Differential Expression of Genes Associated With Degradation Enhancement of Imazethapyr in Barnyardgrass ( Echinochloa crus-galli )

The understanding of mechanism of herbicide resistance in weeds is essential for adequate or innovative weed management practices. The aim of this study was to identify and analyze the expression of genes related to degradation enhancement of imazethapyr in barnyardgrass (Echinochloa crus-galli L. Beauv.). One susceptible (SUSSP01) and two populations previouslly identified as resistant to imazethapyr (ARRGR01 and PALMS01) were used. Gene expression of CYP and GST, the translation initiating factor eIF4B, and ALS genes were evaluated after imazethapyr spraying. A reference gene stability analysis was carried out, wherein the genes 18S and actin showed to be more stable in response to the population and herbicide treatment. The gene expression analysis was performed by qRT-PCR. There was no difference in the relative expression of the ALS gene. The CYP81A6 and GSTF1 genes showed higher relative expression in the resistant populations. The CYP81A6 gene had expression 9.61 and 8.44 higher in the resistant populations ARRGR01 and PALMS01, respectively, in comparison with the untreated susceptible population. The expression of this gene was induced by spraying the herbicide imazethapyr. The GSTF1 gene showed higher relative expression in PALMS01 population, reaching 12.30 times higher in plants treated with imazethapyr in relation to untreated susceptible population. The expression of eIF4B gene in the resistant populations treated with imazethapyr was about six times higher than observed in susceptible population. The high relative expression of CYP81A6 and GSTF1 genes indicate the importance of degradation enhancement for the resistance of barnyargrass to imazethapyr.


Introduction
The resistance of weeds to herbicides is related to the occurrence of two primary mechanisms of resistance grouped as target site resistance (TSR) and non target site resistance (NTSR) (Powles & Yu, 2010).The TSR is associated mainly with mutation in the target enzyme encoding gene and increase activity of the target enzyme.NTSR mechanisms encompass the resistance caused by lower uptake and variation of herbicide translocation, rapid necrosis caused by reactive oxygen species, herbicide sequestration in the vacuole, and degradation enhancement of the herbicide (Sammons & Gaines, 2014;Yu & Powles, 2014;Nandula et al., 2015).
Barnyardgrass (Echinochloa crus-galli L. Beauv.) is a major weed of irrigated rice fields around the world (Chauhan & Johnson, 2011).This weed can cause 21-79% yield rice losses, depending on weed density, rice cultivar and irrigation management (Bajwa et al., 2015), and demands intensive use of different methods of control.Herbicides are the most used method for controlling this weed, and acetolactate synthase (ALS) inhibitors, such as imidazolinones, are widely used.The availability of imidazolinone-resistant rice cultivars (Clearfield® rice) have allowed the repetitive use of these herbicides, selecting resistant barnyardgrass populations.In southern Brazil, the mechanism of resistance of these populations involves both types: mutations in the ALS enconding gene and increased metabolism by detoxifying enzymes (Matzenbacher, Bortoly, Kalsing, & Merotto Jr., 2015).In this study the effect of degradation enhancement as the mechanim of resistance was inferred through P450 inhibitors.However, the genetic regulation of this process is unknown in these populations.
The involvement of CYP and GST genes has been associated with herbicide resistance in weeds and selectivity in crops.The CYP81A is one of the most important gene subfamilies associated with the metabolization of herbicides in plants.The CYP81A6 gene has already been related to the metabolism of bensulfuron-methyl and sulfonylureas in rice (Pan et al., 2006;Liu et al., 2012).Similarly, a CYP81 gene is involved in the resistance of A. japonicus to the herbicide fenoxaprop-P-ehtyl (Chen, Xu, Zhang, Bai, & Dong, 2018).In E. phyllopogon, the higher expressions of the CYP81A12 and CYP81A21 genes are associated with resistance to the ALS-inhibiting herbicides penoxsulam and bensulfuron-methyl (Iwakami et al., 2014a).In this species, previous spraying of bispyribac-sodium, another ALS inhibitor, induced the expression of CYP71AK2 and CYP72A254 genes in plants resistant to these herbicides (Iwakami et al., 2014b).In rice and Arabidopsis thaliana, the CYP72A31 gene confers tolerance to bispyribac-sodium (Saika et al., 2014).GST genes encode enzymes that catalyze conjugation reactions of herbicides with more soluble molecules, decreasing the phytotoxicity of the compounds (Yuan et al., 2007),.These enzymes also have other functions in herbicide detoxification, such as peroxidase activity and stress signaling (Dixon et al., 2002;Powles & Yu, 2010;Cummins et al., 2013).The GSTF1 gene conferred greater tolerance to the herbicides chlorotoluron and fenoxaprop-P-ethyl in A. myosuroides and L. rigidum, causing multiple resistance (Cummins et al., 2013).In rice, overexpression of the GSTL1 or GSTL2 genes led to greater tolerance to chlorsulfuron and glyphosate (Hu, Qv, Xiao, & Huang, 2009;Hu, 2014).Likewise, the greater expression of the GST1 gene has been related with resistance of E. crus-galli plants to quinclorac (Li et al., 2013).
The qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is useful to access the gene expression, which is associated with enzyme activity.In this procedure it is necessary to analyze the stability of reference genes used for calculating the relative expression of the target gene.A number of studies demonstrate that the expression of reference genes widely used in this type of study can vary considerably with experimental conditions, tissues and species (Thellin et al., 1999;Stürzenbaum & Kille, 2001).The analysis of the stability in the expression of reference genes in weeds responding to herbicide stress has already been performed in A. myosuroides for the acetyl CoA carboxylase (ACCase) inhibitors herbicides (Petit, Permin, Heydel, & Délye, 2012), and in L. rigidum for ALS inhibitors herbicides (Duhoux & Délye, 2013).In these studies, the most stable reference gene for A. myosuroides and L. rigidum were genes coding for tubulin and CAP proteins, respectively.The stability of reference genes for barnyardgrass under stress by herbicides has not been investigated yet.
The objectives of this study were to evaluate the stability of reference genes and the expression of CYP and GST genes in imidazolinone-susceptible and -resistant barnyardgrass populations exposed to imazethapyr.

