Three fenoxaprop-P-ethyl-resistant B. syzigachne populations possessing both NTSR and TSR and one susceptible population (S) were used in this study (Pan et al., 2016). These populations were collected from the following locations: S from Feidong in Anhui Province, where fenoxaprop-P-ethyl was never applied; R1 from Xinlucun in Jiangsu Province with 10 years of fenoxaprop-P-ethyl use history; R2 from Lijiacun in Jiangsu Province, with 6 years of fenoxaprop-P-ethyl use; and R3 from Guocun in Jiangsu Province, with 5 years of fenoxaprop-P-ethyl use. These locations were more than 118 km apart from each other.

In our previous studies, we only compared the differences in gene expression between one resistant and one susceptible population, which might yield few “false positive” alleles that show differential expression between populations, but irrelevant to NTSR. Therefore, in the present study, we used three fenoxaprop-P-ethyl-resistant populations with different genetic backgrounds to minimize “false positives.”

Seeds were planted in pots filled with a 2:1 (wt/wt) mixture of sand and loam soil (pH 5.6; organic matter content 1.4%). All pots were placed in a greenhouse at 20°C/15°C day/night under a photoperiod of 12 h/12 h.

Tobacco (Nicotiana benthamiana) plants were grown in vermiculite containing Murashige and Skoog salt nutritional liquid in a growth chamber (light:dark period of 12:12 h at 25°C:18°C). Six-week-old tobacco plants were used for Agrobacterium tumefaciens-mediated transient expression as described (Chen et al., 2008).

The rice (Oryza sativa L.) wild type (WT) and miR397-overexpressing transgenic (OXmiR397) plants were kindly provided by Prof. Yueqin Chen from the Sun Yat-sen University (Zhang et al., 2013). The details about the rice plants were described (Zhang et al., 2013). Rice plants were grown in the culture solution (Zhang et al., 2013) supplemented with fenoxaprop-P-ethyl in a glasshouse with a day/night cycle of 12 h/12 h at 30°C/24°C.

From the B. syzigachne mRNA sequence library (PRJNA290808) and miRNA sequence library (PRJNA290416) established in our laboratory, the B. syzigachne laccase gene (accession number: KP952245) and the B. syzigachne miR397 (sequence: 5′-ATTGAGTGCAGCGTTGATGAA-3′) were detected, and they were named as bsy-Laccase and bsy-miR397, respectively.

In total 300 plants from each of the three populations were cultivated to the tillering stage in 3-inch pots at day/night temperature of 20°C/15°C under a photoperiod of 12 h/12 h. Two tillers were collected from each plant: one for resistance/susceptibility analysis and the other for seed production. Resistance/susceptibility tested to fenoxaprop-P-ethyl was tested at the recommended field dose (62.0 g a.i.⋅ha^-1), and survival assessed 3 weeks after treatment. The confirmed resistant tillers were placed in a pollen mask to produce progenies. In total, 15 susceptible and 285 resistant individuals were confirmed in R1 population; 15 susceptible and 285 resistant individuals in R2 population; and 16 susceptible and 284 resistant individuals in R3 population. The resistance tillers were self-pollinated in isolation to produce seeds of progeny populations. Thus, three resistance-enriched populations (R1, R2, and R3) were generated (Pan et al., 2016). With this method, three “purified” progeny R populations were obtained (thus minimizing genetic variation unrelated to fenoxaprop-P-ethyl resistance).

Plants from each population were cultivated up to the 3- to 4-leaf stage under the experimental conditions described above. Fenoxaprop-P-ethyl was applied at one-eighth of the recommended dose to allow survival of susceptible plants for gene expression analysis (Pan et al., 2016). The expression of candidate genes was measured in 30 untreated and 30 fenoxaprop-P-ethyl-treated plants for each fenoxaprop-P-ethyl dose at 24, 48, and 72 h. Total RNA was extracted using TRIzol (Invitrogen Inc., Carlsbad, CA, United States) following the manufacturer’s protocol. The quality and purity of RNA were assessed using the Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States). Each RNA sample (10 μg) was subjected to reverse transcription using the Prime Script RT Reagent Kit (TaKaRa, Dalian, China) to produce cDNA. Small RNAs were reverse transcribed to cDNA using the Prime Script^TM miRNA qPCR Starter Kit (Version 2.0, TaKaRa, Dalian, China). The absence of gDNA contamination was confirmed by lack of amplification of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the primers containing an intron sequence.

