Time-Dependent Effects of Bentazon Application on the Key Antioxidant Enzymes of Soybean and Common Ragweed

sustainability

Article Time-Dependent Eﬀects of Bentazon Application on the Key Antioxidant Enzymes of Soybean and Common Ragweed

Zalán Czékus 1,2,Máté Farkas 1,László Bakacsy 1 , Attila Ördög 1 , Ágnes Gallé 1 and Péter Poór 1,*

1 Department of Plant Biology, University of Szeged, H 6726 Szeged, Közép fasor 52, Hungary; − [email protected] (Z.C.); [email protected] (M.F.); [email protected] (L.B.); [email protected] (A.Ö.); [email protected] (Á.G.) 2 Doctoral School of Biology, University of Szeged, H 6726 Szeged, Közép fasor 52, Hungary − * Correspondence: [email protected]

Received: 20 March 2020; Accepted: 7 May 2020; Published: 9 May 2020

Abstract: The presence or absence of light is one of the most significant environmental factors affecting plant growth and defence. Therefore, the selection of the most appropriate time of application may maximize the benefits of photosynthetic inhibitors. In this work, the concentration and daytime or night-time-dependent effects of bentazon were tested in soybean and common 1 ragweed. The recommended dose (1440 g ha− ) and also half the recommended dose significantly reduced the maximum quantum yield (Fv/Fm) and increased H2O2 levels in common ragweed. Interestingly, bentazon did not change Fv/Fm in soybean. The activity of superoxide dismutase changed in a dose-dependent manner only in common ragweed. The activity of ascorbate peroxidase, catalase and glutathione S-transferase (GST), as well as the contents of ascorbate (AsA) and glutathione (GSH) did not change significantly in this plant species. In soybean, alterations in H2O2 levels were lower but GST and APX activity, as well as AsA and GSH levels were higher compared to common ragweed. At the same time, the rate of lipid peroxidation and ion leakage increased upon bentazon, and were higher in the light phase-treated leaves in the case of both plant species. These results can contribute to optimizing the effects and uses of herbicides in agriculture.

Keywords: antioxidants; Basagran®; chlorophyll a ﬂuorescence parameters; dark; herbicide; light; night; reactive oxygen species

1. Introduction Soybean (Glycine max L.) is a very signiﬁcant crop of the developing world because this plant is an essential source of protein for animal feeds and many packaged meals [1]. USA (119.51 million tons), Brazil (114.59 million tons) and Argentina (54.97 million tons) were the most important soybean producers in 2017 (FAOSTAT; http://www.fao.org/faostat/en). Sustained and increased productivity, improved quality of soybeans, (bio) controlled weed and pest management and new, sustainable and environmentally friendly methodology and technology are needed in the future [2–4]. The results of basic and applied researches can serve sustainable agriculture management especially in the case of using herbicides that protect the ecosystem and biodiversity from excessive and harmful application. Bentazon (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide; CAS number: 25057-89-0) is the main component of the mercantile herbicide Basagran® (BASF Chemicals, Ludwigshafen, Germany) and belongs to the benzothiadiazoles chemical group. Basagran® is a contact and selective herbicide applied to limit broad-leaved and some grassy weeds in pre- and post-emergence on

Sustainability 2020, 12, 3872; doi:10.3390/su12093872 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 3872 2 of 20 soybean [5–7]. Among many weeds of soybean, common ragweed (Ambrosia artemisiifolia L.) is one of the most signiﬁcant [7–9]. Interference of common ragweed with the growth of other crops results in variable yield losses depending upon the density, the type of infested plant species and the time of emergence relative to the harvested plant [9]. It was observed that common ragweed is very competitive in soybean culture and was able to cause ca 70% yield losses [10]. Therefore, the optimization of the treatments against common ragweed plants is essential to control yield losses of soybean culture. In sensitive plant tissues, bentazon inhibits the photosynthetic electron flow in chloroplasts thus reducing the photosynthetic activity. Bentazon competes with quinone B (QB) for its binding site in the reaction centre of Photosystem II (PSII), thus decreasing the rate of the electron flow as QA is unable to reoxidize. Light-induced PSII blockage by bentazon promotes the generation of triplet-chlorophyll 3 1 ( Chl) and the formation of singlet oxygen ( O2) upon reaction with molecular oxygen [11–13]. Bentazon-promoted oxidative stress damages the proteins and membranes of photosynthetic cells and induces cell death in weeds [12,14–16]. At the same time, bentazon seems to induce oxidative stress expressed as enhanced free radical production and lipid peroxidation, as well as significant induction of components of the antioxidant system in crops, respectively [15,16]. Crops survival under stress conditions depends on the plant defence system via xenobiotic detoxification in tolerant species, a process which may go on in parallel with cell defence reactions to alleviate oxidative stress depending on the antioxidant capacity [17,18]. Thus investigation and understanding of the role of the antioxidant system in herbicide-tolerant crops such as soybean could provide relevant information on practical purpose. Inhibition of chloroplastic electron transport, e.g., by herbicides, results in rapid (within a millisecond time range) and high production of reactive oxygen species (ROS) [11]. Biphasic ROS burst was described in the plant cell death process, where the first maximum of ROS was measured within minutes after the initial stimulus and the second, more intense burst occurred several hours later. The first ROS burst can originate from energy-organelle sources and both ROS maxima can be dependent on the basic antioxidant capacity and the activation of antioxidant enzymes [19]. ROS production and oxidative stress resulting in the rapid damage of lipids, carbohydrates, proteins and nucleic acids thus can inhibit active plant defence and induced cell death [20,21]. To control and minimalize the toxic levels of ROS and to protect cells in the case of harmful oxidative stress conditions, plants possess several enzymatic and non-enzymatic antioxidants. Three key antioxidant enzymes play an essential role in these processes. One of the first is the superoxide dismutase (SOD; EC 1.15.1.1), which catalyses the dismutation (disproportionation) of O to O and H O . At the same time, H O can be degraded • 2− 2 2 2 2 2 by catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11), the second and third main antioxidant enzymes for H2O[18]. Therefore, the activated antioxidant defence system can substantially and rapidly contribute to cell survival by alleviating the primary and secondary oxidative stress effects of herbicides besides activating other xenobiotic detoxification pathways in plants. This was confirmed in both weeds and soybean, where antioxidant enzymes were significantly activated after xenobiotic exposure such as oxyfluorfen [22], linuron [23], dimethenamid [23], glyphosate [24] and paraquat [25]. These results verified the presence of oxidative stress and the significance of antioxidants in defence reactions in the cells of both sensitive and tolerant plants. Interestingly, the activity of these antioxidant enzymes is highly dependent on the circadian clock and the light or dark conditions [26]. It was earlier observed that the activity of SOD, CAT and APX, as well as the content of APX substrate ascorbate (AsA) rose during the light phase of the day and lowered during the night in various plant species. The highest activity at the end of the light phase can be twice as much as at dawn [27–31]. The other key enzyme, which plays a role in the detoxiﬁcation of various xenobiotics is the glutathione S-transferase (GST) [32]. GST activity and glutathione (GSH) levels also display circadian- and light-dependent patterns with a maximum late in the light and early dark phase [32–35]. Therefore, the selected daytime or night-time, and the presence or absence of daylight can be fundamental in case of the herbicide application on crops and weeds. Consequently, Sustainability 2020, 12, 3872 3 of 20

Sustainability 2020, 12, x FOR PEER REVIEW 3 of 20 selection of the most appropriate time of application may maximize the beneﬁts derived from bentazon, aand photosynthetic the key antioxidant inhibitor. enzymes (SOD, CAT, APX), levels of antioxidants (AsA, GSH) and GST in soybeanThe main and common aim of our ragweed work is to plants detect. In and the compare centre the of these daytime- investigations or night-time-dependent was also the effects question of differentwhether concentrationsbentazon application of the photosynthesis in lower concentration inhibitor bentazon could provoke on the defence activity ofresponses photosynthesis in the non and- thetarget key soybean antioxidant and cell enzymes death (SOD,in the CAT,target APX), common levels ragweed of antioxidants plants in (AsA,different GSH) light and or GSTdark in periods soybean of andthe day. common ragweed plants. In the centre of these investigations was also the question whether bentazon application in lower concentration could provoke defence responses in the non-target soybean and2. Materials cell death and in theMethods target common ragweed plants in different light or dark periods of the day.

