Rapid Reduction of Herbicide Susceptibility in Junglerice by Recurrent Selection with Sublethal Dose of Herbicides and Heat Stress

: Global climate change, speciﬁcally rising temperature, can alter the molecular physiology of weedy plants. These changes a ﬀ ect herbicide e ﬃ cacy and weed management. This research aimed to investigate the combined e ﬀ ect of heat stress (HS) and sublethal doses of herbicides (four active ingredients) on adaptive gene expression and e ﬃ cacy of herbicide on Echinochloa colona (L.) Link (junglerice). Three factors were evaluated; factor A was E. colona generation (G0-original population from susceptible standard; G1 and G2 were progenies of recurrent selection), factor B was herbicide treatment (ﬂorpyrauxifen-benzyl, glufosinate-ammonium, imazethapyr, quinclorac and nontreated check) and factor C was HS (30 and 45 ◦ C). The herbicides were applied at 0.125 × the recommended dose. Recurrent exposure to HS, combined with sublethal doses of herbicides, favors the selection of plants less susceptible to the herbicide. Upregulation of defense (antioxidant) genes ( APX : Ascorbate peroxidase ), herbicide detoxiﬁcation genes ( CYP450 family : Cytochrome P450 ), stress acclimation genes ( HSP : Heat shock protein , TPP : Trehalose phosphate phosphatase and TPS : Trehalose phosphate synthase ) and genes related to herbicide conjugation ( UGT : UDP Glucosyltransferase ) was signiﬁcant. The positive regulation of these genes may promote increased tolerance of E. colona to these herbicides.


Introduction
Accelerated world population growth means intensifying demand for food, both in quantity and quality [1]. Food security is compromised, directly or indirectly, by the increasing complexity of pest problems (including weeds), limitations in production inputs, variable availability of technology and infrastructure-all of which are impacted by climate change [2][3][4][5][6]. Worldwide, the genus Echinochloa (mainly, E. colona, E. crus-galli and E. phyllopogon) includes the most troublesome weeds in rice [7,8]. E. colona also infests many major crops including corn, soybean, sorghum, cotton and sugarcane [9]. E. colona can cause between 1.5% and 100% yield losses in rice mainly due its rapid Preliminary experiments were conducted to determine the sublethal dose that would allow plants to survive and produce seeds. The experiment was conducted in the greenhouse at the University of Arkansas, Fayetteville, AR, USA with a 14-h photoperiod under a day/night temperature regime of 30 °C/21 °C. The pots were placed in trays and subirrigated until the seedlings reached the 2-to 3-leaf stage for treatment. The plants were then submitted to optimal temperature (30 °C) in the greenhouse and high temperature (45 °C) in the growth chamber for 7 d when the plants were sprayed with the respective herbicide treatments ( Table 1). The heat-stressed plants were returned to (45 °C) for seven more days, after which all plants were grown under optimal temperature. The herbicides were applied in a spray chamber equipped with a flat fan spray nozzle (TeeJet spray nozzles; Spraying Systems Co., Wheaton, IL, USA), at a pressure of 221 kPa and a volume of 187 L ha -1 . After herbicide application, the plants were returned to the greenhouse or to the growth chamber. Watering by subirrigation was resumed after 24 h. Three weeks after herbicide submitted to selection by low herbicides rates and heat stress starting with the parental population (G0) and selected (G1 and G2) progenies. Herbicide treatments were: nontreated (no herbicide), florpyrauxifen-benzyl, glufosinate-ammonium, imazethapyr and quinclorac, all applied at 0.125× the recommended dose. T0 (prior to herbicide application) and T1 (12 h after herbicide application) represent the timings of leaf tissue collection for RNA extraction and subsequent gene expression.
Preliminary experiments were conducted to determine the sublethal dose that would allow plants to survive and produce seeds. The experiment was conducted in the greenhouse at the University of Arkansas, Fayetteville, AR, USA with a 14-h photoperiod under a day/night temperature regime of 30 • C/21 • C. The pots were placed in trays and subirrigated until the seedlings reached the 2-to 3-leaf stage for treatment. The plants were then submitted to optimal temperature (30 • C) in the greenhouse and high temperature (45 • C) in the growth chamber for 7 d when the plants were sprayed with the respective herbicide treatments ( Table 1). The heat-stressed plants were returned to (45 • C) for seven more days, after which all plants were grown under optimal temperature. The herbicides were applied in a spray chamber equipped with a flat fan spray nozzle (TeeJet spray nozzles; Spraying Systems Co., Wheaton, IL, USA), at a pressure of 221 kPa and a volume of 187 L ha -1 . After herbicide application, the plants were returned to the greenhouse or to the growth chamber. Watering by subirrigation was Agronomy 2020, 10, 1761 4 of 19 resumed after 24 h. Three weeks after herbicide application, the herbicide effect was evaluated visually on a scale of 0% (no symptoms) to 100% (dead). Prior to flowering, the plants were separated spatially to prevent cross-pollination. At maturity, seeds were harvested and bulked for each treatment. During each selection cycle, a separate group of six plants, without herbicide treatment, was grown to produce generations of plants exposed only to either optimal or high temperature. This procedure was repeated over three cycles.

