Effects of ZJ0273 on barley and growth recovery of herbicide-stressed seedlings through application of branched-chain amino acids

Effects of ZJ0273 on barley and growth recovery of herbicide-stressed seedlings through application of branched-chain amino acids 除草剂丙酯草醚对大麦的影响和支链氨基酸对其生长的恢复作用 1 first-author 2 3 3 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:n.ullah@uq.edu.auwjzhou@zju.edu.cn †E-mail:n.ullah@uq.edu.auwjzhou@zju.edu.cn n.ullah@uq.edu.au 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:n.ullah@uq.edu.auwjzhou@zju.edu.cn †E-mail:n.ullah@uq.edu.auwjzhou@zju.edu.cn n.ullah@uq.edu.au Zhejiang Key Lab of Plant Secondary Metabolism and Regulation and College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China Zhejiang Key Lab of Plant Secondary Metabolism and Regulation and College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China Zhuji Municipal Agro-Tech Extension Center, Zhuji 311800, China Zhuji Municipal Agro-Tech Extension Center, Zhuji 311800, China Institute of Crop Science and Zhejiang Key Lab of Crop Germplasm, Zhejiang University, Hangzhou 310058, China Institute of Crop Science and Zhejiang Key Lab of Crop Germplasm, Zhejiang University, Hangzhou 310058, China Queensland Alliance for Agriculture and Food Innovation, Centre for Plant Science, the University of Queensland, Toowoomba, QLD 4350, Australia Queensland Alliance for Agriculture and Food Innovation, Centre for Plant Science, the University of Queensland, Toowoomba, QLD 4350, Australia In this study, we evaluated the effect of the herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273) on barley growth and explored the potential to trigger growth recovery through the application of branched-chain amino acids (BCAAs). Barley plants were foliar-sprayed with various concentrations of ZJ0273 (100, 500, or 1000 mg/L) at the four-leaf stage. Increasing either the herbicide concentration or measurement time after herbicide treatment significantly impaired plant morphological parameters such as plant height and biomass, and affected physiological indexes, i.e. maximal photochemical efficiency ( F v / F m ), quantum yield of photosystem II ( Ф PSII ), net photosynthetic rate ( P n ), and chlorophyll meter value (soil and plant analyzer development (SPAD)). Cellular injury of herbicide-treated plants was also evidenced by increased levels of reactive oxygen species (ROS) and antioxidative enzyme activity. Elevated levels of herbicide significantly reduced the activity of acetolactate synthase (ALS)—a key enzyme in the biosynthesis of BCAAs. In a separate experiment, growth recovery in herbicide-stressed barley plants was studied using various concentrations of BCAAs (10, 50, 100, and 200 mg/L). Increasing BCAA concentration in growth media significantly increased the biomass of herbicide-stressed barley seedlings, but had no significant effect on non-stressed plants. Further, BCAAs (100 mg/L) significantly down-regulated ROS and consequently antioxidant enzyme levels in herbicide-stressed plants. Our results showed that exogenous application of BCAAs could reverse the inhibitory effects of ZJ0273 by restoring protein biosynthesis in barley seedlings. 目的 2 评价除草剂丙酯草醚对大麦生长的影响,探讨外源添加支链氨基酸对其生长的恢复作用. 创新点 2 从除草剂丙酯草醚作用机理出发,阐明了外源添加支链氨基酸对丙酯草醚作用下大麦各项生理和生长指标的缓解作用,为生长调节物质的进一步开发利用提供实验依据. 方法 2 采用水培和大田实验相结合的方法,研究了除草剂丙酯草醚对大麦生长的影响,并通过进一步测定大麦幼苗各项生理指标和支链氨基酸浓度,表明100 mg/L支链氨基酸可显著缓解丙酯草醚对大麦的生理损伤,从而恢复其生长. 结论 2 外源添加支链氨基酸能够缓解除草剂丙酯草醚对大麦生长的抑制作用. Antioxidant enzyme activity Barley Branched-chain amino acid Photosynthetic system Reactive oxygen species ZJ0273 抗氧化酶活性 大麦 支链氨基酸 光合系统 活性氧 丙酯草醚 Introduction 1 Barley ( Hordeum vulgare L.) is an important cereal crop widely cultivated for food and forage purposes throughout the world. Weeds such as Alopecurus aequalis and Malachium aquaticum are the major issue for barley production systems, and can seriously inhibit the growth, yield, and quality of barley crops (Zhang et al., 2000 2000 ). For example, barley yields have been reduced by 28.1% in India (Pandey et al., 1998 1998 ) and 6%–79% in Canada (Watson et al., 2006 2006 ), due to season-long weed competition. In China, weeds in barley production systems are managed partly by crop rotation with rapeseed. This practice leads to an overlapping of weeds in rapeseed and barley fields. In addition, herbicides such as propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273), a broad-spectrum herbicide, are frequently used in oilseed rape production systems in China (Lü et al., 2004 2004 ; Liu et al., 2008 2008 ; Yang et al., 2008 2008 ). Several advantages of this herbicide have been reported such as its high potential for targeting weed plants, low dosage, low mammalian toxicity, broad weeding spectrum and environmental compatibility across Chinese agricultural systems (Chen et al., 2005 2005 ; Zhang et al., 2009 2009 ; Jin et al., 2010 2010 ). Yang et al. ( 2008 2008 ) reported an extensive application of ZJ0273 across Chinese rapeseed fields with about 90% eradication efficiency for monocotyledonous and dicotyledonous weeds. This herbicide when applied at the rate of 100 mg/L can effectively reduce M. aquaticum and A. aequalis in oilseed fields (Zhang et al., 2009 2009 ). In contrast, higher dosages negatively affect the photosynthetic efficiency of oilseed rape (Jin et al., 2010 2010 ). Rapeseed, a dicotyledonous crop, has been found to behave differently from rice, a monocotyledonous crop, in response to ZJ0273 application. Li et al. ( 2009 2009 ) found that monocot species can absorb higher amounts of ZJ0273 than dicots at any given sampling time. This was supported by Guo et al. ( 2011 2011 ), who observed a significant reduction in the volume and number of vacuoles and the mitotic index in ZJ0273-treated barley cells. Acetolactate synthase (ALS), an enzyme that catalyzes the first step of parallel reactions in the biosynthesis of branched-chain amino acids (BCAAs) in plants (Leyval et al., 2003 2003 ), has been found to be the main target of ZJ0273. Previous studies suggested that ZJ0273 arrested weed growth by inhibiting ALS activity (Zhou et al., 2007 2007 ) and thus BCAA synthesis, leading to impaired protein biosynthesis (Liu et al., 2008 2008 ; Tian et al., 2014 2014 ). In plants, changes in the activity of enzymes which catalyze biosynthesis of BCAAs can be reflected in the BCAA content of treated plant leaves (Ortéga and Bastide, 1997 1997 ; Wright et al., 1998 1998 ). Considering the importance of BCAAs in plant defense mechanisms, we proposed that exogenously applied BCAAs may restore growth of herbicide-stressed plants. To our knowledge, little information is available on the morphological or physiological adaptation of barley seedlings to stress induced by ZJ0273. In this paper, we elucidate the biochemical pathways through which the herbicide ZJ0273 damages barley growth and explore the potential of BCAAs to promote growth recovery in herbicide-stressed seedlings. Materials and methods 1 Plant materials and treatments 2 Seeds of barley ( Hordeum vulgare L.) cultivar Zhenongda 3 were obtained from the Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, China. Herbicide ZJ0273 was supplied by the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China. Field trials were conducted at the Zhejiang University farm, Hangzhou, China (30°10′ N, 120°12′ E). Conventional crop management was used throughout the growing period. At the four-leaf stage, barley seedlings were foliar-sprayed once with different concentrations of ZJ0273 (0, 100, 500, or 1000 mg/L). Field trials were conducted to explore the effects of the herbicide on plant growth (plant height, biomass), chlorophyll fluorescence (maximal photochemical efficiency ( F v / F m ), the quantum yield of photosystem II ( Ф PSII ), and non-photochemical quenching (NPQ)), and physiological attributes (concentrations of ALS, glutathione transferases (GSTs), BCAAs, and total amino acids (TAAs)) of the seedlings. The experiment was arranged in a completely randomized design (CRD) with three replicates. A hydroponic experiment was conducted to study growth recovery of ZJ0273-stressed barley seedlings following BCAA application. Mature barley seeds were surface-sterilized with 0.15 g/mL NaClO for 15 min and then washed three times with distilled water. The seeds were arrayed in a germination incubator at a temperature of 20 °C under a 16-h photoperiod (light intensity of 140 μmol/(m 2 ·s)) and high relative humidity (70%‒80%), on a double layer of sterilized filter paper until germination. Seedlings were transplanted into plastic casks containing Hoagland solution, which was continuously aerated with an air pump. Two-week-old plantlets were exposed to