2.2.1. Materials Plant Materials and Methods

2.1. PlantSeeds Materials of soybean (Glycine max L. cv. Pannónia Kincse) and common ragweed (Ambrosia artemisiifolia L.) were germinated at 26 °C for 3 days in the dark. Six healthy soybean and twelve healthySeeds common of soybean ragweed (Glycine plants max L. were cv. Pann grownónia Kincse) in 4 kg and mixture common of ragweed3:1 soil (Ambrosia and sand artemisiifolia (Florimo, L.)MATÉCSA were germinated Kft., Kecel, at 26 Hungary)◦C for 3 days containing in the dark. organic Six matterhealthy (70 soybean m/m%), and N twelve (10,000 healthy mg kg common−1), P2O5 ragweed(1000 mg plants kg−1);were K2O grown(3000 mg in 4 kg kg−1 mixture), and pH of 3:16.4 soil ± 0.5. and They sand were (Florimo, irrigated MAT ÉeveryCSA Kft.,3 days Kecel, with Hungary) 250 mL 1 1 1 containingof ½ Hoagland organic nutrient matter solution (70 m/m%), (5 mM N (10,000Ca(NO3 mg)2, 5 kg mM− ), KNO P2O53,(1000 1 mM mg KH kg2PO− );4, K2 2mMO (3000 MgSO mg4, kg1 μ−M), and pH 6.4 0.5. They were irrigated every 3 days with 250 mL of 1 Hoagland nutrient solution (5 mM Fe–EDTA, 0.04± μM H3BO3, 14.48 μM MnCl2, 0.81 μM ZnCl2, 0.37 μ2M CuCl2, 0.001 μM Na2MoO4) [36]. Ca(NOGrowing3)2 ,conditions 5 mM KNO 3 of, 1 plants mM KH w2POere4 ,controlled. 2 mM MgSO Illumination4, 1 µM Fe–EDTA, was 200 0.04 µ μmolMH 3 mBO−23 ,s 14.48−1 forµ M 12 MnCl h and2, 0.81occurredµM ZnCl from2, 6:00 0.37 µa.m.M CuCl until2 ,18:00 0.001 p.µmM. Na(White2MoO LED4)[36 (5700]. Growing K) illumination conditions with of plants supplementary were controlled. FAR 2 1 IlluminationLEDs; PSI, Drásov, was 200 Czechµmol m Republic− s− for). The 12 h day/night and occurred temperatures from 6:00 a.m. and until the relative 18:00 p.m. humidity (White were LED (570024/22 K)°C illuminationand 55–60%, with respectively supplementary [37]. For FAR the LEDs; experiments, PSI, Drá 5sov,- toCzech 6-week Republic).-old intact The plants day in/night 3–4 temperaturesdeveloped leaf and-level the relativestages were humidity used were. 24/22 ◦C and 55–60%, respectively [37]. For the experiments, 5- to 6-week-old intact plants in 3–4 developed leaf-level stages were used. 2.2. Treatments 2.2. Treatments Leaves of soybean and ragweed plants were sprayed with Basagran® herbicide solution (BASF, ® Ludwigshafen,Leaves of Germany) soybean and diluted ragweed in double plants-distilled were water sprayed to a with final Basagran concentrationherbicide of 720 or solution 1440 g (BASF,ha−1 of Ludwigshafen,active ingredient Germany) bentazon diluted (3-isopropyl in double-distilled-1H-2,1,3-benzothiadiazin water to a final-4(3H concentration)-one 2,2-dioxide) of 720. To or 1 1440investigate g ha− ofthe active daytime ingredient- or night bentazon-time-(3-isopropyl-1 and light-dependentH-2,1,3-benzothiadiazin-4(3 effects of differentH concentrations)-one 2,2-dioxide). of Tobentazon investigate, plants the were daytime- treated or at night-time- several times and, such light-dependent as in the late eafternoonffects of di (5:00fferent p.m.), concentrations in the evening of bentazon,(9:00 p.m.), plants at dawn were (4:00 treated a.m.) at and several in the times, morning such as(8:00 in thea.m.) late [37 afternoon]. Plant responses (5:00 p.m.), were in the detected evening at (9:009:00 a.m. p.m.), in atthe dawn second (4:00 light a.m.) phase and of in the the day morning after each (8:00 treatment a.m.) [37]. (Fig Planture responses 1). As a control were detected treatment, at 9:00spraying a.m. inwith the double second-distilled light phase water of was the dayused after in all each times treatment, the results (Figure of which1). As were a control averaged. treatment, spraying with double-distilled water was used in all times, the results of which were averaged.

Figure 1. Experimental setup of bentazon treatments and time of samplings. Figure 1. Experimental setup of bentazon treatments and time of samplings. 2.3. Measurement of Photosynthetic Activity 2.3. MeasurementTo detect the of chlorophyll Photosynthetica fluorescence Activity parameters in leaves of soybean and common ragweed plants,To pulse detect amplitude the chlorophyll modulation a fluorescence (PAM) chlorophyll parameters fluorometer in leaves (PAM-2000;of soybean and Heinz-Walz, common Eragweedffeltrich, Germany)plants, pulse was used.amplitude Before modulation the measurements (PAM) of chlorophyll the minimal fluorometer fluorescence ( yieldPAM of-2000; the dark-adapted Heinz-Walz, stateEffeltrich, (F0), leaves Germany) were dark-adaptedwas used. Before for 15 the min. measurements After dark-adaptation, of the minimal firstly fluorescence the maximal fluorescenceyield of the indark dark-adapted-adapted state state (F (F0)m, ) leaves was measured were dark and-adapted the following for 15 parameters min. After were dark calculated:-adaptation, The firstly maximal the maximal fluorescence in dark-adapted state (Fm) was measured and the following parameters were calculated: The maximal quantum efficiency of PSII photochemistry (Fv/Fm = (Fm − F0)/Fm), the actual

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quantum efficiency of PSII photochemistry (Fv/Fm = (Fm F )/Fm), the actual quantum yield of PSII − 0 electron transport in the light adapted state (Φ = (Fm’ Fs)/Fm’) and the photochemical quenching − PSII − coefficient (qP = (Fm’ Fs)/(Fm’ F ’)). Finally, the light-induced photoprotection through thermal − − 0 dissipation of energy was determined ªNPQ = (Fm Fm’)/Fm’) [38,39]. − 2.4. Determination of Photosynthetic Pigment Content Twenty-five mg of leaf tissues of soybean and common ragweed plants were homogenised in 1 mL of 100% (v/v) cold acetone, and extracted for one day at 4 ◦C in the dark. Samples were centrifuged (12,000 g for 15 min at 4 C). The pellet was extracted again with 1 mL of cold × ◦ acetone/Tris buffer solution (80:20 v/v, pH = 7.8) for another one day at 4 ◦C in the dark. Supernatants were collected after centrifugation (12,000 g, 15 min, 4 C), and the absorbance was measured by × ◦ spectrophotometer (KONTRON, Milano, Italy). Contents of photosynthetic pigments were calculated based on Lichtenthaler and Wellburn [40].