Real-time PCR was performed using the ABI-7500 Fast Real-Time PCR System (ABI, United States) by using the SYBR^® Premix Ex Taq^TM kit (TaKaRa, Japan).

The relative expression of genes to that of the control was calculated using the 2^-ΔΔCT method (Livak and Schmittgen, 2001). In each experiment, qPCR was conducted on each single plant (creating 30 data points) and repeated at least twice (two technical replicates). Significant differences in expression levels were analyzed using Welch’s t-test (Zheng et al., 2013). Two threshold values—a significant result in the t-test (P < 0.05) and a two-fold change were used to determine either up- or down-regulation. Capsine phosphatase, GAPDH, and ubiquitin were used as internal controls. The qPCR primers were designed based on the bsy-Laccase and bsy-miR397 sequences. The primers used for qRT-PCR are listed in Supplementary Tables S1, S2. The reverse primer 5′-GTGCAGGGTCCGAGGT-3′ was used for bsy-miR397 amplification.

We also included three populations (possessing TSR and in which metabolic resistance might not be involved) for comparison. These three populations were JYDX-R, SHQP-R, and JYSC-R. ACCase sequencing results showed that all plants in JYDX-R contained the Ile-1781-Leu mutation, those in SHQP-R contained the Ile-2041-Asn mutation, and those in JYSC-R contained the Gly-2096-Ala mutation. Next, 50 randomly selected plants from each of these populations were treated with fenoxaprop-P-ethyl + piperonyl butoxide (PBO, a known P450 inhibitor), and 20 randomly selected plants were treated with fenoxaprop-P-ethyl alone. No phenotypic differences were observed between plants subjected to fenoxaprop-P-ethyl alone and fenoxaprop-P-ethyl + PBO. When the three populations were compared with the S population, no difference was found in the structural and expressions of the 15 genes related to metabolic resistance. Our previous studies showed that PBO can partially reverse fenoxaprop resistance and that the 15 genes played a role in metabolic resistance in the three resistant populations R1, R2, and R3 (Pan et al., 2016); thus, populations JYDX-R, SHQP-R, and JYSC-R likely exhibited only TSR. The expression profiles of bsy-miR397 and bsy-Laccase in JYDX-R, SHQP-R, and JYSC-R populations were analyzed as described above.

Laccase was isolated using the protocol of Wang et al. (2004) with slight modifications. Leaf samples of the S, R1, R2, R3 and JYDX-R, SHQP-R, JYSC-R plants were harvested at the 3- to 4-leaf stage. Crude laccase was extracted from the leaf material (200 mg fresh weight) in 1 mL buffer (25 mM MOPS, 200 mM CaCl₂, pH 7.0), followed by centrifugation (16,000 × g, 10 min). Ten microliters of crude enzyme (about 6 μg protein) was added to a 0.5-mL mixture, and the reaction was conducted at 30°C for 30 min. Coomassie brilliant blue method was used to quantify the (total) protein using bovine serum albumin as the standard. Laccase activity prior to and after fenoxaprop treatment (24, 48, and 72 h) was determined colorimetrically by measuring the oxidation rate of 2 mg/mL 2,2-azobis (3-ethylbenzothiazoline-6-sulphonic acid) at 420 nm. The test was performed twice with three replicates per treatment.