hydroponic solution containing the herbicide ZJ0273 (100 mg/L) or no herbicide and with or without BCCAs (0, 10, 50, 100, or 200 mg/L) for 7 d. Then the growth parameters such as plant height and biomass were calculated to evaluate the mitigation effects contributed by BCAAs. Determination of morphological and physiological characteristics 2 In the field trials, the morphological measurements were conducted at 5, 10, and 15 d after treatment (DAT) to estimate the effects of different concentrations of ZJ0273 (0, 100, 500, or 1000 mg/L) on plant growth. Six plants per treatment were harvested for measuring shoot length and fresh and dry weights. Plant samples were then oven-dried at 110 °C for 0.5 h and then at 80 °C until they reached a constant dry weight (Momoh and Zhou, 2001 2001 ). In the hydroponic testing, morphological characteristics were also measured to observe the alleviatory effects of BCAAs. All measurements were made in triplicate. The middle region of the topmost fully expanded barley leaves was used for determining ALS activity following the methods of Yang et al. ( 2000 2000 ) and Leyval et al. ( 2003 2003 ) with some modifications. GST activity was analyzed according to Irzyk and Fuerst ( 1993 1993 ) and Hatton et al. ( 1996 1996 ). The absorbance of the mixture was monitored at 340 nm for 120 s. An extinction coefficient of 9.6 L/(mmol·cm) was used to quantify conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH). Fresh leaf samples (0.2 g) were homogenized in 5 mL potassium phosphate buffer (50 mmol/L, pH 7.8) and then centrifuged at 10 000 g for 20 min at 4 °C. The supernatant was used for analyzing antioxidant enzyme activity, reactive oxygen species (ROS), and malondialdehyde (MDA) content. The method of Zhou et al. ( 1997 1997 ) was used to determine superoxide dismutase (SOD) activity following inhibition of photochemical reduction by nitro blue tetrazolium (NBT). Peroxidase (POD) activity was determined from variation in guaiacol absorbance of the mixture assay at 470 nm (Zhou and Leul, 1998 1998 ). Catalase (CAT) activity was measured at 240 nm according to Cakmak et al. ( 1993 1993 ). Ascorbate peroxide (APX) activity was determined according to Nakano and Asada ( 1981 1981 ) with some modifications. Glutathione reductase (GR) activity was determined according to Jiang and Zhang ( 2002 2002 ). MDA content was measured using a reaction solution (0.5% thiobarbituric acid (TBA) made in 5% trichloroacetic acid (TCA)) (Zhang et al., 2008 2008 ). The extract mixture in buffer solution was heated at 95 °C for 15 min and then immediately cooled on ice. The samples were centrifuged at 4800 g for 10 min and the absorbance of the supernatant was measured at 532 nm. Production of ROS such as H 2 O 2 and O 2 − was determined according to Al-Aghabary et al. ( 2004 2004 ) and Jiang and Zhang ( 2001 2001 ), respectively. The GSH and ascorbic acid (ASA) contents were measured according to Anderson ( 1985 1985 ) and Law et al. ( 1983 1983 ), respectively. Fresh leaf samples (0.2 g) were homogenized with 0.05 g/mL TCA (5 mL) in an ice bath and centrifuged at 4000 g for 10 min. The resultant supernatant was used to measure GSH and ASA contents. Photosynthesis and chlorophyll fluorescence 2 The middle region of the topmost fully expanded leaves was selected for estimating leaf chlorophyll fluorescence using a portable fluorometer (Mini-PAM, Walz, Effeltrich, Germany). The leaves were pre-conditioned in the dark for 30 min, and then minimal fluorescence ( F 0 ) with all photosystem II (PSII) reaction centers opened was determined under weak light (0.04 μmol/(m 2 ∙s)). Maximal fluorescence ( F m ) with all PSII reaction centers closed was measured with a 0.8-s saturating pulse at 5000 μmol/(m 2 ∙s) in dark-adapted leaves. An actinic light (330 μmol/(m 2 ∙s)) was applied until these parameters were at steady state with saturating flashes. From each of these, the value of F m ', the maximum fluorescence in the light, was detected. The steady-state value of fluorescence immediately before a flash of light was termed F t . ( F m − F 0 )/ F m or F v / F m was considered as a measure of the maximum quantum yield of PSII in the dark. NPQ was calculated as ( F m − F m ')/ F m '. The Ф PSII was defined as ( F m '− F t )/ F m ' (Maxwell and Johnson, 2000 2000 ). Chlorophyll meter values (soil and plant analyzer development (SPAD) readings) were measured using a chlorophyll meter (Minolta SPAD-502, Japan). The net photosynthetic rate ( P n ) of leaves was measured using a portable photosynthesis system (Model Li-6400, USA). Each measurement was replicated three times. Determination of endogenous free amino acid content 2 Fresh leaf samples were collected randomly to measure the concentrations of endogenous free amino acids including three BCCAs (valine (Val), leucine (Leu), and isoleucine (ILe)) and a total of 17 amino acids (TAAs) at three different time intervals, i.e. 5, 10, and 15 DAT. The samples were fixed in an oven at 110 °C for 30 min and then at 80 °C until constant weight. Dried samples were ground into powder and sieved through 60-mesh. Free amino acid content was determined according to the method of Zhu and Zhao ( 1994 1994 ). Powder was rubbed in 1 mL of 80% alcohol until volatilization and then homogenized with 1 mL 4% sulfosalicylic acid before extracting using an ultrasonicator for 20 min. The supernatant was collected after centrifugation at 12 000 g for 15 min and mixed with 1 mL 4% sulfosalicylic acid in deposition, and the extraction procedure was repeated. The two supernatants were merged and sterilized with a 0.25-μm filter. Free amino acid content was calculated by an automatic amino acid analyzer (Hitachi, L-8900, Japan). Statistical analysis 2 In these experiments, three replicates were used for each measurement. Data were analyzed using the Statistical Analysis System (SAS). Significant effects were determined by analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (LSD) test to identify homogenous groups within the means at P ≤0.05 level. Results 1 Changes in free amino acid content 2 A significant increase in BCAAs such as Val, Leu, and ILe was recorded in response to increasing the ZJ0273 application rate or the treatment duration in this field trial (Table 1 1 ). BCAAs in barley leaves remained unaffected by the lower herbicide (100 mg/L) level at 5 DAT, but the treated plants experienced a significant increase in BCAAs at 10 and 15 DAT. No significant change in BCAAs was observed when the ZJ0273 treatment rate was increased beyond 100 mg/L at 10 DAT. BCAAs were undetectable in plant tissues under higher ZJ0273 treatments (500 and 1000 mg/L), when treatment duration was prolonged to 15 d. Within a column, means at the same DAT followed by the same letters are not significantly different by the LSD test at P ≤0.05 under completely randomized design. Data were analyzed separately at each measurement time. DAT: days after treatment; Val: valine; Leu: leucine; Ile: isoleucine; TAAs: total amino acids; BCAAs: branched-chain amino acids 2 DAT 2 Concentration of ZJ0273 (mg/L) 4 Concentration of free amino acids (mg/100 mg DW) 2 BCAAs/TAAs (%) Val Leu Ile TAAs 4 5 0 1.816 b 1.977 b 2.023 b 29.450 d 19.75 a 100 1.996 ab 2.231 b 2.244 b 32.921 c 19.66 a 500 1.936 ab 2.256 b 2.318 ab 37.957 b 17.15 c 1000 2.139 a 2.908 a 2.666 a 43.070 a 17.91 b 4 10 0 1.653 b 2.239 b 1.733 b 31.065 c 18.11 b 100 2.167 a 2.660 a 2.342 a 36.059 b 19.88 a 500 2.265 a 2.635 a 2.561 a 40.209 b 18.55 b 1000 2.529 a 2.842 a 2.838 a 46.487 a 17.66 c 4 15 0 1.865 b 2.133 b 2.231 a 32.403 b 19.23 a 100 2.336 a 4.376 a 2.312 a 47.293 a 19.08 a 500 1000 5 TAA content in plant tissues also increased significantly in response to increasing ZJ0273 concentrations. Higher (500 and 1000 mg/L) ZJ0273 concentrations significantly increased TAAs and decreased BCAAs/TAAs compared with untreated controls at 5 and 10 DAT. Under the prolonged treatment duration (15 DAT) of ZJ0273 (500 and 1000 mg/L), TAAs were not detectable due to the death of the plants, suggesting that longtime treatment duration with higher dosages of ZJ0273 (500 and 1000 mg/L) was toxic for barley seedlings. Plant growth response to different concentrations of ZJ0273 2 Fig. 1 1 shows the plant growth in response to the different concentrations of ZJ0273 (0–1000 mg/L) at the fourth-leaf stage of barley. Treatments with ZJ0273 (100–1000 mg/L) significantly inhibited plant growth, as reflected by the reductions in plant height, fresh weight, and dry weight of barley seedlings at 5 DAT. Similar trends were observed at 10 and 15 DAT, compared with controls. Plant development in the controls was obviously better than that