2.5. Determination of H2O2 Content 200 mg of leaf tissues of soybean and common ragweed plants were homogenised with 1 mL of 0.1% (w/v) ice-cold trichloroacetic acid (TCA), then the samples were centrifuged at 12,000 g for 10 min × at 4 ◦C. Then 0.25 mL of 50 mM potassium phosphate buﬀer (pH 7.0) and 0.5 mL of 1 M potassium iodide (KI) were added to 0.25 mL of the supernatant. Samples were measured by spectrophotometer (KONTRON, Milano, Italy) after 10 min at 390 nm. The amount of H2O2 was determined using a standard curve prepared from increasing concentration of H2O2 [41]. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.6. Determination of Activities of Key Antioxidant Enzymes Two hundred and fifty mg of leaf tissue of soybean and common ragweed plants were homogenised with 1 mL of ice-cold extraction buffer (100 mM phosphate buffer (pH 7.0); 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% (w:v) polyvinyl-polypirrolidone (PVPP)), then the homogenate was centrifuged (12,000 g for 20 min at 4 C). This supernatant was used for superoxide dismutase (SOD) and catalase × ◦ (CAT) enzyme activity assays. The extraction for ascorbate peroxidase activity (APX) was performed in the presence of 1 mM ascorbate (AsA). The absorbance of samples was detected by spectrophotometer (KONTRON, Milano, Italy). SOD (EC 1.15.1.1) activity was measured by determining the ability of the enzyme to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) in the presence of riboflavin under light. One unit (U) means the amount of SOD activity causing 50% inhibition of NBT reduction under light. CAT activity (EC 1.11.1.6) was measured by detecting the consumption of H2O2 1 1 at 240 nm for 3 min at 25 ◦C(ε240 = 39.4 mM− cm− ). One unit of CAT activity means the amount 1 of enzyme needed to decompose 1 µmol min− H2O2. APX activity (EC 1.11.1.11) was calculated by 1 1 measuring the decrease in AsA amount at 290 nm for 3 min at 25 ◦C(ε 290 = 2.8 mM− cm− ). One unit 1 of APX activity means the amount of enzyme needed to oxidize 1 µmol min− AsA [42]. All enzyme 1 activities were expressed as U mg− protein. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Soluble protein concentration in samples was measured by the method of Bradford [43] using bovine serum albumin (BSA) as a standard.

2.7. Measurements of Ascorbate (AsA) and Glutathione (GSH) Levels Two hundred and ﬁfty mg of leaves were homogenised with 1 mL of 5% (w/v) TCA. Samples were centrifuged at 12,000 g for 20 min at 4 C. To assay total AsA, 10 mM dithiothreitol (DTT) was added × ◦ to the mixture and the excess DTT was eliminated by using 0.5% (w/v) N-ethylmaleimide (NEM). AsA concentrations were measured in the mixture of 10% (w/v) TCA, 43% (w/v) H3PO4, 4% bipyridyl, 3% (w/v) FeCl3 using spectrophotometer (KONTRON, Milano, Italy) at 525 nm [42]. GSH concentration was detected using an enzymatic assay containing 100 mM phosphate buﬀer (pH 7.5), 1 mM 5,50-dithiobis(2-nitrobenzoic acid) (DTNB), 1 mM NADPH, 1 U of glutathione reductase Sustainability 2020, 12, 3872 5 of 20

(baker’s yeast, Sigma-Aldrich) and 20 µL of the supernatant in 1 mL volume. GSH concentrations were measured spectrophotometrically at 412 nm [42]. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.8. Determination of Glutathione S-Transferase (GST) Activity Two hundred and fifty mg of leaf tissues of soybean and common ragweed plants were homogenised in 1 mL of 100 mM phosphate buffer (pH 7.0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% (w:v) polyvinyl-polypirrolidone (PVPP) on ice, then the homogenate was centrifuged at 12,000 g for 20 min at 4 C. The supernatant was used for the determination of GST × ◦ (EC 2.5.1.18) activity by using 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) as substrate [44]. GST activity was determined after the addition of CDNB to the mixture based on the increment at 340 nm for 3 min spectrophotometry (KONTRON, Milano, Italy). One unit of GST activity 1 1 is the amount of the enzyme that produces 1 µmol conjugated product in 1 min (ε340 = 9.6 mM− cm− ). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Soluble protein concentration in samples was measured by the method of Bradford [43] using bovine serum albumin (BSA) as a standard.

2.9. Determination of Malondialdehyde (MDA) Content One hundred mg of leaf tissues of soybean and common ragweed plants were homogenized with 1 mL of 0.1% TCA. Before the centrifugation (12,000 g, 20 min, 4 C) 0.1 mL of 4% butylated × ◦ hydroxytoluene (BHT) was added to the samples; 0.5 mL of supernatant was added to 2 mL 0.5% thiobarbituric acid (TBA) dissolved in 20% TCA and incubated in boiling water for 30 min. The absorbance of the mixture was detected at 532 nm and the non-speciﬁc absorbance was measured at 600 nm using a spectrophotometer (KONTRON, Milano, Italy). Malondialdehyde (MDA) content was 1 1 determined based on the extinction coeﬃcient of 155 mM− cm− [45]. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.10. Determination of Electrolyte Leakage (EL) and Leaf Fresh Mass (FM) Firstly, leaf fresh mass per individual plant was determined using a laboratory scale (Mettler-Toledo; Greifensee, Switzerland). For detection of electrolyte leakage (EL) from leaves, 50 mg of leaf tissues of soybean and common ragweed plants were incubated in 20 mL double-distilled water for 2 h at 25 ◦C in darkness. Then the conductivity of water was determined (C1) with a conductivity meter (HANNA Instruments, Woonsocket, RI, USA). To determine the total conductivity (C2), the samples were boiled for 40 min. EL was calculated based on the actual conductivity (C1) as a percentage of total conductivity (C2) (EL (%) = (C /C ) 100) [46]. 1 2 × 2.11. Statistical Analysis Each experiment contains at least three biological replicates (at least three plants per treatment) and the entire experiment was conducted three times. Results are expressed as mean SE calculated by ± Microsoft Excel 2016. Statistical analysis was accomplished using Sigma plot 11.0 software (SPSS Science Software, Erkrath, Germany). Differences between the treatments in the case of each plant species were statistically analyzed by one-way ANOVA using Tukey’s test. The means of each treatment were significant if p 0.05. ≤ 3. Results First of all, the daytime- and night-time-dependent effects of different bentazon concentrations were tested on the photosynthetic activity of both soybean and common ragweed plants based on the measurements of chlorophyll a fluorescence parameters. The maximal quantum yield of PSII Sustainability 2020, 12, 3872 6 of 20

