## 3 Discussion  
In the present study, we analyzed the microRNA-mediated regulation of enzymes involved in herbicide detoxification in _Echinchloa oryzicola_ (Vasinger) Vasinger (late-watergrass). The aim was to understand some manifestations of herbicide resistance not related to the commonly known mechanisms (i.e., TSR). In particular, we wanted to explore the role of sncRNAs in this phenomenon. For this purpose, the expression of cytochromes P450 monooxygenase, glutathione S-transferase, and eukaryotic translation initiation factor 4B and that of miRNAs able to target mRNA of the same genes was analyzed before and after profoxydim treatment in susceptible (SUS) and resistant (RES1 and RES2) biotypes. Our results highlight that some of the miRNAs we identified can affect profoxydim resistance in _E. oryzicola_ collected from rice fields in the Lombardy region (Po Valley, northern Italy). Herbicide application triggered the transcription of some miRNAs which down-regulated the expression of target genes, reducing their detoxification ability. Otherwise, when herbicide spraying did not stimulate the transcription of miRNAs, the target gene mRNAs could be translated into proteins, leading to herbicide detoxification. It is known that epigenetics participates in stress responses. For example, chromatin modifications have been described when plants are exposed to stressful environments [28]. The role of sncRNAs in the adaptation of plants to chemical control (i.e., herbicide application) has only been considered recently. For example, [PERSON] et al. (2016) analyzed how miRNAs regulated the expression of genes involved in resistance to fenoxaprop-P-ethyl in _Beckmannia syzigaclne_ ([PERSON]). [PERSON] [45]. [PERSON] et al. (2015) recorded a significantly altered expression of several miRNA families upon treatment of maize (_Z. mays_) with glyphosate [46]. [PERSON] et al. (2022) investigated in _E. crus-galli_ the ability of some miRNAs to affect the expression of CYP450, GST, and _elF4B_, all involved in bispyribac-Na (ALS inhibitor) detoxification [36]. In light of these first observations, in the present study, we wanted to search for an epigenetic miRNA-mediated response to profoxydim (ACCase inhibitor) treatment in _E. oryzicola_. Figure 3 graphically summarizes the results regarding the expression of the studied miRNAs and of their target genes after the application of profoxydim in susceptible (SUS) and resistant (RES1 and RES2) biotypes. The expression of _ata-miR866c-5p, ath-miR847_, osa-miR85538, gra-miR7487c, osa-miR66a-5c,_ and _gra-miR8759_ was stimulated in the SUS biotype after herbicide application. On the other hand, reduced expression of their respective target genes (_CYP72A122, CYP71 AK2, EcGST, CYP72A254, CYP81A12_, and _elF4B_) was observed (Figure 3A). Considering the RES biotypes, the expression of _ata-miR866c-5p, ath-miR847, osa-miR8538_, and _gra-miR87487c_ was highly stimulated after profoxydim administration, and the expression of their target genes (_CYP72A122, CYP71 AK2, EcGST, CYP72A254_) was reduced (Figure 3B,C). The expression of the remaining miRNAs (_osa-miR82099-5p, ath-miR396b, osa-miR8395f, osa-miR8396a-5p,_osa-miRR166a-5p, osa-miRR166d-5p, gra-miRR8759, and gma-miRR396f)_ was not stimulated by the herbicide, and the expression of their target genes (CYP81A24, CYP81A22, CYP81A6, CYP81A63, CYP81A12, CYP81A21, _elF4B_, and _GSTF1_) did not appear to reduce after herbicide application. Hence, after profoxydim treatment, the expression of some genes (CYP450, 81A subfamily) was higher in the RES biotypes (Figure 3B,C) than in the susceptible one (Figure 3A). Our findings are in agreement with [PERSON] et al., 2014 [18] and with [PERSON] et al., 2019 [17], who demonstrated the involvement of CYP450 of the 81A subfamily in the detoxification of ALS and ACCase inhibitors.