Plant Material
The source of susceptible population was at Engenheiro Coelho, SP (SUSSP01) and the resistant populations were at Arroio Grande-RS (ARRGR01) and Palmares do Sul-RS (PALMS01).Barnyardgrass resistant populations were collected from paddy fields of South Brazil, where escapees of control with imidazolinone herbicides had occurred and historic use of Clearfield®-rice cultivars was known.The susceptible population SUSSP01 was originally from an area where no herbicides had been applied before and efficient control had been obtained during previous pot-studies using imazethapyr.The PALMS01 population is resistant to imidazolinones due to the mutation Ser653Asn in ALS, but enhanced metabolism was also identified trough P450 inhibitors (Dalazen, Pisoni, Rafaeli, & Merotto Jr., in press).Meanwhile, in the population ARRGR01 there was absence of ALS-gene mutation associated with herbicide resistance (Matzenbacher et al., 2015).
The seed dormancy overcoming was performed by immersing the seeds in KNO 3 solution (0.2%) at a temperature of 25 °C until germination (radicle emission), which occurred approximately four days after immersion.The seeds were then rinsed in distilled water and placed in Petri dishes incubated at 25 °C until the emergence of the first leaf.Then, seedlings were transplanted into 200 ml pots filled with a mixture of ultisol and organic compound at a ratio of 10:1, and the mineral fertilizer (05-20-20 NPK complex) at 2.5 g kg -1 .Plants were maintained in greenhouse with temperatures ranging from 25 to 27 °C, relative humidity of about 70% and photoperiod of 14/10 hours (day/night).
The herbicide imazethapyr was sprayed at the label dose of 106 g ha -1 plus adjuvant (Dash 0.5% v/v).The spray treatments were applied in plants with three to four leaves stage using an automated spray chamber with nozzle TJ8002E, spray pressure of 2.89 bar and speed of 1.16 m s -1 , resulting in a spray volume of 200 L ha -1 .
Leaves of barnyardgrass plants treated and untreated with imazethapyr were analyzed.The samples were collected before (T0, unreated control) and 24 hours after herbicide spraying (T24).Three biological replicates were used.The samples were collected in a 1.5 mL microtube and immediately conditioned in liquid nitrogen (LN 2 ).The samples were stored in an ultrafreezer (-85 °C) until the RNA extractions.

RNA Extraction and cDNA Synthesis
RNA extraction was performed using the Trizol® method (Invitrogen).The RNA was quantified in a spectrophotometer (Genesys 2 TM , Thermo Spectronic) at a wavelength of 260 nm and diluted in RNAse free water at 1 μg μL -1 .Each sample was purified with DNAse® I (Invitrogen) in an amount of 1 μg of total RNA, as per the manufacturer's recommendation.The next step consisted of obtaining the cDNA strand from RNA through SuperScrip® III reverse transcriptase (Invitrogen) in the amount of 1 μg of RNA using polidT primers.

Candidate Genes and Primer Design
The reference genes (Table B1) evaluated for stability analysis were selected from the study of Duhoux and Délye (2013), including actin, CAP (catabolite activator protein), EF1 (elongation factor 1), rubisco, ubiquitin, 18S (18S ribosomal RNA) and 28S (28S ribosomal RNA).The candidate genes CYP and GST were chosen based on a large literature review (Table B2).The analyzed genes were CYP81A6, CYP81A12, CYP81A21, CYP71C30, CYP71AK2, CYP72A254, CYP72A3, GSTF1, and GSTL1.In addition to these genes, the expression of ALS and eIF4B (translation initiator factor) was considered.The eIF4B gene is present in the genus Echinochloa (Iwakami et al., 2014b) and its involvement in the detoxification of xenobiotics in other eukaryotic organisms has been reported (Kim et al., 2011).
The primers sequences were designed by the program Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) from the most conserved region of each sequence obtained in the Genbank (http://www.ncbi.nlm.nih.gov/genbank).For each gene, at least three primer pairs were designed based on annealing temperature around 60 °C, size close to 20 bp and expected fragment size around 100 bp.