photochemistry (Fv/Fm) did not change in soybean plants one light cycle later after bentazon treatments. In contrast to these results, Fv/Fm significantly decreased after bentazon concentrations were applied to both in common ragweed plants, independently of the time of the treatments (Figure2A). Similar to changes in the Fv/Fm parameter, there were no significant changes in parameters of the minimal fluorescence yield of the dark-adapted state (F0; Figure2B) and the maximal fluorescence in the dark-adapted state (Fm; Figure2C) after none of the bentazon treatments in leaves of soybean plants. In contrast to this observation, F0 significantly increased (Figure2B) and F m significantly decreased (Fig. 2C) in common ragweed plants after bentazon application at all times. In addition, the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) significantly declined in 1 soybean plants after the application of 1440 g ha− bentazon at 5:00 p.m. and 4:00 a.m. Moreover, a significant decline in ΦPSII was measured in common ragweed plants exposed to both bentazon concentrations at every time point (Figure2D). The photochemical quenching coe fficient (qP) also decreased significantly in common ragweed plants after bentazon exposure in all cases. In soybean 1 leaves, qP decreased significantly after the treatments with 1440 g ha− bentazon at 5:00 p.m, as well as at 4:00 a.m. (Figure2E). The non-photochemical quenching (NPQ) parameter was basically higher in common ragweed leaves, which were reduced by the herbicide treatments in this plant species similarly in the case of ΦPSII and qP. However, bentazon did not influence significantly the NPQ in 1 1 the concentration of 720 g ha− in soybean but it was decreased in the case of 1440 g ha− at 5:00 p.m. (Figure2F). Sustainability 2020, 12, x FOR PEER REVIEW 7 of 20

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Figure 2. Changes in the chlorophyll a fluorescence parameters (Fv/Fm (A); F0 (B); Fm (C); фPSII (D); Figure 2. Changes in the chlorophyll a fluorescence parameters (Fv/Fm (A); F0 (B); Fm (C); фPSII (D); qP (E); NPQ (F)) in leaves of soybean (white columns) and common ragweed plants (grey columns) qP (E); NPQ (F)) in leaves of soybean1 (white columns) and common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha− bentazon at different times (at the end of light cycle (5:00 p.m.), treated foliar with 720 or 1440 g ha−1 bentazon at different times (at the end of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning (8:00 a.m.)). Measurements were disposed at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning (8:00 a.m.)). Measurements were disposed of in the morning at the second light phase of the day (9:00 a.m.) after the treatments. Means SE, n = 3. of in the morning at the second light phase of the day (9:00 a.m.) after the treatments. Means± ± SE, n Means marked with different letters are significantly different at p 0.05 as determined by Tukey’s test = 3. Means marked with different letters are significantly different ≤at p ≤ 0.05 as determined by Tukey’s (upper case letters show the different effects in soybean and lower case letters show the different effects test (upper case letters show the different effects in soybean and lower case letters show the different in common ragweed plants). effects in common ragweed plants). In contrast to the significant changes in chlorophyll a fluorescence parameters of soybean and commonIn contrast ragweed to the plants, significant bentazon changes treatments in chlorophyll did not changea fluorescence significantly parameters either of the soybean content and of chlorophyllcommon ragweeda + b (Figure plants,3A) bentazon or the carotenoidstreatments content did not (Figure change3B) siingnificantly soybean and either common the content ragweed of leaveschlorophyll during a + this b (Fig timeure period. 3A) or the carotenoids content (Figure 3B) in soybean and common ragweed leaves during this time period.

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Figure 3. Changes in the chlorophyll a + b (A) and carotenoids (B) content in leaves of soybean Figure 3. Changes in the chlorophyll a + b (A) and carotenoids (B) content in leaves of soybean (white (white columns) and common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha 1 columns) and common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha−1 bentazon− bentazon at different times (at the end of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) at different times (at the end of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in and in the morning (8:00 a.m.)). Measurements were disposed of in the morning at the second light the morning (8:00 a.m.)). Measurements were disposed of in the morning at the second light phase of phase of the day (9:00 a.m.) after the treatments. Means SE, n = 3. Means marked with different the day (9:00 a.m.) after the treatments. Means ± SE, n =± 3. Means marked with different letters are letters are significantly different at p 0.05 as determined by Tukey’s test (upper case letters show the significantly different at p ≤ 0.05 as≤ determined by Tukey’s test (upper case letters show the different different effects in soybean and lower case letters show the different effects in common ragweed plants). effects in soybean and lower case letters show the different effects in common ragweed plants). Effects of oxidative stress induced by bentazon treatments were detected by the measurements of Effects of oxidative stress induced by bentazon treatments were detected by the measurements H2O2 content in leaves of soybean and common ragweed plants. H2O2 levels were basically higher in of H2O2 content in leaves of soybean and common ragweed plants. H2O2 levels were basically higher common ragweed compared to soybean leaves but bentazon application caused much higher H O 2 2 in common ragweed compared to soybean leaves but bentazon application1 caused much higher H2O2 generation in common ragweed plants. Bentazon in 1440 g ha− concentration induced significant generation in common ragweed plants. Bentazon in 1440 g ha−1 concentration induced significant H2O2 production in soybean leaves treated at all times except at 8:00 a.m. In addition, the lower H2O2 production in soybean leaves treated at all times except at 8:00 a.m. In addition, the lower herbicide concentration did not elevate it significantly in the case of the treatment at night at 9:00 p.m. herbicide concentration did not elevate it significantly in the case of the treatment at night at 9:00 p.m. (Figure4). The application of bentazon induced significant increase in H 2O2 levels in common ragweed (Figure 4). The application of bentazon induced significant increase in H2O2 levels in common leaves compared to the control, which was the highest in the late afternoon (5:00 p.m.)-treated leaves ragweed leaves compared to the control, which was the1 highest in the late afternoon (5:00 p.m.)- while H2O2 content was the lowest in the case of 720 g ha− bentazon application in the night (Figure4). treated leaves while H2O2 content was the lowest in the case of 720 g ha−1 bentazon application in the night (Figure 4).

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Figure 4. Changes in the H2O2 content in leaves of soybean (white columns) and common ragweed Figure 4. Changes in the H2O2 content in leaves of soybean1 (white columns) and common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha− bentazon at different times (at the end plants (grey columns) treated foliar with 720 or 1440 g ha−1 bentazon at different times (at the end of of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning (8:00 a.m.)). light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning (8:00 a.m.)). Measurements were disposed of in the morning at the second light phase of the day (9:00 a.m.) after theMeasurements treatments. Meanswere disposedSE, n = of3. in Means the morning marked at with the second different light letters phase are of significantly the day (9:00 di ffa.m.)erent after at ± pthe0.05 treatments. as determined Means by± SE, Tukey’s n = 3. testMeans (upper marked case with letters different show theletters diff erentare significantly effects in soybean different and at p ≤ lower≤ 0.05 case as determined letters show by the Tukey’s different test effects (upper in common case letters ragweed show plants). the different effects in soybean and lower case letters show the different effects in common ragweed plants).

