Comprehensive evaluation of fluroxypyr herbicide on physiological parameters of spring hybrid millet

Plant and experimental design

Fluroxypyr (20%, emulsifiable concentrate) was provided by Dow AgroScience Co. (Jiangsu, China). Foxtail millet (S. italica L. cv. Zhangza 5) was supplied by the Zhangjiakou Academy of Agricultural Sciences of Hebei Province, China. The seeds were uniformly sown in a plastic pot (130 mm diameter) containing a mixture of sand and soil (1:2, v:v). Calcareous cinnamon soil, with a pH of 7.85, 24.49 g kg⁻¹ organic matter, 51.92 mg kg⁻¹ total nitrogen, 24.13 mg kg⁻¹ available phosphorus, and 183.6 mg kg⁻¹ rapidly-available potassium was used in this work. At the three-leaf stage, the foxtail millet seedlings were thinned and maintained at 10 uniform plants per pot.

The experiment was conducted as a randomized complete block design with three replications. After growing to the five-leaf stage, foxtail millet seedlings were treated with 0, 0.5, 1, 2, and 4 L ai ha⁻¹ fluroxypyr by using a laboratory pot-sprayer equipped with a nozzle and previously calibrated to deliver 450 L ha⁻¹. The manufacturer recommended an effective dose of one L ai ha⁻¹ for field application. After herbicide treatment for 5 and 10 days, the physiological indices of all foxtail millet seedlings were analyzed.

Measurement of growth parameters

The plant height, length and width of leaves were measured with a ruler. Leaf area was calculated using the following equation: leaf area = 0.75 × leaf length × leaf width. Leaf width was considered the widest part of the penultimate leaf.

Determination of superoxide generation rate and H₂O₂ content

Fresh foxtail millet leaves (0.1 g) were homogenized with two mL of 65 mmol L⁻¹ sodium phosphate buffer (pH 7.8) and centrifuged at 10,000 rpm for 10 min. The supernatant (one mL) was mixed with one mL of 65 mmol L⁻¹ sodium phosphate buffer (pH 7.8) and 0.2 mL of 10 mmol L⁻¹ hydroxyl ammonium chloride. The resulting supernatant was incubated 25 °C for 20 min, and the above reaction mixture (one mL) was mixed with one mL of 4-aminobenzene sulfonic acid (17 mmol L⁻¹) and one mL of α-naphthylamine (seven mmol L⁻¹), then incubated at 30 °C for 30 min. The absorbance at 530 nm was measured with a 756C-UV-VIS spectrophotometer (Shanghai Spectrum Instruments Co. Ltd, Shanghai, China) (Elstner & Heupel, 1976).

Fresh foxtail millet leaves (0.1 g) were homogenized with five mL of chilled acetone in an ice bath and centrifuged at 4,000 rpm for 15 min. The supernatant (one mL) was mixed with 0.1 mL of 20% TiCl₄ concentrated hydrochloric acid and 0.2 mL of concentrated ammonium hydroxide. The reaction mixture was centrifuged at 10,000 rpm for 10 min and the precipitate was dissolved in three mL H₂SO₄ (one mol L^–1). The absorbance of the supernatant at 410 nm was monitored with a 756C-UV-VIS spectrophotometer (Zhang et al., 2012).

Determination of antioxidant enzyme activities

Fresh foxtail millet leaves (0.1 g) were homogenized with two mL of sodium phosphate buffer (pH 7.0, containing 0.1 mmol L⁻¹ EDTA and 1% PVP (w/v)) in an ice bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant was extracted and used to measure the activities of superoxide (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). The samples were measured with a 756C-UV-VIS spectrophotometer.