Determination of Susceptibility Level to Herbicides
The original population (G0) and selected progenies (G1 and G2) were subjected to their respective heat treatment (30 or 45 • C) assignment, as implemented in the previous experiments, for 7 d and sprayed with a range of herbicide doses when the plants were at the 2-to 3-leaf stage, with two plants per treatment. The herbicide rates were 0×, 0.0625×, 0.125×, 0.25×, 0.5×, 0.75×, 1.0×, 1.5×, 2.0× and 4.0× the recommended dose. The herbicide treatments were applied as described previously. After herbicide application, the plants were returned to the greenhouse or growth chamber and were subirrigated again 24 h later. At three weeks after herbicide application, the herbicide effect was evaluated visually on a scale of 0% (no symptoms) to 100% (dead). Once the homoscedasticity and the normal distribution were verified, the data were subjected to analysis of variance (ANOVA). Dose response data were analyzed using the 'drc' package in R v. 3.3.0 (CRC Press Taylor & Francis Group: Boca Raton, FL, USA) [28,29]. The three-parameter log-logistic model in Equation (1) was used: where Y is the response (% control); d is asymptotic value of Y at the upper limit; b is the slope of the curve around e (ED 50 : the herbicide rate giving response halfway between d and the lower asymptotic limit, which was set to 0) and x is the herbicide rate. Susceptibility index (SI) was calculated as the ratio of the ED 50 of the G1 and G2 divided by the ED 50 of the G0 at 30 • C.

Plant Material
The control sample was composed of leaf tissues of G0 plants without herbicide treatment, grown under optimal temperature (30 • C), cultivated for three cycles, at the same period as the other plants to produce G2. In other words, the control samples were G0 plants that were cultured under optimal temperature without herbicide treatment. For all treatments, leaf tissues were collected before herbicide application (T0) and 12 h after herbicide application (T1). The tissues were flash-frozen in liquid nitrogen and stored in −80 • C until processed.

RNA Extraction and cDNA Synthesis
The RNA extraction process was performed using the reagent PureLinK TM (Plant RNA Reagent-Invitrogen TM , Carlsbad, CA, USA), following the manufacturer's recommendations. The quantity and quality were assessed by agarose gel electrophoresis (1% w/v). The amount and purity of RNA were determined using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA was treated with DNase and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

qRT-PCR Assay
The qRT-PCR was conducted to determine changes in expression of NTSR candidate genes relative to the reference genes. The qRT-PCR was performed following MIQE guidelines [30], using specific oligonucleotides for target genes and the reference genes ACT1, UBQ5 and EF1-α ( Table 2). The qRT-PCR experiments were conducted in a total volume of 10 µL containing 5.0 µL of iTaq™ Universal SYBR ® Green Supermix (Bio-Rad, Hercules, CA, USA), 0.5 µL primer forward (10 mM), 0.5 µL primer reverse (10 mM), 1 µL cDNA (1:5 dilution) and 3.0 µL of water. Two biological replicates and two technical replicates were performed for each treatment and primer pair. The amplification efficiency was determined for each primer pair and melt-curve analysis were performed. The cycle conditions were amplified for one cycle of 95 • C for 5 min, followed by 39 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 1 min and extension at 72 • C for 30 s and a final dissociation curve of denaturation at 95 • C for 5 s, followed by cooling to 70 • C for 1 min and gradual heating at 0.11 • C steps to 95 • C and cooling 40 • C for 30 s. For each gene analyzed, UBQ5 was used as an endogenous control to quantify cDNA abundance, after the stability analysis of the expression data using DataAssist™ v3.0 Software (Applied biosystems, Life Technologies, Carlsbad, CA, USA).
Cycle threshold (Ct) during the reaction cycles, and relative gene expression values were calculated using the 2 −∆∆Ct method [36]. Gene expression data were analyzed using MultiExperiment Viewer (MeV) software (The Institute for Genomic Research (TIGR), J. Craig Venter Institute, USA) and presented as a heat map diagram using the harvest stage as a baseline [37]. mRNA abundance of each gene from the G0 population (control from susceptible standard) served as the baseline for determining relative RNA levels, in the G2 population treatment.