The main antioxidant enzymes playing a crucial role in H2O2 metabolism were further analysed upon herbicideThe main exposure.antioxidant First enzymes of all, theplaying activity a crucial of SOD, role which in H catalyzes2O2 metabolism the dismutation were further of Oanalysed¯ into • 2 upon herbicide exposure. First of all, the activity of SOD, which catalyzes the dismutation of •O2¯ molecular oxygen and H2O2, was measured in the leaf tissues. Both bentazon treatments increased the SODinto activity molecular in soybean oxygen plants and H and2O2,the was highest measured activity in was the measured leaf tissues. in leaves Both bentazon treated at treatments 5:00 p.m. increased the SOD1 activity in soybean plants and the highest activity was measured in leaves treated with 1440 g ha− bentazon (Figure5A). In contrast to soybean plants, SOD activity was significantly 1 −1 1 higherat 5:00 for p.m. 1440 with g ha 1440− compared g ha bentazon to 720 g(Fig ha−urebentazon 5A). In treatmentscontrast to insoybean common plants, ragweed. SOD Applicationactivity was significantly higher for 14401 g ha−1 compared to 720 g ha−1 bentazon treatments in common ragweed. of bentazon in 1440 g ha− in the late and early light phase of the day (at 5:00 p.m. and 8:00 a.m.) causedApplication two maxima of bentazon in SOD in activities1440 g ha in−1 commonin the late ragweed and early leaves light (Figure phase 5ofA). the day (at 5:00 p.m. and 8:00 a.m.) caused two maxima in SOD activities in common ragweed leaves (Figure 5A). The activity of CAT enzyme catalyzing the decomposition of H2O2 to H2O and O2, did not display any significantThe activity changes of CAT in enzyme either soybean catalyz oring common the decomposition ragweed leaves of H but2O2 to it wasH2Oelevated and O2, did slightly not display in the nightany significant (at 9:00 p.m.)-treated changes in commoneither soybean ragweed or plantscommon (Figure ragweed5B). leaves but it was elevated slightly in the night (at 9:00 p.m.)-treated common ragweed plants (Figure 5B). In contrast to CAT, the activity of APX, the other H2O2-detoxificating enzyme using AsA as co-substrate,In contrast significantly to CAT, the increased activity inof soybeanAPX, the leavesother H in2O case2-detoxificating of treatments enzyme in all using times AsA except as atco- dawnsubstrate, (Figure significantly5C). At the increased same time, in significant soybean leaves concentration-dependent in case of treatments e ffinects all oftimes bentazon except were at dawn not detected(Figure on5C). APX At in the this same plant time, species. significant Surprisingly, concentration bentazon-dependent did not cause effects any significant of bentazon changes were notin APXdetected activity on inAPX common in this ragweedplant species. leaves Surprisingly, (Figure5C). bentazon did not cause any significant changes in APX activity in common ragweed leaves (Figure 5C).

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Figure 5. Changes in the activity of superoxide dismutase (SOD; A), catalase (CAT; B) and ascorbate peroxidaseFigure 5. (APX;ChangesC) inin leavesthe activity of soybean of superoxide (white columns) dismutase and (SOD; common A), ragweedcatalase (CAT; plants B (grey) and columns) ascorbate peroxidase (APX; C) in leaves of1 soybean (white columns) and common ragweed plants (grey treated foliar with 720 or 1440 g ha− bentazon at different times (at the end of light cycle (5:00 p.m.), −1 atcolumns) night (9:00 treated p.m.), atfoliar dawn with (4:00 720 a.m.) or 1440 and g in ha the morningbentazon (8:00at different a.m.)). Measurementstimes (at the end were of disposedlight cycle of(5:00 in the p.m.), morning at night at the (9:00 second p.m.), light at phase dawn of (4:00 the day a.m.) (9:00 and a.m.) in the after morning the treatments. (8:00 a.m.) Means). MeasurementsSE, n = 3. ± Meanswere markeddisposed with of in di fftheerent morning letters at are the significantly second light di ffphaseerent of at pthe 0.05day as(9:00 determined a.m.) after by the Tukey’s treatm testents. ≤ (upperMeans case ± SE,letters n = show 3. Means the di ff markederent eff ectswith in different soybean lettersand lower are case significantly letters show different the di ff aterent p ≤e ff0.05ects as indetermined common ragweed by Tukey’s plants). test (upper case letters show the different effects in soybean and lower case letters show the different effects in common ragweed plants). 1 As shown in Figure6 neither 720 nor 1440 g ha − bentazon treatment caused any significant changesAs in shown total AsA in contentFigure 6 in neither the leaves 720 of nor soybean 1440 plants g ha−1 (Figurebentazon6A). treatment However, caused in common any ragweedsignificant plantschanges treated in totalwith any AsA bentazon content concentrations, in the leaves of AsA soybean contents plants decreased (Figure significantly, 6A). However, especially in common in the caseragweed of the herbicide plants treated application with any at 5:00 bentazon p.m. and concentrations, 9:00 p.m. (Figure AsA6A). contents Both AsA decreased and GSH significantly, contents wereespecially lower inin leavesthe case of commonof the herbicide ragweed application compared at to 5:00 soybean p.m. plants and 9:00 (Figure p.m.6 ).(Fig Levelsure 6A). of GSH Both also AsA didand not GSH change contents in soybean were lower leaves in (Figureleaves of6B). common In contrast ragweed to this, compared GSH levels to soybean decreased plants with (Fig 1440ure g 6). 1 1 haLevels− bentazon of GSH treatment also did at no5:00t change p.m. and in with soybean 720 g leaves ha− bentazon (Figure application6B). In contrast at 8:00 to a.m. this in, GSH leaves levels of common ragweed plants (Figure6B).

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decreased with 1440 g ha−1 bentazon treatment at 5:00 p.m. and with 720 g ha−1 bentazon application at 8:00 a.m. in leaves of common ragweed plants (Figure 6B). Sustainability 2020, 12, 3872 11 of 20

FigureFigure 6. 6.Changes Changes in in the the ascorbate ascorbate (AsA; (AsA;A )A and) and glutathione glutathione (GSH; (GSH;B )B content) content in in leaves leaves of of soybean soybean 1 (white(white columns) columns) and and common common ragweed ragweed plants plants (grey (grey columns) columns) treated treated foliar foliar with with 720 720 or or 1440 1440 g hag −ha−1 bentazonbentazon at diat ffdifferenterent times times [at the[at endthe end of light of light cycle cycle (5:00 (5:00 p.m.), p.m.), at night at (9:00night p.m.),(9:00 atp.m.), dawn at (4:00dawn a.m.) (4:00 anda.m.) in theand morning in the morning (8:00 a.m.)]. (8:00 Measurementsa.m.)]. Measurements were disposed were disposed of in the of morning in the morning at the second at the second light phase of the day (9:00 a.m.) after the treatments. Means SE, n = 3. Means marked with different light phase of the day (9:00 a.m.) after the treatments. Means± ± SE, n = 3. Means marked with different letters are significantly different at p 0.05 as determined by Tukey’s test (upper case letters show the letters are significantly different at≤ p ≤ 0.05 as determined by Tukey’s test (upper case letters show the diffdifferenterent eff ects effects in soybean in soybean and lower and lower case letters case letters show theshow diff erentthe different effects in effects common in ragweedcommon plants). ragweed plants). Not only the key antioxidant enzymes but also the activity of the key detoxifying enzyme, GST was determinedNot only in the the leaves key antioxidant of these plant enzymes species. but GST also activity the activity was basically of the key higher detoxi in soybeanfying enzyme, compared GST towas common determined ragweed in leavesthe leaves (Figure of 7these). Enzyme plant activityspecies. of GST GST activity was elevated was basically by bentazon higher application in soybean incompared both concentrations to common and ragweed the significantly leaves (Fig highesture 7). levels Enzyme were activity measured of GST in the was soybean elevated leaves by bentazon treated inapplication the light phase in both of theconcentrations day (at 5:00 and p.m. the and significantly 8:00 a.m.). highest GST activity levels were also measured increased in significantly the soybean 1 inleaves common treated ragweed in the leaves light phase but only of the in theday case (at 5:00 of treatment p.m. and at8:00 8:00 a.m.). a.m. GST for 720activity g ha also− bentazon increased (Figuresignificantly7). in common ragweed leaves but only in the case of treatment at 8:00 a.m. for 720 g ha−1 bentazon (Figure 7).