in all treated plantlets. The growth of barley was obviously suppressed under the stress of 100 mg/L ZJ0273 compared with the control. With the successive increases of ZJ0273 concentrations from 100 to 1000 mg/L, the shoot length and biomass of barley dramatically declined. However, the barley plantlets did not show a significant difference between 500 and 1000 mg/L ZJ0273 for shoot length or biomass at all DAT (Fig. 1 1 ). When plants were treated with 1000 mg/L of ZJ0273, 24.9%, 22.2%, and 27.9% reductions in shoot length were observed at 5, 10, and 15 DAT, respectively, compared with controls. Prolonging the DAT from 5 to 15 led to growth recovery under the lower dosage of ZJ0273 (100 mg/L), as reflected by the shoot length, fresh weight, and dry weight. However, these trends were not observed with the application of higher dosages of ZJ0273 (500 and 1000 mg/L). Thus, 100 mg/L ZJ0273 was relatively safe for the barley seedlings. Measurements were made at 5, 10, and 15 d after treatment (DAT). Values are expressed as mean±standard error (SE), n =3. Means followed by the same letters did not differ significantly by the LSD test at P ≤0.05 Chlorophyll fluorescence and photosynthesis 2 Increasing ZJ0237 rates of application significantly reduced maximal photochemical efficiency ( F v / F m ) of barley leaves at 1 DAT, and this reduction grew in the subsequent measurements (1–9 DAT; Table 2 2 ). The maximum reduction in F v / F m was observed in response to 1000 mg/L ZJ0273, which caused a 14.1% reduction (compared with controls) in F v / F m at 9 DAT. Changes in leaf Ф PSII in response to ZJ0237 application followed a similar pattern to that of F v / F m . Although 100 mg/L ZJ0273 had no significant effect on Ф PSII at 1 DAT, a steep reduction in leaf Ф PSII was recorded under 1000 mg/L ZJ0273 with increasing application period. For example, Ф PSII was reduced by 13.6% at 1 DAT and by 46.4% at 9 DAT (compared with controls). In contrast to F v / F m , a significant increase in NPQ of barley leaves was observed in response to increasing the ZJ0237 application rate or the time after treatment, although the lower herbicide concentration (100 mg/L) had no significant effect on NPQ at 1 DAT. The 1000 mg/L ZJ0273 treatment increased NPQ by 72.8% at 1 DAT and by 124.4% at 9 DAT, compared with controls. No significant differences in the NPQ of the plants were detected between treatments of 500 or 1000 mg/L of ZJ0237 when measured at 5–9 DAT (Table 2 2 ). Within a column, means at the same DAT followed by the same letters are not significantly different by the LSD test at P ≤0.05 under completely randomized design. Data were analyzed separately at each measurement time. DAT: days after treatment; F v / F m : maximal photochemical efficiency; Ф PSII : quantum yield of PSII; NPQ: non-photochemical quenching DAT Concentration of ZJ0273 (mg/L) F v / F m Ф PSII NPQ 1 0 0.816 a 0.550 a 0.180 b 100 0.808 b 0.534 a 0.195 b 500 0.805 bc 0.478 b 0.316 a 1000 0.800 c 0.475 b 0.311 a 3 0 0.813 a 0.511 a 0.185 d 100 0.794 b 0.445 b 0.283 c 500 0.788 b 0.417 c 0.397 b 1000 0.785 b 0.409 c 0.433 a 5 0 0.815 a 0.517 a 0.181 c 100 0.799 b 0.421 b 0.336 b 500 0.762 c 0.364 c 0.411 a 1000 0.758 c 0.361 c 0.435 a 7 0 0.816 a 0.539 a 0.209 c 100 0.780 b 0.382 b 0.400 b 500 0.750 c 0.352 c 0.443 a 1000 0.725 d 0.338 c 0.440 a 9 0 0.817 a 0.552 a 0.205 c 100 0.755 b 0.428 b 0.427 b 500 0.729 c 0.326 c 0.448 ab 1000 0.702 c 0.296 d 0.460 a SPAD values of barley leaves declined significantly with each successive increase in the ZJ0273 application rate (Table 3 3 ). In addition, reductions in leaf SPAD were observed as the time after treatment was increased. For example, addition of 1000 mg/L ZJ0273 caused reductions in leaf SPAD of 57.7% at 20 DAT and 65.9% at 30 DAT, compared with controls. Damage to leaf greenness resulted in reductions in leaf P n under increasing ZJ0273 concentrations and increasing measurement time after treatment. The maximum reduction in leaf P n was observed under 1000 mg/L ZJ0273, causing a 75.6% reduction compared with the control at 30 DAT (Table 3 3 ). Within a column, means at the same DAT followed by the same letters are not significantly different by the LSD test at P ≤0.05 under completely randomized design. Data were analyzed separately at each measurement time. Values are expressed as mean±SE ( n =3). DAT: days after treatment; SPAD: chlorophyll meter value (soil and plant analyzer development); P n : net photosynthetic rate DAT Concentration of ZJ0273 (mg/L) SPAD P n 20 0 36.37±0.96 a 24.11±1.32 a 100 22.37±1.72 b 16.11±1.98 b 500 18.07±1.16 c 12.54±0.32 c 1000 15.37±0.71 d 6.88±0.92 d 30 0 36.90±1.10 a 24.54±1.17 a 100 19.90±0.26 b 12.69±0.34 b 500 15.70±1.00 c 9.49±0.77 c 1000 12.57±1.39 d 6.00±0.38 d Endogenous enzyme activity 2 No significant effect of the lower ZJ0273 concentration (100 mg/L) was observed on ALS activity of barley leaves at 5 or 10 DAT (Table 4 4 ), although higher application rates significantly reduced ALS activity. ALS activity was significantly reduced in response to all ZJ0273 application rates when measured at 15 and 20 DAT. With successive increases in ZJ0273 concentrations from 100 to 1000 mg/L, ALS activity dramatically decreased. When plants were treated with 1000 mg/L of ZJ0273, the ALS activity of barley leaves was reduced by 28.3% at 5 DAT, 36.3% at 10 DAT, 50.2% at 15 DAT, and 57.7% at 20 DAT compared with controls. Within a column, means at the same DAT followed by the same letters are not significantly different by the LSD test at P ≤0.05 under completely randomized design. Data were analyzed separately at each measurement time. Values are expressed as mean±SE ( n =3). DAT: days after treatment; ALS: acetolactate synthase; GST: glutathione transferase; OD 530 : optical density at 530 nm; FW: fresh weight DAT ZJ0273 (mg/L) ALS activity (OD 530 /(g FW·min)) GST activity (nmol/(min·mg protein)) 5 0 1.87±0.16 a 5.85±0.31 c 100 1.83±0.36 a 7.76±0.76 b 500 1.62±0.41 b 11.30±0.81 a 1000 1.34±0.35 c 11.83±1.48 a 10 0 1.90±0.45 a 6.61±0.53 b 100 1.81±0.42 a 8.05±0.66 b 500 1.59±0.24 b 12.84±1.07 a 1000 1.21±0.52 c 14.04±2.14 a 15 0 2.07±0.21 a 6.59±0.50 d 100 1.69±0.19 b 10.41±0.61 c 500 1.52±0.38 c 13.86±1.12 b 1000 1.03±0.41 d 16.05±0.86 a 20 0 2.15±0.22 a 6.62±0.46 d 100 1.65±0.25 b 15.40±1.04 c 500 1.47±0.19 c 17.93±0.94 b 1000 0.91±0.17 d 20.90±1.61 a GST activity of barley leaves increased in response to increasing herbicide application rates, although no significant change in GST activity was observed by increasing ZJ0273 concentrations from 500 to 1000 mg/L at 5 or 10 DAT (Table 4 4 ). Maximum GST activity (20.90 nmol/(min·mg protein)) was observed under 1000 mg/L ZJ0273 at 20 DAT, when it was 215.7% greater than that of the control leaves. Growth of ZJ0273-stressed seedlings in response to exogenously applied branched-chain amino acids 2 A significant reduction in shoot length, fresh and dry weights was recorded in barley seedlings in response to 100 mg/L of ZJ0273 (Table 5 5 ). Plants treated with 100 mg/L ZJ0273 alone experienced maximum damage in this hydroponic study, showing reductions of 31.3% in shoot length, 30.2% in fresh weight, and 25.0% in dry weight, compared with controls. Exogenously applied BCAAs (10–200 mg/L) in the nutrient solution significantly increased the growth of barley seedlings in terms of shoot length and fresh and dry weights. Application of BCAAs to herbicide-free media also promoted growth of barley seedlings, but the effect on shoot length or dry weight was not significant compared with the control (non-BCAA, non-ZJ0237 treated). Maximum growth (shoot length, fresh and dry weights) of herbicide-stressed seedlings was observed with 200 mg/L of BCAAs. Plants treated with 200 mg/L BCAAs alone showed no significant change in growth. BCAAs significantly promoted barley growth when applied to ZJ0273 (100 mg/L)-stressed plants, causing increases of 37.0% in shoot length, 38.1% in fresh weight, and 33.9% in dry weight, compared with the 100 mg/L ZJ0273 treatment alone. The fresh and dry weights of the plants remained unaffected when treated with 100 mg/L ZJ0273+200 mg/L BCAAs. No significant changes occurred in the shoot growth of stressed seedlings with 100 or 200 mg/L of BCAAs, suggesting that 100 mg/L of BCAAs could be the best concentration for protecting barley seedlings from herbicide stress. Thus, we selected this concentration for the subsequent physiology experiments. Within a column, means followed by the same letters are not significantly different by the LSD test at P ≤0.05 under completely randomized design. Z0: control; Z100: 100 mg/L ZJ0273; aa10: 10 mg/L BCAAs (valine, leucine, isoleucine); aa50: 