## 2 Results
### Expression of CYP81A and miRNAs Targeting Their Transcripts
Expression of _CYP7 IA_, _CYP7 IA_, _EcGST_, _GSTF1_, _and_ eIF4B_ and of the miRNAs Targeting Their Transcripts  
Results concerning cytochromes P450 belonging to the 71A and 72A subfamilies, _EcGST_, _GSTF1_, _elF4B_, and the miRNAs targeting their transcripts are reported in Figure 2. Significant changes in the expression levels (fold change \(>\) 2) of genes and miRNAs are identified with a \(p\) < 0.05. The expression of _CYP7 IAK2_ (Figure 2A) showed very low levels (\(\leq\)1.00) in the SUS, RES1, and RES2 biotypes before profoxydim application and further decreased after spraying. Meanwhile, after herbicide treatment, _ath-miR847_ transcription significantly rose in the SUS (from 1.02 \(\pm\) 0.26 to 2.82 \(\pm\) 0.63), RES1 (from 0.69 \(\pm\) 0.21 to 6.58 \(\pm\) 2.30), and RES2 (from 2.76 \(\pm\) 1.25 to 15.25 \(\pm\) 6.32) biotypes. Low values of _CYP7 IA122_ expression (Figure 2B) were recorded in the SUS, RES1, and RES2 biotypes both before and after profoxydim administration (\(\leq\)1.00), while _athi-miR66c-5p_ transcription significantly rose (_p_ < 0.05) in SUS (from 1.02 \(\pm\) 0.26 to 4.79 \(\pm\) 1.45), RES1 (from 0.87 \(\pm\) 0.28 to 51.91 \(\pm\) 17.90), and RES2 (from 1.25 \(\pm\) 0.39 to 8.93 \(\pm\) 1.18) after herbicide application. Similar expression profiles were shown for _CYP7 IA254_ and _gra-miR7486c_ (Figure 2C), with the _CYP7 IA254_ mRNA being under-expressed in the SUS, RES1, and RES2 biotypes after herbicide application (\(\leq\)1.00 fold) and _gra-miR7486c_ being overexpressed in SUS (1.00 \(\pm\) 0.06 vs. 2.39 \(\pm\) 0.17; \(p\) < 0.05), RES1 (0.96 \(\pm\) 0.12 vs. 1.08 \(\pm\) 0.30; \(p\) > 0.05), and RES2 (1.91 \(\pm\) 0.15 vs. 3.41 \(\pm\) 0.26; \(p\) < 0.05) after herbicide application. _EcGST_ (Figure 2D) showed low expression values in all of the analyzed biotypes, both before and after profoxydim treatment (\(\leq\)1.00), with its expression further decreasing after spraying. _Osa-miR_85538 expression displayed appreciable values even before treatment, and significantly increased (\(p<0.05\)) after herbicide administration in SUS (from 1.04 \(\pm\) 0.38 to 3.64 \(\pm\) 0.53), RES1 (from 1.23 \(\pm\) 0.34 to 7.58 \(\pm\) 0.80), and RES2 (from 0.91 \(\pm\) 0.39 to 2.12 \(\pm\) 0.90). In the SUS biotype, both _GSTF1_ mRNA and _gma-miR_8396f_ expression (Figure 2E) were found to be unaffected by herbicide treatment, while in resistant biotypes, the expression of _GSTF1_ mRNA significantly increased (\(p<0.05\)) in RES1 (from 0.29 \(\pm\) 0.006 to 2.97 \(\pm\) 0.19)  
Figure 2: Expression levels of the miRNAs studied (red) and the mRNA of their target genes (blue) in susceptible (SUS) and resistant (RES1 and RES2) biotypes of _Echinchloa orgzicola_ before (BT) and after (AT) profoxydim treatment. Mean and standard error values are indicated as bar plots and error bars, respectively. The expressions of genes and miRNAs were normalized with those of the respective internal references (_b-Actin_ and _non-coding small nuclear RNA U6_). **(A)**_CYP1 AK2_ and _ath-miR847_; **(B)**_CYP2A122_ and _ata-miR866c-5p; **(C)**_CYP7A2A54_ and _ga-miR87487c; **(D)**_EcGST_ and _osa-miR85538_; **(E)**_GSTF1_ and _gma-miR8396f; **(F)**_elF4B_ and _gma-miR8759. \(t\)_-test:_ *:_ \(p<0.05\). and in RES2 (from 0.01 \(\pm\) 0.001 to 4.65 \(\pm\) 0.62) after profoxydim application. Meanwhile, _gma-miR8396_ expression significantly decreased after herbicide spraying (6.28 \(\pm\) 0.73 vs. 2.97 \(\pm\) 0.79 for RES1 and 2.32 \(\pm\) 0.13 vs. 0.43 \(\pm\) 0.01 for RES2). In Figure 2F, the expression profiles of _elF4B_ and _gra-miR8759_ are reported. It can be observed that, in the SUS biotype, the expression of _elF4B_ did not vary after treatment, while it significantly increased in RES1 (0.26 \(\pm\) 0.03 vs. 3.06 \(\pm\) 0.34) and RES2 (0.03 \(\pm\) 0.003 vs. 4.85 \(\pm\) 0.90).