Determination of Susceptibility Level to Herbicides
Dose response assays were performed to determine the level of sensitivity after the herbicide-susceptible Echinochloa colona parental population was treated with sublethal doses of florpyrauxifen-benzyl, glufosinate-ammonium, imazethapyr or quinclorac under optimal or high temperature, over three consecutive cycles. Recurrent exposure to high temperature stress, combined with low-dose herbicide stress, reduced the sensitivity of E. colona to the herbicides tested (Table 3). Under optimal temperature (30 • C) the ED 50 increased minimally in general between G0 and G2 with all herbicides tested. Specifically, the ED 50 increased by 0.12 with florpyrauxifen-benzyl ( (Table 3). Table 3. Parameter estimates (b, d, ED 50 and SI) for the control of Echinochloa colona with various herbicides across cycles of exposure to high temperature stress and sublethal herbicide dose, three weeks after herbicide treatment. The data were fitted with a three-parameter log-logistic regression model in Equation (1).    Under high-temperature stress (45 • C), the ED 50 increased significantly between G0 and G2 with all herbicides (Table 3). These increases were higher compared to the ED 50 increases under optimal temperature across generations. The ED 50 Figure 2J-L). The increase in tolerance was 10-37 times higher under high temperature stress compared to optimal temperature.

Florpyrauxifen-Benzyl
The relative expression of some target genes increased in G2 compared to G0, after recurrent exposure to a low dose of florpyrauxifen-benzyl, with or without heat stress. The APX gene was constitutively expressed but was upregulated in G2 12 h after florpyrauxifen-benzyl application ( Figure 3). The expression of CYP72A15 in G2 plants selected with florpyrauxifen-benzyl was not different between plants kept at 30 • C and those in 45 • C for seven days, without florpyrauxifen-benzyl treatment. Upon treatment with florpyrauxifen-benzyl, CYP72A15 was induced 4.5-fold in G2 plants kept at high temperature and downregulated (−0.1-fold) in optimal temperature condition. Florpyrauxifen-benzyl induced the expression of CYP72A14, but the induction was greater under optimal temperature (4.2-fold) than at high temperature (3.2-fold). The other two genes of the cytochrome P450 family showed a similar profile (Figure 3) without florpyrauxifen-benzyl treatment. CYP709B1 and CYP709B2 were not differentially expressed under 30 • C, but both genes were highly induced at 45 • C (6.1-and 8.4-fold, respectively). Both genes were downregulated after treatment with florpyrauxifen-benzyl (4.9-and 2.0-fold, respectively).
The HSP10 and HSP15 genes are members of the heat shock protein family. The greatest induction of HSP10 occurred at 30 • C with values of 0.9-fold and 3.1-fold at T0 and T1, respectively ( Figure 3). HSP10 was not induced under heat stress, either before or after treatment with florpyrauxifen-benzyl. Similar to HSP10, HSP15 was induced 12 h after florpyrauxifen-benzyl application with values of 7.7-fold and 3.8-fold at 30 • C and 45 • C, respectively.
Concerning the TPS gene, induction of 1.6-fold was detected when plants were heat-stressed, and plants which received heat and florpyrauxifen-benzyl treatment (12 h) a 0.2-fold expression was detected. These findings indicated the possible involvement of the TPS gene with abiotic stress tolerance. For the TPP gene the highest value was observed at 30 • C after the application of florpyrauxifen-benzyl (2.7-fold); however, at 45 • C before the application it was 1.6 and 0.8 in T1. The UGT gene, which is related to herbicide conjugation, increased its expression from −1.4 in T0 to 3.9 in T1 at 30 • C and from 2.3 in T0 to 3.7 in T1 at 45 • C (Figure 3).