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Figure 7. Changes in the glutathione S-transferase (GST) activity in leaves of soybean (white columns) Figureand common 7. Changes ragweed in the glutathione plants (grey S-transferase columns) treated (GST) activity foliar with in leaves 720 of or soybean 1440 g (whiteha−1 bentazon columns) at Figure 7. Changes in the glutathione S-transferase (GST) activity in leaves of soybean (white columns) anddifferent common times ragweed (at the plants end of (grey light columns) cycle (5:00 treated p.m.), foliar at night with (9:00 720 p.m.), or 1440 at g dawn ha 1 bentazon (4:00 a.m.) at and different in the and common ragweed plants (grey columns) treated foliar with 720 or 1440− g ha−1 bentazon at timesmorning (at the (8:00 end a.m.) of light). Measurements cycle (5:00 p.m.), were at disposed night (9:00 of p.m.),in the atmorning dawn (4:00at the a.m.) second and light in the phase morning of the different times (at the end of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in the (8:00day a.m.)). (9:00 a.m.) Measurements after the treatments. were disposed Means of ± in SE, the n morning = 3. Means at the marked second with light dif phaseferent of letters the day are morning (8:00 a.m.)). Measurements were disposed of in the morning at the second light phase of the (9:00significantly a.m.) after different the treatments. at p ≤ 0.05 Means as determinedSE, n = 3. by Means Tukey’s marked test (upper with di ffcaseerent letters letters show are significantly the different day (9:00 a.m.) after the treatments.± Means ± SE, n = 3. Means marked with different letters are dieffectsfferent in at soybeanp 0.05 and as determinedlower case letters by Tukey’s show the test different (upper caseeffects letters in common show the ragweed different plants). effects in significantly ≤different at p ≤ 0.05 as determined by Tukey’s test (upper case letters show the different soybean and lower case letters show the different effects in common ragweed plants). effects in soybean and lower case letters show the different effects in common ragweed plants). The effects of oxidative stress on lipids were determined based on the measurements of MDA contentThe einff ectsthe leaves. of oxidative MDA stress content on increased lipids were significantly determined after based 1440 on g the ha measurements−1 bentazon treatment of MDA in The effects of oxidative stress on lipids were determined based on the1 measurements of MDA contentsoybean in plants, the leaves. especially MDA in content the case increased of treatments significantly in the after light 1440 phase g ha(at− 5:00bentazon p.m. and treatment 8:00 a.m.) in content in the leaves. MDA content increased significantly after 1440 g ha−1 bentazon treatment in soybean(Figure 8). plants, Similar especially tendencies in thewere case found of treatments in the case inof common the light rag phaseweed (at, but 5:00 in p.m. the case and of 8:00 this a.m.) plant soybean plants, especially in the case of treatments in the light phase (at 5:00 p.m. and 8:00 a.m.) (Figurespecies8)., the Similar 720 g tendencies ha−1 bentazon were was found able in theto increase case of common the rate ragweed,of the lipid but peroxidation in the case of in this the plant light (Figure 8). Similar tendencies1 were found in the case of common ragweed, but in the case of this plant species,phase too, the based 720 g on ha− thebentazon changes wasin MDA able content to increase (Figure the 8). rate The of thelowest lipid levels peroxidation in MDA content in the light were species, the 720 g ha−1 bentazon was able to increase the rate of the lipid peroxidation in the light phasemeasured too, based in case on of the bentazon changes treatments in MDA content in the dark (Figure (at.8 9:00). The p.m. lowest and 4:00 levels a.m.). in MDA content were measuredphase too, in based case ofon bentazon the changes treatments in MDA in content the dark (Fig (at.ure 9:00 8). p.m.The lowest and 4:00 levels a.m.). in MDA content were measured in case of bentazon treatments in the dark (at. 9:00 p.m. and 4:00 a.m.).

Figure 8. Changes in the malondialdehyde (MDA) content in leaves of soybean (white columns) and Figure 8. Changes in the malondialdehyde (MDA) content in leaves of soybean1 (white columns) and common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha− bentazon at different common ragweed plants (grey columns) treated foliar with 720 or 1440 g ha−1 bentazon at different timesFigure (at 8. the Changes end of lightin the cycle malondialdehyde (5:00 p.m.), at night(MDA) (9:00 content p.m.), in at leaves dawn of (4:00 soybean a.m.) (white and in columns) the morning and times (at the end of light cycle (5:00 p.m.), at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning (8:00common a.m.)). ragweed Measurements plants (grey were columns) disposed treated of in thefoliar morning with 720 at theor 1440 second g ha light−1 bentazon phase ofat thedifferent day (9:00(8:00 a.m.) a.m.) after). Measurements the treatments. were Means disposedSE, nof= in3. the Means morning marked at the with second different light letters phase are of significantlythe day (9:00 times (at the end of light cycle (5:00 p.m.),± at night (9:00 p.m.), at dawn (4:00 a.m.) and in the morning dia.m.)fferent after at pthe 0.05treatments. as determined Means ± by SE, Tukey’s n = 3. Means test (upper marked case with letters different show theletters diff areerent significantly effects in (8:00 a.m.)). Measurements≤ were disposed of in the morning at the second light phase of the day (9:00 soybean and lower case letters show the different effects in common ragweed plants). a.m.) after the treatments. Means ± SE, n = 3. Means marked with different letters are significantly

Sustainability 2020, 12, x FOR PEER REVIEW 13 of 20

different at p ≤ 0.05 as determined by Tukey’s test (upper case letters show the different effects in soybean and lower case letters show the different effects in common ragweed plants). Sustainability 2020, 12, 3872 13 of 20 The cell viability based on the measurement of electrolyte leakage (EL) changed significantly upon 1440 g ha−1 bentazon treatment in soybean plants treated in the light phase of the day (at 5:00 The cell viability based on the measurement of electrolyte leakage (EL) changed significantly upon p.m. and 8:00 a.m.) (Figure 9A). In parallel, the same concentration of bentazon caused the highest EL 1440 g ha 1 bentazon treatment in soybean plants treated in the light phase of the day (at 5:00 p.m. and from common− ragweed leaves at the same day times but significant differences between the two 8:00 a.m.) (Figure9A). In parallel, the same concentration of bentazon caused the highest EL from applied herbicide concentrations were not detected (Figure 9A). common ragweed leaves at the same day times but significant differences between the two applied The leaf fresh mass per plant significantly decreased in common ragweed plants upon all herbicide concentrations were not detected (Figure9A). bentazon treatments except at 9:00 p.m. with 720 g ha−1 bentazon treatment but it did not change in The leaf fresh mass per plant significantly decreased in common ragweed plants upon all bentazon soybean (Figure 9B). 1 treatments except at 9:00 p.m. with 720 g ha− bentazon treatment but it did not change in soybean (Figure9B).

