2010)
(Siobhan A. Braybrook and CrisKuhlemeier, 2010)
(Siobhan A. Braybrook and CrisKuhlemeier, 2010)
(Siobhan A. Braybrook and CrisKuhlemeier, 2010)
Figure 14 | A comparison of the miR390/TAS3/ta-siARF pathway in leaf of Arabidopsis and rice
with in the flower of Gloxinia.
Conclusion
Using a computational pipeline, we identified 157 miRNAs in the developing petals of Gloxinia. Among them, our analysis revealed that miR157 and miR390 show dorsoventrally differentially expression. Further, the target genes of Ssp-miR157 and Ss-miR390 were validated through PARE analysis. Consistent to our expectation, the expression patterns of these target genes show negative correlation with the expression levels of their corresponding miRNAs. Thus, the present study revealed a regulatory network of miRNAs and may further facilitate the investigation of the functional importance of miRNA-mediated gene regulation during the establishment of differential dorsal and ventral petal morphogenesis.