The bsy-miR397 and bsy-Laccase co-transformation in tobacco leaves was used to validate the interaction between bsy-miR397 and bsy-Laccase genes. A full-length (1,740 bp) cDNA of bsy-Laccase was RT-PCR amplified from B. syzigachne by using the primers listed in Supplementary Table S3, and the sequence-confirmed PCR fragment was inserted into the pEarleyGate vector 202 under the control of a 35S promoter (Earley et al., 2006). The amiR-bsy-miR397 was constructed using a previously described method (Warthmann et al., 2008). The primers for amiR-bsy-miR397 amplification are listed in Supplementary Table S3. A 21-mer sequence was used to replace the endogenous miRNA and miRNA^∗ in the plasmid pRS300 (Schwab et al., 2006) designed using the Web MicroRNA Designer platform. The resulting amiRNA was cloned into pEarleyGate vector 201 under the control of a 35S promoter. All constructs were sequenced to confirm the intended construction and designs. Descriptions of the amiRNAs, their target sequences, and genes are listed in Supplementary Table S4.

The Agrobacterium strain GV3101 was used to transfect the recombinant vectors into tobacco leaf cells. The strains containing different recombinant vectors (35S:bsy-miR397, 35S:bsy-Laccase, and 35S:Os-miR164-control) were cultured to OD₆₀₀ = 0.8 at 28°C in a shaker at 220 rpm. Each cultured sample was collected and diluted to OD₆₀₀ = 0.5 with MgCl₂ and acetosyringone. The 35S:bsy-laccase and 35S:bsy-miR397 samples were mixed in equal volumes, and the mixture was adjusted to OD₆₀₀ = 1.0 and used to test the cleavage function of bsy-miR397. In the tobacco assay, laccase needs to be co-expressed with other non-effect miRNAs as control. As Os-miR164 has no effect on laccase, we co-expressed Os-miR164 and laccase to test our hypothesis. Subsequently, 100 μL of each treatment sample was used to infiltrate the tobacco leaves. The agrobacteria were infiltrated into the tobacco leaves, and treated leaves were harvested 2 days later. Total RNAs were extracted for qRT-PCR. Tobacco 18S rRNA expression was used as an internal standard for normalization. Primers used to detect the expression of bsy-Laccase and bsy-miR397 in these plants are listed in Supplementary Tables S1, S2.

Wild type and OXmiR397-overexpressing transgenic rice seeds were planted in pots containing commercial potting soil (Zhang et al., 2013) and maintained in a well-watered state in a greenhouse. When the seedlings reached the 3- to 4-leaf stage, fenoxaprop-P-ethyl was applied at the recommended field dose (62 g a.i.⋅ha^-1) to the WT and OXmiR397 rice lines. Whether miR397/laccase is involved specifically in fenoxaprop-P-ethyl resistance was determined by including another ACCase-inhibiting herbicide haloxyfop-R-methyl (recommended field dose, 40.5 g a.i.⋅ha^-1). Three weeks after treatment, the root length, plant height, and fresh weight were determined for each plant.

The target for OXmiR397 in rice is LOC_Os05g38420 (OsLAC) (Jeong et al., 2011; Zhang et al., 2013). The OsLAC expression and laccase activity were quantified in rice as described above. The primers were OsLAC-F (5′-GAGGAGGTGCCCATCATGTTC-3′) and OsLAC-R (5′-CCTTCAGCTTAAACGTGTCTTGG-3′) were used for quantification of OsLAC expression.

Laccase expression has been shown to be induced by 20 mM CuSO₄ (Zhu and Ding, 2003; Jin et al., 2008). This approach was also used to determine the role of miR397/laccase in fenoxaprop-P-ethyl resistance in B. syzigachne. Induction of the bsy-Laccase expression in B. syzigachne by 20 mM CuSO₄ was confirmed by comparing the expression level of bsy-Laccase with and without CuSO₄ treatment.