FigureFigure 9. 9.Changes Changes in in the the electrolyte electrolyte leakage leakage (EL; (ELA; A) and) and leaf leaf fresh fresh mass mass (B (B) of) of soybean soybean (white (white columns) columns) 1 andand common common ragweed ragweed plants plants (grey (grey columns) columns) treated treated foliar with foliar 720 with or 1440 720 g or ha − 1440bentazon g ha−1 atbentazon different at timesdifferent (at the times end ( ofat lightthe end cycle of (5:00light p.m.),cycle (5:00 at night p.m.), (9:00 at p.m.),night (9:00 at dawn p.m.), (4:00 at dawn a.m.) and(4:00 in a.m.) the morningand in the (8:00morning a.m.)) (8:00 after a.m.) one week.) after Measurementsone week. Measurements were disposed were of indisposed the morning of in the at the morning second at light the phasesecond of the day (9:00 a.m.) after the treatments. Means SE, n = 3. Means marked with different letters are light phase of the day (9:00 a.m.) after the treatments.± Means ± SE, n = 3. Means marked with different significantly different at p 0.05 as determined by Tukey’s test (upper case letters show the different letters are significantly different≤ at p ≤ 0.05 as determined by Tukey’s test (upper case letters show the effects in soybean and lower case letters show the different effects in common ragweed plants). different effects in soybean and lower case letters show the different effects in common ragweed 4. Discussionplants).

4. DiscussionWeed control costs billions of US dollars worldwide; thus, optimization of the herbicide application is one of the key questions in agriculture and other related industries [47]. Use of herbicides is essential Weed control costs billions of US dollars worldwide; thus, optimization of the herbicide for limiting and eliminating various weed species but at the same time it possesses many problems and application is one of the key questions in agriculture and other related industries [47]. Use of risks; thus, their application must be minimized to account for the desired economic and environmental herbicides is essential for limiting and eliminating various weed species but at the same time it impacts [48]. However, there are strict regulations worldwide for herbicide usage [49]. In order to possesses many problems and risks; thus, their application must be minimized to account for the ensure sustainable and successful weed control and eﬀective elimination of weeds, new and innovative strategies, technologies and methods must be developed. Results of basic and applied sciences can signiﬁcantly serve these aims and provide information for a practical purpose. Sustainability 2020, 12, 3872 14 of 20

Herbicides, which belong to photosynthesis inhibitors based on their mode of action (Groups 5–7), can provide the possibility to optimize their efficiency on plants by the selection of the daytime or night-time of their application. These herbicides, such as bentazon, inhibit the photosynthetic electron transport by binding to the QB-binding site of the D1 protein complex located in the chloroplast thylakoid membrane, which results in plants being unable to reoxidize QA, resulting in triplet chlorophyll 3 1 − ( Chl) generation, which forms singlet oxygen ( O2) upon reacting with molecular oxygen [13]. Therefore, high ROS production generated by inhibited electron transport in chloroplasts requires the presence of light. The herbicide treatment-caused ROS production and oxidative stress result in the damage of lipids, carbohydrates, proteins and nucleic acids inducing cell death in weeds [20,21]. The antioxidant system of plants plays a crucial role in the elimination and detoxification of high ROS levels protecting plants from lethal damage [50,51]. However, the activity of antioxidant enzymes and levels of non-enzymatic antioxidants are also highly dependent on light and dark conditions and on the circadian rhythm [18]. It is also known that phytotoxicity is less prevalent under low light conditions than under strong sunlight in the case of some herbicides [52,53]. Therefore, a well-chosen time of the herbicide application, especially at dawn, where the basic antioxidant enzyme activities are low, can be essential in optimizing the use and effect of herbicides on crops and weeds. In contrast, the herbicide application late in the light period, where the basic antioxidant enzyme activities are high, can delay the toxic effects of several herbicides on weeds. Bentazon treatments generally decreased all of the chlorophyll a fluorescence parameters independently of the time of the application in common ragweed plants, confirming the selective photosynthesis inhibitory effects of this chemical on this weed species. Both the increase in F0 and the decrease in Fm contributed to the decrease in Fv/Fm in common ragweed plants. This significant decrease in Fm, as well as in Fv/Fm parameters indicates that PSII cooperativity is inhibited and a large proportion of the PSII reaction centre is damaged [54]. It can be concluded that multiple effects of bentazon treatments (decrease in Fv/Fm and ΦPSII, as well as the inhibition of the increase of F0 and NPQ) resulted in chronic photoinhibition in leaves of common ragweed plants [55]. Interestingly, 1 others found that 50 nmol l− bentazon reduced Fv/Fm and ΦPSII after 18 days but increased F0 only after 25 days in the non-target pea plants suggesting that this herbicide has a long and time-dependent effect on the photosynthetic activity [56]. It is well known that the uptake of bentazon through the cell membranes is a rapid process [57]; thus, it can act on photosynthesis despite the metabolization to less phytotoxic glucosyl conjugates [58]. In contrast to common ragweed, Fv/Fm did not change but ΦPSII decreased after bentazon treatments in the light phase of the day and at dawn in soybean, suggesting that bentazon reduced the photosynthetic activity in soybean but did not cause significant photoinhibition at this time point in soybean [59]. The effect of bentazon on the photosynthesis of soybean plants is dependent on the concentration of the treatments based on the changes in qP and NPQ, which decreased with the higher bentazon concentration. Surprisingly, the chlorophyll 1 a fluorescence parameters did not change significantly after 720 g ha− bentazon treatment at night (9:00 p.m.) in soybean leaves, suggesting a higher degree of basic defence processes in the early dark phase of the day in this plant species and underlining the significance of night-time in herbicide application to crops. Excess energy in PSII under light condition can lead to the production of ROS, which can destroy the electron transport components and PSII structure, causing more ROS production and initiation of cell death [60,61]. In accordance, photoinhibition in common ragweed plants and the significant decrease in NPQ after all bentazon treatments cannot dissipate excessive pressure accumulated in PSII reaction centres and cannot protect PSII under light conditions, which could result in high ROS production in chloroplasts of herbicide-treated leaves [62]. Interestingly, the carotenoid content did not change significantly during this time period after herbicide application, the pigments of which can scavenge singlet oxygen under stress condition [63]. Earlier, it was found that bentazon treatments induced a rapid production of ROS within 24 hours in cyanobacterium (Synechococcous elongatus PCC7942) [14], peanut leaves (Arachis hypogaea L.) [16] and common sage (Salvia officinalis Sustainability 2020, 12, 3872 15 of 20