Glufosinate-Ammonium
The APX gene was induced 12 h after glufosinate-ammonium application with values ranging from 0.6-to 1.4-fold and −1.2to 0.4-fold at 30 and 45 • C, respectively ( Figure 3). With HSP genes, specifically HSP15 the expression level did not change (8.9-fold at 30 • C and 8.6-fold at 45 • C), after glufosinate-ammonium treatment.
The TPS and TPP genes were significantly downregulated 12 h after glufosinate-ammonium treatment under 45 • C, while UGT was upregulated under the same treatment ( Figure 3). This indicates possible use of the substrate from the trehalose biosynthesis pathway for conjugation with the herbicide. The expression of CYP450 genes did not change between G0 and G2, nor was it affected by glufosinate-ammonium treatment under optimal temperature conditions. However, the CYP450 genes CYP709B1, CYP709B2, CYP72A14 and CYP72A15 were upregulated under high temperature 12 h after glufosinate-ammonium treatment, also indicating a possible attempt to detoxify the herbicide.

Imazethapyr
Imazethapyr induced the expression of APX (3.2-fold) 12 h after treatment under optimal temperature, but suppressed APX under high temperature (−1.2-fold; Figure 3). The heat shock protein genes, HSP10 and HSP15, were induced by imazethapyr, under optimal and high temperatures, specifically HSP15 with expression greater than 7.2-fold at 30 • C and 6.4-fold at 45 • C.
CYP709B1 and CYP709B2 were highly expressed at 45 • C in G2 plants before herbicide (4.8-and 6.5-fold, respectively), but expression was significantly reduced 12 h after imazethapyr application (0.6-and 1.4-fold; Figure 3). The same gene expression pattern was observed at optimal temperature with CYP709B1 expression being downregulated from 2.8-to 1.7-fold and CYP709B2 expression being downregulated from 5.0-to 3.3-fold after imazethapyr treatment. However, CYP72A14 and CYP72A15 were induced 12 h after imazethapyr treatment at 30 • C, indicating the possible involvement of these two genes with imazethapyr detoxification under optimal temperature conditions. As observed with CYP709B1 and CYP709B2, the CYP72A14 and CYP72A15 genes were also downregulated after imazethapyr treatment at high temperature.

Quinclorac
The APX antioxidant enzyme-encoding gene expression did not change 12 h after quinclorac treatment under optimal temperature, but was downregulated (from 0.8-to −0.6-fold) under high temperature (Figure 3). On the other hand, HSP10 and HSP15 were upregulated after quinclorac treatment mainly under optimal temperature. The combination of quinclorac plus high temperature also leads to an upregulation of HSP genes; however, lower transcript accumulation was detected compared to optimal temperature. These results indicate that heat shock proteins are most likely to play a role in mitigating the effect of quinclorac.
Under optimal temperature, TPS, TPP and UGT genes were induced 12 h after quinclorac application, showing 5.9-, 3.8-and 4.1-fold expressions, respectively (Figure 3). This indicated the possible role of these genes in coping with quinclorac stress. However, these genes were downregulated after the plants were exposed to quinclorac and heat stress treatment, showing −1.4-, −1.7and −0.8-fold expression, respectively. Similarly, CYP450-encoding genes were induced after quinclorac application under optimal temperature and downregulated at 45 • C.
In general, this study highlighted some candidate genes related to herbicide detoxification and/or stress tolerance mechanisms that help promote the adaptive evolution of plants such as E. colona to heat stress and herbicides. Some candidate genes are; APX, CYP72A14, CYP72A15, HSP15, TPS and UGT for florpyrauxifen-benzyl-benzyl; APX, CYP709B2, CYP72A14, CYP72A15, HSP10, HSP15 and UGT for glufosinate-ammonium; HSP10, HSP15, TPS and UGT for imazethapyr and HSP10 and HSP15 for quinclorac.