qRT-PCR Analysis
The obtained cDNA was amplified by real-time PCR using the SYBR Green® kit.qRT-PCR analysis was performed with the 7300 Real-Time PCR System (Applied Biosystems) on 96-well plates PCR-96M2-HS-C® (Axygen) with a sealer MicroAmp® Optical Adhesive Film (Applied Biosystems).
The reactions were carried out in a final volume of 20 μL, consisting of 10 μL of the cDNA sample (diluted 1:100) in Mili-Q water; 10 μL of the constituents of the reaction composed of 2 μL 10X buffer, 0.5 μL dNTPs (10 μM of each nucleotide), 1.2 μL of MgCl 2 solution (50 mM), 2 μL of SYBR Green® (Invitrogen) diluted 1:100 (prepared at the time of use from diluted 100X solution), 0.2 μL of ROX Reference Dye, 0.1 μL Taq Platinum® (Invitrogen) and 0.4 μL of the combination of forward and reverse primers.
The amplification steps included an initial cycle of 95 °C for 5 min, followed by a 40-cycle sequence: started at 94 °C for 15 sec, 60 °C for 10 sec, 72 °C for 15 sec and 60 °C for 35 sec, and a final denaturation cycle of 95 °C for 15 sec, 60 °C for 60 sec, 95 °C for 15 sec and 60 °C for 15 sec.

Reference Genes Stability Analysis
Analysis of the stability of the reference genes was performed from the Ct values obtained in the qRT-PCR reaction (Wang, Ma, Huang, & Zhang, 2015).The software RefFinder (http://fulxie.0fees.us/?ckattempt=1) was used, which is based on the algorithms geNorm, Normfinder, BestKeper and Delta Ct method.The stability coefficients (SC) were classified in each of the algorithms and, finally, a comprehensive classification of all the algorithms was determined by the program RefFinder.A lower SC value indicate greater the stability of the reference gene.The two genes with lower stability coefficients were used as reference genes in calculations of relative expression of genes related to resistance to the herbicide imazethapyr.jas.ccsenet.

Analys
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The expre evaluated greater tra from 23.35 actin, CAP Five algorithms were used to determine the stability of the reference genes.The Delta Ct method determines the stability of the genes according to the variation of the Ct value (Silver et al., 2006).In the geNorm method, the stability coefficient is called the M-value, which is calculated according to the variation of Ct eliminating the two less stable genes in the first round of the calculation.In this analysis, the lower the M-value the greater the stability of the reference gene (Vandesompele et al., 2002).The NormFinder method calculates the stability coefficient S and the standard deviation (SD).Reference genes are considerable stable when both S and SD values are low (Andersen, Jensen, & Orntoft, 2004).In the BestKeeper method the variation of the Ct values and the standard deviation (SD) of each gene are used for the calculation of stability.Genes with SD < 1 are considered stable and ordered based on the correlation between the Ct value and the geometric mean of the Ct values of all values with SD < 1 (BestKeeper index).The candidate genes with the highest correlation with the BestKeeper index are considered to be the most stable (Pfaffl, Tichopad, Prgomet, & Neuvians, 2004).Finally, the RefFinder method uses an integrated way the other methods of analysis, classifying them comprehensively.Based on the ordering of the methods described above, the RefFinder method assigns an appropriate weight to an individual gene and calculates the geometric mean of its weights for the comprehensive classification of candidate reference genes (Xie, Xiao, Chen, Xu, & Zhang, 2012).The stability analysis of the reference genes indicated low variations of the classification and stability among the methods used (Table 1 and Appendix A).For all the methods used, the 18S and actin genes presented higher stability.Therefore, these genes were used in the relative expression calculations of the genes possibly related to the herbicide resistance.Note.18S: 18S ribosomal RNA; Act: actin; Ubi: ubiquitin; 28S: 28 S ribosomal RNA; EF1: elongation factor 1; CAP: catabolite activator protein; Rub: Rubisco.

Expression Analysis of the ALS Gene
The relative expression of the ALS gene was not altered by the evaluated treatments (Figure 2).The expression in both resistant populations ARRGR01 and PALMS01 was similar to that observed in SUSSP01 susceptible population.Spraying of imazethapyr had no effect on the relative expression of the ALS gene.This indicated that the greater expression or the greater number of copies of the ALS gene is not a mechanism of resistance to imazethapyr in these populations of barnyardgrass. jas.ccsenet.

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In  Note.† Primers sequences designed from NCBI number using the software Primer3Plus.§ Primer sequence available in the reference.

Table 1 .
Stability classification of reference genes in leaves of imidazolinone-susceptible and -resistant barnyardgrass (Echinochloa crus-galli), treated and untreated with imazethapyr

Genes and primers analyzed in the researchTable B1 .
Reference genes and primers sequences used for the qRT-PCR analysis Note.† Act: actin; CAP: catabolite activator protein; EF1: elongation factor 1; Rub: Rubisco; Ubi: ubiquitin; 18S: 18S ribosomal RNA; 28S: 28 S ribosomal RNA.TableB2.DNA sequences of the primers used for real-time RT-PCR analysis of CYP and GST genes