Seeds from the S and three B. syzigachne populations possessing both TSR and NTSR were germinated using the method described previously (Pan et al., 2015b). The 3-week-old (at the 3- to 4-leaf stage) plants were treated with fenoxaprop-P-ethyl alone, and 20 mM CuSO₄ plus fenoxaprop-P-ethyl using a laboratory sprayer as described previously (Pan et al., 2015b). The experiments were conducted twice in a completely randomized design with four replicates per treatment. Herbicide application rates were selected around the GR₅₀ value of each population. Fenoxaprop-P-ethyl was applied at 0, 5, 10, 20, 40, 80, and 160 g a.i. ha^-1 to the susceptible population (S) and at 0, 80, 160, 320, 640, 1280, and 2560 g a.i. ha^-1 to the resistant populations (R1, R2, and R3). CuSO₄ was applied 1 h before fenoxaprop-P-ethyl application. Three weeks after treatment, the above-ground material was harvested, and fresh weights were determined.

The experiments with fenoxaprop-P-ethyl and 20 mM CuSO₄ plus fenoxaprop-P-ethyl treatments in JYDX-R, SHQP-R, and JYSC-R populations were conducted as described above.

MiR397 has been implicated in the network for lignin biosynthesis via putative interactions with several transcription factors (TFs) and laccases in Populus trichocarpa (Lu et al., 2013). The possible mechanism of miR397/laccase in regulating plant resistance to fenoxaprop-P-ethyl was assessed by constructing B. syzigachne RNA-seq libraries using RNA from the S population and three R populations (R1, R2, and R3) (Pan et al., 2016). The four libraries were assayed for quality and quantity and sequenced by Beijing BioMarker Technologies (Beijing, China). The mRNA-Seq libraries were constructed and sequence data were analyzed, assembled, and annotated as described by Pan et al. (2016). Gene names of specific TFs were assigned based on the best Basic Local Alignment Search Tool hit (highest score), excluding any uninformative descriptions.

The results of the comparison between S and R libraries suggested that RSEM could be used to quantify the reads identified as TFs (Li and Dewey, 2011). Transcript levels were quantified by counting reads per kilobase of exon model per million mapped reads (RPKM) (Mortazavi et al., 2008). The RPKM method of read density reflects the molar concentration of a transcript by normalizing for RNA length and the total number of reads. The EBSeq software was used to determine P-values for the samples with no biological replicates. EBSeq employs an empirical Bayes hierarchical model (Leng et al., 2013). A two-fold change, a P-value of <0.01, and a minimum RPKM of 2 in at least one of the treatments were used as the criteria to classify a response as up- or down-regulation. TFs were found to be consistently up- or down-regulated in R1, R2, and R3.

The downstream effect of laccase down-regulation on the up-regulated pathways that contribute to NTSR against fenoxaprop were further confirmed by conducting additional experiments that detected the expressions of these TFs and oxidase/peroxidase genes in WT and OXmiR397 rice. Primers used for detecting their expression are listed in Supplementary Table S5.

Quantitative PCR was used to analyze the expression of bsy-miR397 and bsy-Laccase in 30 plants each of the S, R1, R2, R3, and JYDX-R, SHQP-R, JYSC-R population. Expression of bsy-miR397 was significantly higher in the 30 plants each of the R1, R2, and R3 populations than in the S population, whereas the expression of bsy-Laccase was significantly higher in the S plants than in the R1, R2, and R3 plants (Figure 1). The expression of bsy-miR397 was 8–9 times higher in plants of the R1, R2, and R3 populations than in the S population (Figure 1). In contrast, the expression of bsy-Laccase was 8–9-times lower in the plants of the R1, R2, and R3 populations than in the S population (Figure 1). Notably, we found that in JYDX-R, SHQP-R, and JYSC-R plants that likely exhibited only TSR, the expression patterns of bsy-miR397 and bsy-Laccase were consistent with that in the S plants (Figure 1).