Heat Stress Reduces Susceptibility of Junglerice to Herbicides
Temperature governs plant growth and development, herbicide uptake and translocation and herbicide detoxification and protection processes thereby influencing herbicide efficacy and evolution of weed resistance to herbicides [26,[38][39][40]. This research supports our hypothesis that recurrent selection of E. colona with a sublethal dose of herbicides under high temperature increases plant tolerance to the herbicide, with transgenerational effects. The increase in the level of tolerance to the herbicides tested was still below the recommended dose (Table 3), but the accelerated evolution of higher tolerance under high temperature was apparent. The 'resistance evolution rate' observed here was not higher than was reported for either Lolium rigidum with low doses of the herbicide diclofop-methyl for two generations or Raphanus raphanistrum following four generations of low doses of 2,4-D selection [16,18]. It is important to highlight that E. colona is a self-pollinating species and evolution may be slower due to the low crossing rate, unlike allogamous species where genetic traits of resistance can accumulate rapidly during crossing and in successive generations from surviving plants [15,41,42]. Moreover, previous studies also suggested that abiotic stresses, such as herbicides (propanil, quinclorac, imazethapyr and cyhalofop), elevated temperature (26/38 • C night/day) and elevated CO 2 concentration (700 ± 50 mmol·mol −1 ) favor the selection of Echinochloa spp. plants resistant to herbicides [14,24], corroborating the results reported in this study.