50 mg/L BCAAs; aa100: 100 mg/L BCAAS; aa200: 200 mg/L BCAAs Concentration (mg/L) Shoot length (cm) Fresh weight (g/10 plants) Dry weight (g/10 plants)  Z0 19.40 a 2.186 bcd 0.220 abc  Z0+aa10 19.46 a 2.223 bc 0.223 ab  Z0+aa50 19.49 a 2.338 ab 0.230 a  Z0+aa100 19.51 a 2.398 a 0.239 a  Z0+aa200 19.77 a 2.469 a 0.236 a  Z100 13.32 e 1.526 f 0.165 e  Z100+aa10 14.78 d 1.715 e 0.191 d  Z100+aa50 16.03 c 1.814 e 0.200 cd  Z100+aa100 18.08 b 2.050 d 0.210 bcd  Z100+aa200 18.25 b 2.108 cd 0.221 abc ZJ0273 treatment (100 mg/L) increased the generation of ROS such as O 2 − and H 2 O 2 in barley leaf tissues (Fig. 2 2 ). In contrast, addition of 100 mg/L BCAAs to the solution significantly decreased ROS generation in herbicide-treated seedlings. BCAA application caused reductions of 36.4% in O 2 − and 24.4% in H 2 O 2 content of herbicide-stressed plants, compared with ZJ0273 treatment alone. BCAAs applied to herbicide-free media had no significant effect on leaf O 2 − or H 2 O 2 content. Values are expressed as mean±SE ( n =3). Means followed by the same letters did not differ significantly by the LSD test at P ≤0.05. CK: control, no ZJ0273; aa: BCAAs; FW: fresh weight ASA and GSH content in leaf tissues increased significantly in response to 100 mg/L ZJ0273 and decreased when BCAAs were added to the growth media. BCAAs caused reductions of 20.2% in ASA content and 10.1% in GSH content of the leaves of herbicide-treated seedlings, compared with ZJ0273 treatment alone. The effect of BCAAs on herbicide-free seedlings was non-significant (Fig. 3 3 ). Values are expressed as mean±SE ( n =3). Means followed by the same letters did not significantly differ by the LSD test at P ≤0.05. CK: control, no ZJ0273; aa: BCAAs; FW: fresh weight The activity of various antioxidant enzymes (POD, SOD, CAT, APX, and GR) and leaf peroxidation (MDA) increased significantly in response to ZJ0273 treatment (Fig. 4 4 ). BCAAs applied exogenously to herbicide-stressed seedlings, significantly reduced antioxidant enzyme activity and MDA content. For example, BCAAs applied to herbicide-stressed plants decreased the activity of POD by 19.4%, SOD by 35.3%, CAT by 17.2%, and APX by 48.6%, compared with ZJ0273 treatment alone, although GR activity remained unaffected. No significant effect of BCAA application was observed on antioxidant enzymes or the MDA content of barley seedlings in herbicide-free media, except for SOD and APX activity, which increased significantly under BCAA treatment under both herbicide-stressed and herbicide-free conditions. (a) Peroxidase (POD) activity; (b) Superoxide dismutase (SOD) activity; (c) Catalase (CAT) activity; (d) Ascorbate peroxide (APX) activity; (e) Glutathione reductase (GR) activity; (f) Malondialdehyde (MDA) content. Values are expressed as mean±SE ( n =3). Means followed by the same letters did not significantly differ by the LSD test at P ≤0.05. CK: control, no ZJ0273; aa: BCAAs; FW: fresh weight Discussion 1 The first objective of these experiments was to identify the mechanism by which herbicide ZJ0273 inhibits barley growth. A significant reduction in SPAD and chlorophyll fluorescence of herbicide-stressed leaves indicated that ZJ0273 can impair chlorophyll structure and function. Ralph et al. ( 2005 2005 ) indicated that inhibition curves of chlorophyll fluorescence parameters varied with different herbicide concentrations. Damage to photosynthetic machinery leads to an overall inhibition of photosynthesis and biomass production (Anderson, 1985 1985 ; Cobb and Kirkwood, 2000 2000 ; Abbaspoor et al., 2006 2006 ; Ali et al., 2018 2018 ). Various biochemical pathways for stress-induced P n reduction have been proposed. The interrupted biosynthesis of proteins associated with the carbon assimilation process has been suggested by Halliwell and Gutteridge ( 1999 1999 ) and Romero-Puertas et al. ( 2004 2004 ). Similarly, Orcaray et al. ( 2010 2010 ) indicated that glyphosate can inhibit biosynthesis of aromatic amino acids by blocking the shikimate pathway and arresting the photosynthetic process in leaf tissues. Herbicide-induced chlorophyll impairment in this study could have been associated with an increased production of ROS and lipid membrane damage (MDA production), which are the major causes of cellular injury in plants. In contrast to a significant recovery in leaf P n at 28 DAT