L.) [15]. Based on our results, application of bentazon caused significantly higher H2O2 generation, 1 which was lower in case of 720 g L ha− bentazon treatment at night in common ragweed similarly to in soybean plants. These results can confirm the significant role of the presence of light and the active photosynthesis in ROS production [64] at the time of the application of this herbicide. Moreover, these results confirm also the significance of basic activities of antioxidant enzymes depending on the circadian rhythm in the detoxification of ROS in this process [65]. Plants evolved antioxidant systems that keep the generation of ROS below a harmful level [50,51]. SOD means the first element of this system, which can dismutate O ¯ into O and H O [66]. Earlier • 2 2 2 2 it was found that SOD activity was the highest late in the light phase and decreased during the night in tomato (Solanum lycopersicum L.) leaves [27,31]. SOD activity was significantly induced by bentazon treatment in both plant species. It was the highest in leaves of soybean after the treatment at 5:00 p.m. with bentazon and in common ragweed plants after the treatment in the late and early light phase of the day (at 5:00 p.m. and 8:00 a.m.). These changes in SOD activity are in good accordance with H2O2 1 results, especially in the case of 720 g ha− bentazon treatment. It shows also strong correspondence with the results of Radwan et al. [16], who measured significant increase in SOD activity of peanut leaves after Basagran® treatments. These results also confirm that both oxidative stress and antioxidant defence responses are induced by bentazon in herbicide-tolerant plant species, such as soybean. Thus the effective defence of soybean, in parallel with other detoxification pathways, can depend on the antioxidant defence system, which is regulated by the circadian clock and the light or dark conditions [26], providing a practical way to maximize the herbicide application. H2O2 can be mostly degraded by the two key antioxidant enzymes, CAT and APX. APX has a higher affinity to hydrogen peroxide and can detoxify it in low concentrations, whereas CAT shows a high reaction rate but a low affinity to hydrogen peroxide [18]. In addition, the regulation of these antioxidant enzymes also shows circadian and/or diurnal rhythm, as well as a light dependency [28,29]. Surprisingly, the activity of CAT did not change significantly upon bentazon treatments in either soybean or common ragweed leaves but it was slightly elevated in the night-treated common ragweed plants. Interestingly, CAT activity neither changed in Synechococcous elongatus [14] but it increased in peanut [16] and common sage plants [15]. In contrast to CAT, APX activity significantly rose in soybean leaves, except in dawn-treated plants, independently of the applied bentazon concentrations. APX activity also increased in peanut [16] and common sage plants [15]. Surprisingly, bentazon treatments did not change APX activity in common ragweed plants contributing to the high H2O2 levels in these herbicide-treated weeds. In parallel with antioxidant enzymes, AsA and GSH levels show also diurnal changes in plants [28,33]. Interestingly bentazon treatments did not cause a significant decrease in total AsA and GSH contents in leaves of soybean plants, which can effectively contribute to the detoxification of toxic ROS in this plant species [50], where these antioxidant contents were basically higher compared to common ragweed plants. In contrast to soybean, AsA content decreased after herbicide application, especially in the case of the treatments at 5:00 p.m. and at 9:00 p.m. in common ragweed 1 leaves. GSH also decreased after 1440 g ha− bentazon treatment at 5:00 p.m. in this plant species. These significant changes with bentazon can negatively influence the ROS-mediated toxic effects in tissues of common ragweed. GST also plays a key role in plant detoxification system, which enzyme shows circadian- and light-dependent activity with a maximum late in the light and early dark period [34]. GST also contributes to the detoxification of various herbicides, depending on their chemical structure [67,68]. Earlier it was found that GST activity by time- and concentration-dependent manner increased in cyanobacterium (Anabaena cylindrica) after bentazon exposure [12]. Based on our investigations, the activity of GST was basically higher in soybean plants and it was significantly elevated by bentazon treatments, especially in the light phase-treated leaves (at 5:00 p.m. and 8:00 a.m.). Surprisingly, GST activity was increased only in 8:00 a.m.-treated common ragweed leaves. These results suggest that GST could not moderate cell death induction in common ragweed leaves. Sustainability 2020, 12, 3872 16 of 20

Changes in MDA content show the herbicide-induced effects of oxidative stress on lipids. Bentazon induced high degree of lipid peroxidation in various crops and weeds [12,14–16]. MDA contents 1 increased significantly after 1440 g ha− bentazon treatments in the leaves of soybean, especially in case of the light phase treatments similarly to the common ragweed plants. The lowest levels in MDA content were measured after bentazon treatments in the dark. Based on our result, herbicide-induced lipid peroxidation is not only dependent on the herbicide’s concentration rate and type, and plant species as earlier described [15,69–71], but also on the time of its application. Oxidative damage of lipids can contribute to loss of cell membrane integrity and cell death in plants [20]. EL, which is a cell viability test based on the integrity of cell membranes, increased after bentazon treatments at high concentration in the light phase of the day (at 5:00 p.m. and 8:00 a.m.) in both investigated plant species but leaf fresh mass per plants decreased significantly only in common ragweed plants confirming the lethal effects of herbicide application on this species. In contrast to changes in EL in soybean leaves, this plant species outgrows the harmful effects of bentazon one week later (data not shown). Earlier same effects of bentazon on EL were detected in Anabaena cylindrical [12]. At the same time, lower concentration of bentazon exposure also significantly elevated EL in common ragweed plants and significant differences between the two applied herbicide concentrations were not detected, suggesting that bentazon in lower concentration at well-chosen times can effectively kill common ragweed plants.

5. Conclusions This work provides firstly a comparative biochemical and physiological analysis and comprehensive study of photosynthetic activity, antioxidants and GST activity in soybean and common ragweed plants exposed to different concentrations of the herbicide bentazon. The daytime- or night-time-dependent effects of bentazon application on the photosynthetic activity and defence responses in the weed-crop model system were specially analysed. According to our results it can be concluded that changes in photosynthetic activity, as well as plant defence responses and/or cell death induced by bentazon are not only dependent on the applied herbicide concentration and plant species but also on the time of the application. Here we firstly demonstrated that treatments with bentazon in the early dark period of the day can delay the lethal effects of this herbicide based on the measurements in H2O2 content, SOD activity, MDA content and EL. Our results indicate that bentazon in lower concentration is also effective for cell death induction in the late dark phase (at dawn) and early morning in common ragweed plants where the EL and MDA content significantly increased and leaf fresh mass per plants decreased, while defence responses of soybean plants were highly activated at this time point based on the changes in APX and GST activity; moreover, EL and leaf fresh mass per plant did not change. Furthermore, these results indicate that the antioxidant defence system contributes to cell survival by limiting the oxidative stress-triggering effects of bentazon besides other xenobiotic detoxification pathways in both sensitive and tolerant plant species; thus, these result can provide relevant information in the aspect of the time- and concentration-dependent practical application of photosynthesis inhibitor herbicides.

Author Contributions: Conceptualization, P.P. and Á.G.; Investigation, Z.C., M.F., L.B., A.Ö.; Writing—original draft preparation, Z.C. and P.P.; Writing—review and editing, Z.C., A.Ö., Á.G. and P.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the grants from the National Research, Development and Innovation Office of Hungary—NKFIH (NKFIH FK 124871) and by the UNKP-19-4-SZTE-86 New National Excellence Program of the Ministry of Human Capacities. Péter Poór was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Acknowledgments: We are grateful to Tibor Janda for his kind help to the experiments. In addition, we thank Bécs Attiláné for professional technical assistance. Conflicts of Interest: No conflict of interest is declared. Sustainability 2020, 12, 3872 17 of 20

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