Gene Expression Profile Reflects the Transgenerational Reduces Susceptibility Memory
In the plant-environment system there is a dynamic signaling and response process resulting from the metabolic capacity that results in a series of molecular, physiological, biochemical and morphological changes [43][44][45][46]. At the molecular level, the complex signaling system reprograms gene expression, prioritizing stress-responsive genes so that response in plants can be characterized as increased tolerance to a subsequent stressful event [47][48][49][50]. In the literature there are suggestions that epigenetic regulation is also involved in the evolution of resistance, based on the response of oxidative stress arising from herbicides, as is seen with abiotic stresses in plants [51][52][53].
The ability to upregulate genes to detoxify a range of chemicals in a plant is a result of increased metabolism that is directly affecting increased tolerance and, eventually resistance, to herbicides. Such increased metabolism is strongly influenced by climate change, which can accelerate the adaptation process in both crops and weeds [54][55][56][57][58]. The complexity of the system and the data scarcity on weed species make it difficult to understand the herbicide metabolism mechanisms, as there is a great variability of candidate genes and processes that may be involved [59].
During the development of an organism, epigenetic mechanisms establish patterns of gene expression to ensure adequate differentiation, adaptation to environmental changes and future responsiveness due to previous exposure to particular conditions over the short and long term, even affecting subsequent generations [60,61]. In this context, the APX enzyme plays an essential role in the antioxidant defense system, acting to prevent oxidative damage caused by ROS (reactive oxygen species) [62,63]. The APX gene has been reported to respond to thermal stress, endowing protection from herbicide effects, as corroborated by the increased expression seen in the current study (Figure 3). This response was previously reported after florpyrauxifen-benzyl and glufosinate-ammonium application, indicating a potential mechanism for adaptation to abiotic stress related to the mechanisms of action (MOAs) of these active ingredients [62,[64][65][66]. On the other hand, a reduction in APX expression observed 12 h after quinclorac application was also observed in the shoots of quinclorac-sensitive Echinochloa oryzicola [40,67]. This reduction indicates that the adaptation of Echinochloa colona at low quinclorac use rates and high temperature is not a result of APX expression.
Herbicide detoxification is a complex metabolic process involving herbicide transformation, conjugation and exportation for final processing, coordinated by enzymes and regulatory genes [55,68]. Herbicide metabolism studies indicate that tolerance or resistance is mostly due to increased rates of herbicide detoxification, in both crops and weeds, and generally involves the activity of cytochrome P450 monooxygenases [12,14,[69][70][71][72][73]. In this study, the expression of CYP72A14 and CYP72A15 increased when exposed to florpyrauxifen-benzyl-benzyl, while CYP709B1, CYP709B2, CYP72A14 and CYP72A15 were induced by glufosinate-ammonium treatment, especially when subjected to high temperature stress. Cytochrome P450s are phase 1 detoxification enzymes. The induction of the genes mentioned, by florpyrauxifen-benzyl and glufosinate, indicates some level of metabolic degradation of these herbicides in susceptible E. colona, although such a process would not occur fast enough to prevent plant death. However, higher induction of these genes after herbicide treatment under high temperature indicates that the degradation process could occur slightly faster, effecting some elevation in tolerance to the herbicide. Recurrent exposure to these conditions apparently has some transgenerational footprint; thus, we observed increased ED 50 values in G2 plants under high temperature relative to G0. Herbicide metabolism mediated by increased CYP450 expression was reported in resistant plants such as Alopecurus aequalis, Arabidopsis, Echinochloa phyllopogon, Glycine max, Lolium rigidum and Oryza sativa [73][74][75][76][77].
The reduction of CYP450 gene expression observed with imazethapyr and quinclorac may be due to a variety of reasons. First, the mechanisms by which E. colona copes with these herbicides do not involve these specific CytP450 genes. The CYP450 family is large and diverse (350,000 cytochrome P450 sequences) [78,79]. It is possible that other CytP450 genes are involved, which are not included in the selection of primary target genes. Dalazen et al. [80] demonstrated the importance of degradation enhancement for resistance of Echinochloa crus-galli to imazethapyr in terms of relatively high expression of CYP81A6 and GSTF1 genes. Wright et al. [59] studying a biotype of junglerice with resistance to four herbicides (imazamox, fenoxaprop-P-ethyl, quinclorac and propanil) demonstrated that for two of these herbicides (imazamox and quinclorac) the resistance was reduced when the herbicide was applied with a cytochrome P450 inhibitor (malathion). Therefore, the differential expression analysis and qPCR were performed, but did not identify any cytochrome P450s as differentially expressed; they did identify a GST and kinase as being significantly upregulated. Other coping mechanisms might be involved, as demonstrated by Chayapakdee et al. [81] where Echinochloa phyllopogon resistance to quinclorac was associated with reduced ethylene synthesis rather than enhanced cyanide detoxification by β-cyanoalanine. On the other hand, the EcCAS gene mutation and higher gene expression may enhance the activity of β-CAS to avoid the accumulation of toxic cyanide in resistant populations, thus contributing to the resistance mechanism of E. crus-galli var. zelayensis to quinclorac [82]. Thus, there are many possibilities involved in the pathways of tolerance and resistance of Echinochloa spp. to herbicides. Plants are exposed to various biotic and abiotic conditions that, when exceeding the limits of fluctuations, can be deemed as stress [83,84]. The action of HSPs can be activated under various stress conditions including osmotic and oxidative stress, drought, salinity, extreme temperatures, pesticide response and multiple stress resistance, among others [25,[85][86][87][88][89]. The upregulation of HSP 12 h after application of all herbicides under both temperature treatments (30 and 45 • C), especially HSP15, indicates possible contribution to the adaptation to herbicides with different MOAs and thermotolerance. HSPs are molecular chaperones particularly important for plant survival, playing a large role in many processes that regulate protein molding/folding, localization, accumulation and degradation, and assisting in cellular homeostasis under ideal and adverse conditions [35,90,91].