The time-response expression profiles of bsy-miR397 and bsy-Laccase were determined in 30 plants each of the S, R1, R2, R3 and JYDX-R, SHQP-R, JYSC-R populations at 24, 48, and 72 h after fenoxaprop-P-ethyl treatment. In the S and JYDX-R, SHQP-R, JYSC-R plants, the expression of bsy-Laccase and bsy-miR397 remained unchanged (Figure 2). However, in the R1, R2, and R3 plants, after fenoxaprop-P-ethyl treatment, the expression of bsy-miR397 was up-regulated, whereas bsy-Laccase down-regulated (Figure 2). This was consistent with the expression of these two genes in the untreated R1, R2, and R3 plants. These data indicate a negative correlation between bsy-miR397 and bsy-Laccase expression both constitutively and in response to fenoxaprop-P-ethyl treatment in B. syzigachne R1, R2, and R3 populations.

Consistent with bsy-Laccase expression in B. syzigachne, a higher level (5.8- to 8.6-fold) of laccase activity was detected in the S than in R1, R2, and R3 plants (P < 0.05; Figure 3A). These results are consistent with those for bsy-Laccase gene expression, suggesting that laccase might play a role in fenoxaprop-P-ethyl resistance of B. syzigachne.

However, after fenoxaprop-P-ethyl treatment, laccase activity in R1, R2, and R3 plants decreased relative to that in the untreated R1, R2, and R3 plants (Figure 3B), whereas in the S plants laccase activity did not change considerably. We found that in JYDX-R, SHQP-R, and JYSC-R plants, laccase activity were consistent with that in the S plants either before or after fenoxaprop-P-ethyl treatment (Figure 3). These results are also consistent with the bsy-Laccase expression pattern in S and R1, R2, and R3 plants after fenoxaprop-P-ethyl treatment.

In rice, the expression of laccase genes is known to be regulated by miRNA397 (Zhang et al., 2013). The target gene of bsy-miR397 is predicted to be bsy-Laccase from http://www.mirbase.org, and the 714- to 734-bp region on the bsy-Laccase gene might be a target site for bsy-miR397. Hence, we hypothesized that bsy-Laccase gene could be cleaved by bsy-miR397. Since co-expression in tobacco is one of the best ways to validate the interaction between an miRNA and its predicted target (Qi et al., 2012), we co-expressed the bsy-miR397 and bsy-Laccase genes in tobacco.

Quantitative RT-PCR analyses revealed that the bsy-Laccase expression in tobacco carrying 35S:bsy-Laccase/35S:bsy-miR397 was 3.6-fold lower than that in tobacco plants carrying only 35S:bsy-Laccase (Supplementary Figure S1). These results indicate that bsy-Laccase expression was repressed by bsy-miR397 in transformed tobacco carrying 35S:bsy-Laccase/35S:bsy-miR397, and bsy-Laccase might be the target gene of bsy-miR397.

As miR397 is conserved across most dicotyledon and monocotyledon species, and fenoxaprop-P-ethyl has no activity in broadleaf tobacco, we attempted to further investigate the role of miR397/laccase in fenoxaprop-P-ethyl resistance by using OXmiR397-overexpressing transgenic rice and the corresponding WT lines.

Rice OXmiR397 is known to regulate laccase. To determine if this regulation has an impact on herbicide tolerance, we treated WT and OXmiR397-overexpressing transgenic rice with the field rate of fenoxaprop-P-ethyl (62 g a.i. ha^-1), with untreated rice plants as a control. Seven days after fenoxaprop-P-ethyl treatment, WT rice plants started to show phytotoxicity symptoms such as leaf withering and reduced plant height, whereas OXmiR397-overexpressing plants showed no visual damage. Three weeks after treatment, WT rice plants showed severe tissue damage, whereas OXmiR397-overexpressing plants remained largely healthy and green (Figure 4). In addition, OXmiR397-overexpressing plants exhibited significantly greater root length and plant height (Figure 4 and Table 1) as well as overall fresh weight than the WT rice (Figure 4 and Table 1). Six weeks after fenoxaprop-P-ethyl treatment, WT plants died, whereas OXmiR397-overexpressing plants only showed slight tissue damage (which occurred about a week later than the WT plants and never recovered). As expected, OXmiR397-overexpressing plants had lower levels of OsLAC expression and laccase activity than the WT plants (Figure 5). These results indicated that miR397/laccase regulation can also provide protection from fenoxaprop-P-ethyl damage in OXmiR397-overexpressing transgenic rice, suggesting that miR397/laccase might be involved in fenoxaprop-P-ethyl NTSR.