in 100 and 500 mg/L ZJ0273-treated Brassica plants (Jin et al., 2010 2010 ), no photosynthesis recovery in barley seedlings suggested a relatively high herbicide sensitivity for monocotyledons. Although stressed barley plants up-regulated defense-related primary antioxidants (POD, SOD, CAT, APX, and GR) and secondary antioxidants (ASA and GSH), no significant growth recovery was observed in this study. This suggests that ZJ0273 can cause permanent cellular damage in barley seedlings and the plants are unable to mitigate this damage through internal antioxidative defense mechanisms. Up-regulation of antioxidant enzymes has also been linked with increased cellular injury to ZJ0273-treated Brassica plants (Law et al., 1983 1983 ; Dixon et al., 1998 1998 ; Frankart et al., 2003 2003 ; Jin et al., 2010 2010 ). Studies of the key enzymes in the biosynthesis of natural products have been important in revealing the mechanism pathways (Yang et al., 2012 2012 ; Liang et al., 2013 2013 ; Xia et al., 2015 2015 , 2016 2016 ; Zhang et al., 2017 2017 ). Herbicide-induced growth suppression in barley seedlings in this study was associated with inhibited activity of ALS, an enzyme associated with BCAA production. Previous studies indicated that ALS inhibitors can block the biosynthesis of BCAAs and cell division, and eventually cause plant death (Ray, 1984 1984 ; Shaner and Singh, 1993 1993 ). Chen et al. ( 2005 2005 ) suggested that ZJ0273 can inhibit ALS activity by altering the biosynthesis of amino acids. Similarly, using near-infrared spectroscopy (NIRS) techniques, Tian et al. ( 2014 2014 ) confirmed a gradual reduction in free amino acids (BCAAs and TAAs) and ALS activity in oilseed rape leaves in response to increasing ZJ0273 concentrations. Growth inhibition in ALS inhibitor-treated plants has been linked with a dramatic decrease in specific BCAAs (Rost and Reynolds, 1985 1985 ; Hofgen et al., 1995 1995 ). In contrast, a significant increase in BCAA content in this study under herbicide application could be the result of protein transformation to satisfy new protein requirements (Royuela et al., 2000 2000 ; Orcaray et al., 2010 2010 ). Similarly, Royuela et al. ( 2000 2000 ) and Liu et al. ( 2008 2008 ) suggested an increase in BCAA content in response to herbicide application as a result of degradation of some proteins to maintain the balance of new protein synthesis. A significant increase in BCAAs/TAAs of ZJ0273-stressed barley leaves in this study supports the suggestion that amino acid composition and thus protein biosynthesis had been altered. The second objective of this study was to examine the growth recovery of herbicide-stressed barley seedlings following BCAA application. A significant growth recovery in ZJ0273-stressed barley was achieved in response to BCAA application. No significant effect of BCAAs on unstressed plants suggested that the primary role of these amino acids was to restore growth of stressed plants without affecting normal plant growth patterns. Exogenously applied amino acids had already been found to be effective in alleviating the negative effects of ALS inhibitors in pea (Rost and Reynolds, 1985 1985 ) and corn (Shaner and Reider, 1986 1986 ). Therefore, the herbicide ZJ0273 may block the biosynthetic pathway of BCAAs by inhibiting ALS, and exogenous application of BCAAs may compensate for the shortage of BCAAs induced by the herbicide (Tang et al., 2006 2006 ; Xia et al., 2006 2006 ). Conclusions 1 In this study, we confirmed that tolerance to herbicide increased with the growth of barley. The lower dosage (100 mg/L) of ZJ0273 was safer for barley than the higher dosages (500 and 1000 mg/L). 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Chin J Chromatogr , 1994 . 12 ( 1 ): 20 - 24 . (in Chinese) . Analysis of amino acids by high liquid chromatography Compliance with ethics guidelines Ling XU, Jian-yao SHOU, Rafaqat Ali GILL, Xiang GUO, Ullah NAJEEB, and Wei-jun ZHOU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1700375 Project supported by the Zhejiang Science and Technology Department (Nos. LGN18C130007 and 2016C02050-8), the Jiangsu Collaborative Innovation Center for Modern Crop Production, and the Science Foundation of Zhejiang Sci-Tech University (No. 14042216-Y), China 2017-07-25 2018-01-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 20