In response to several environmental stresses, another process important to plant adaptation is the trehalose biosynthetic pathway and trehalose accumulation. Here, the TPP gene was upregulated at 30 • C in response to all herbicides, but was downregulated after herbicide treatment at 45 • C.
This suggests the use of trehalose substrate for another purpose that does not need to flow through the action of TPP [34,[91][92][93]. Another component of the trehalose pathway, TPS, was also induced under high temperature stress after application of florpyrauxifen-benzyl and imazethapyr. The trehalose pathway could have a significant role in the evolution of NTSR mechanisms to florpyrauxifen-benzyl and imazethapyr in E. colona. However, TPS expression declined after application of glufosinate-ammonium and quinclorac at 45 • C. Thus, TPP and TPS gene expression appears to be inhibited by high temperature and the application of glufosinate-ammonium and quinclorac, indicating that these genes are not involved in tolerance to thermal stress combined with these particular herbicides. These results corroborate previous reports about HSPs and trehalose being important in plant adaptation and tolerance to various environmental stresses [94][95][96][97][98][99][100][101].
UDP-glucosyltransferases (UGT) is another group of genes involved in xenobiotic detoxification, which is also associated with auxin synthesis, signaling and response [86]. UGT may play a role in plant response to florpyrauxifen-benzyl as it was induced by the herbicide under both temperatures ( Figure 3). Increased UGT expression was also observed after glufosinate-ammonium and imazethapyr application. Enzymes in the glycosyltransferase family have been reported to play a role in phase II of xenobiotic detoxification, specifically herbicide metabolism, and are associated with abiotic stress tolerance [55,69,102,103].
The study of adaptation mechanisms to environmental and herbicide stress in Echinochloa colona is important since plants are constantly exposed to such stresses. Concurrent exposure to environmental stress can facilitate the evolution of tolerance to new herbicides and different MOAs, as demonstrated by Benedetti et al. [104] where observed that recurrent selection by a herbicide sublethal dose and drought stress results in rapid reduction of herbicide sensitivity in junglerice. This research provides information on the reduction of sensitivity to herbicides under high temperature and, thus, the imminent acceleration of weed resistance evolution to herbicides under heat stress ( Figure 4). However, information is still needed on the genetic, biochemical or physiological mechanisms that endow resistance. Moreover, it would be valuable to study the combination of different stress factors such as herbicides and temperatures to understand responses to multiple stress factors.
Agronomy 2020, 10, x FOR PEER REVIEW 13 of 19 and imazethapyr in E. colona. However, TPS expression declined after application of glufosinateammonium and quinclorac at 45 °C. Thus, TPP and TPS gene expression appears to be inhibited by high temperature and the application of glufosinate-ammonium and quinclorac, indicating that these genes are not involved in tolerance to thermal stress combined with these particular herbicides. These results corroborate previous reports about HSPs and trehalose being important in plant adaptation and tolerance to various environmental stresses [94][95][96][97][98][99][100][101]. UDP-glucosyltransferases (UGT) is another group of genes involved in xenobiotic detoxification, which is also associated with auxin synthesis, signaling and response [86]. UGT may play a role in plant response to florpyrauxifen-benzyl as it was induced by the herbicide under both temperatures (Figure 3). Increased UGT expression was also observed after glufosinate-ammonium and imazethapyr application. Enzymes in the glycosyltransferase family have been reported to play a role in phase II of xenobiotic detoxification, specifically herbicide metabolism, and are associated with abiotic stress tolerance [55,69,102,103].
The study of adaptation mechanisms to environmental and herbicide stress in Echinochloa colona is important since plants are constantly exposed to such stresses. Concurrent exposure to environmental stress can facilitate the evolution of tolerance to new herbicides and different MOAs, as demonstrated by Benedetti et al. [104] where observed that recurrent selection by a herbicide sublethal dose and drought stress results in rapid reduction of herbicide sensitivity in junglerice. This research provides information on the reduction of sensitivity to herbicides under high temperature and, thus, the imminent acceleration of weed resistance evolution to herbicides under heat stress ( Figure 4). However, information is still needed on the genetic, biochemical or physiological mechanisms that endow resistance. Moreover, it would be valuable to study the combination of different stress factors such as herbicides and temperatures to understand responses to multiple stress factors.
The increased expression of genes related to stress signaling and herbicide metabolism, appear to be involved in response to mediate the negative effects by herbicide activity and heat stress, and can be a facilitator of adaptation of E. colona (Figure 4). Research on E. colona populations that already evolved resistance to these herbicides would be useful in providing further understanding of adaptation mechanisms to environmental stresses.

Conclusions
The joint effect of recurrent selection with a sublethal dose of herbicide and heat stress reduced junglerice susceptibility, due to adaptive expression of metabolism-and protection-related genes in E. colona with a transgenerational effect. The increased expression of genes related to stress signaling and herbicide metabolism, appear to be involved in response to mediate the negative effects by herbicide activity and heat stress, and can be a facilitator of adaptation of E. colona (Figure 4). Research on E. colona populations that already evolved resistance to these herbicides would be useful in providing further understanding of adaptation mechanisms to environmental stresses.

Conclusions
The joint effect of recurrent selection with a sublethal dose of herbicide and heat stress reduced junglerice susceptibility, due to adaptive expression of metabolism-and protection-related genes in E. colona with a transgenerational effect.
The significant upregulation of defense (antioxidant) genes (APX: Ascorbate peroxidase), herbicide detoxification genes (CYP450 family: Cytochrome P450), stress acclimation genes (HSP: Heat shock protein, TPP: Trehalose phosphate phosphatase and TPS: Trehalose phosphate synthase) and genes related to herbicide conjugation (UGT: UDP Glucosyltransferase) may promote reduction in sensitivity of E. colona to herbicides under climate change.