In contrast, 3 weeks after treatment with another ACCase herbicide haloxyfop-R-methyl at the recommended field dose, OXmiR397-overexpressing rice and WT plants all exhibited significantly smaller root length and plant height than that without haloxyfop-R-methyl treatment (Table 2). Six weeks after treatment, they all died. This result suggested that the miR397/laccase pathway might not be a general defense mechanism against all herbicides, but likely specific to fenoxaprop-P-ethyl.

In this experiment, 3-week-old (at the 3- to 4-leaf stage) B. syzigachne plants were used. Treatment with 20 mM CuSO₄ alone did not have a visual effect on B. syzigachne plants (Table 3). However, bsy-Laccase expression was significantly induced 1 h after CuSO₄ treatment in all populations (Figure 6). Fenoxaprop-P-ethyl treatment in combination with 20 mM CuSO₄ increased herbicide toxicity in the three B. syzigachne R populations, with a slight (but significant) reduction of the GR₅₀ (the effective rate of herbicide causing 50% inhibition in plant fresh weight) value (Table 3), whereas the GR₅₀ value of the S population did not change significantly. These results indicate that the activation of bsy-Laccase by CuSO₄ in fenoxaprop-P-ethyl-resistant B. syzigachne can have an impact on resistance.

However, CuSO₄ treatment did not increase fenoxaprop-P-ethyl toxicity in JYDX-R, SHQP-R, and JYSC-R populations which mainly exhibited TSR (Table 4), indicating that laccase might play a role in B. syzigachne R1, R2, and R3 populations.

From the RNA-seq libraries established in our previous study (Pan et al., 2016), we found 587 genes that were annotated as TFs. Of these, four TFs were consistently down-regulated in R1, R2, and R3, whereas the rest of them did not exhibit consistent changes in expression. The expression of two myeloblastosis (MYB) family genes (bsy-MYB39 and bsy-MYB2) and two auxin response factor (ARF) genes (bsy-ARF5 and bsy-ARF8) remained significantly lower in the R1, R2, and R3 plants than in the S plants (Table 5). In particular, the expression of bsy-MYB39, bsy-MYB2, bsy-ARF5, and bsy-ARF8 was about 3–6 times lower in the R than in the S plants (Table 5).

To further confirm the downstream effect of laccase down-regulation on the up-regulated pathways that contributes to NTSR against fenoxaprop, we analyzed the expression of the four TFs and four oxidase/peroxidase genes in WT and OXmiR397 rice. The results showed that the expression of Os-peroxidase 66 and Os-ARF8 remained unchanged between WT and OXmiR397 rice. However, the expression levels of Os-ARF5, Os-MYB2, and Os-MYB39 were significantly lower in the OXmiR397 rice, whereas those of Os-l-ascorbate oxidase, Os-ubiquinol oxidase 1, and Os-peroxidase 1 were significantly higher (Figure 7). In particular, the expression of Os-MYB2 (Figure 7A), Os-MYB39 (Figure 7B), and Os-ARF5 (Figure 7C) was, respectively, 6.2, 3.5, and 3.0 times lower in OXmiR397 rice than in WT rice. In contrast, the expression of Os-l-ascorbate oxidase (Figure 7D), Os-ubiquinol oxidase 1 (Figure 7E), and Os-peroxidase (Figure 7F) was, respectively, 4.1-, 4.4-, and 2.2-fold higher in OXmiR397 rice than in WT rice.

The relative expression levels of these three TFs and three oxidase/peroxidase genes are consistent with those in B. syzigachne, indicating that ARF5, MYB2, MYB39, l-ascorbate oxidase, ubiquinol oxidase 1, and peroxidase 1 might be involved in the regulatory network with bsy-miR397/bsy-Laccase.