Superoxide activity was analyzed by using the nitro blue tetrazolium (NBT) method. The reaction mixture (five mL) consisted of phosphate buffer (50 mmol L⁻¹, pH 7.8), L-methionine (13 mmol L⁻¹), NBT (0.075 mmol L⁻¹), EDTA (0.1 mmol L⁻¹), riboflavin (0.002 mmol L⁻¹), and 20 μL of enzyme extract. The reaction mixture was then illuminated under 4000 lx for 15 min at 25 °C. One unit of activity corresponds to the amount of protein required to inhibit 50% of the initial reduction of NBT under light conditions. The absorbance at 560 nm was measured, and a non-irradiated complete reaction mixture served as the control. For POD activity determination, 20 µL of enzyme extract was added into three mL of reaction liquid containing three mL of sodium phosphate buffer (100 mmol L⁻¹, pH 6.0), 19 µL of guaiacol, and 28 µL of 30% H₂O₂. POD activity was measured by the changes in the absorbance of the reaction solution at 470 nm for 3 min. For CAT activity determination, the reaction liquid consisted of 2.7 mL of Tris-HCl (50 mmol L⁻¹, pH 7.0), 50 µL of H₂O₂ (200 mmol L⁻¹), and 20 µL of enzyme extract. Enzyme activity was continuously determined at 240 nm for 3 min following H₂O₂ decomposition. In accordance with the method of APX activity determination by Nakano & Asada (1981), the reaction mixture consisted of three mL sodium phosphate buffer (50 mmol L⁻¹, pH 7.0), 0.4 mL of EDTA (0.3 mmol L⁻¹), one mL of ascorbate (0.9 mmol L⁻¹), and one mL of enzyme extract. The reaction mixture was then incubated for 5 min at 25 °C. The above reaction mixture was mixed with 0.5 mL of H₂O₂ (0.25 mmol L⁻¹), and H₂O₂ decomposition was measured by the decline in absorbance at 290 nm for 1 min.

Glutathione reductase (GR) activity was analyzed according to Halliwell & Foyer (1978). Fresh foxtail millet leaves (0.1 g) were ground in an ice bath with two mL of Tris-HCl (pH 7.5, containing 1% PVP and 0.1 mmol L⁻¹ EDTA), and centrifuged at 10,000 rpm for 15 min at 4 °C. The enzyme extract (150 μL) was added into three mL of reaction liquid including Tris-HCl (pH 7.5), MgCl₂ (three mmol L⁻¹), GSSG (0.5 mmol L⁻¹), and NADPH (0.15 mmol L⁻¹). Enzyme activity was calculated by the change in absorbance at 340 nm for 3 min.

Determination of photosynthetic gas exchange and chlorophyll

Photosynthetic rate (P_N), transpiration rate (E), and stomatal conductance (G_s) were measured by CI-340 portable photosynthesis system (CID Bio-Science, Inc., Camas, WA, USA) from 9:30 to 10:30 am. The photosynthetically active radiation (PAR) at the leaf surface was approximately 1,000 ± 50 µmol/m²/s, the temperature of the leaf chamber was 30 ± 2 °C, and the ambient CO₂ concentration was 380 ± 50 µmol/mol.

To measure photosynthetic pigments, fresh leaves (0.1 g) were soaked in 10 mL of ethanol (96%, v/v) and stored in the dark for 24 h. The absorbance of the supernatants was measured at 649 and 665 nm with a 756C-UV-VIS spectrophotometer (Lichtenthaler, 1987).

Measurement of chlorophyll fluorescence and P₇₀₀ parameters

The chlorophyll fluorescence and P₇₀₀ parameters were simultaneously detected using a luminoscope (Dual PAM-100, WALZ, Effeltrich, Germany) (Pfündel, Klughammer & Schreiber, 2008). After dark adaptation for 30 min, the kinetics of chlorophyll fluorescence induction and P₇₀₀ oxidation were simultaneously detected according to the “Fluo + P₇₀₀” analysis mode.

First, the minimal fluorescence (F₀) was detected under weak light (seven µmol/m²/s). Subsequently, the maximum fluorescence (F_m) was determined by the saturation pulse (4,000 μmol/m² /s, 800 ms) method. The slow induction curve was calculated as follows: F_v/F_m = (F_m − F₀)/F_m, ETR_II = PAR × 0.84 × 0.5 × Y_II, Y_II = (F′_m – F)/F′_m, Y_NO = 1/(NPQ + 1 + qL (F_m/F₀ − 1)), Y_NPQ = 1 − Y_II − Y_NO, where F_v/F_m and Y_II are the maximum PSII quantum yield and effective PSII quantum yield, respectively, and Y_NPQ and Y_NO are the regulated energy dissipation and non-regulated energy dissipation, respectively.

P₇₀₀ oxidation was monitored by the changes in the transmittance signals at 875 and 830 nm (Klughammer & Schreiber, 2008). The maximal P₇₀₀ (P_m) was denoted by the maximal P₇₀₀ signal reduction to full oxidation. Y_NA, the nonphotochemical quantum yield of photosystem I (PSI), is a measure of the acceptor-side limitation and calculated according to the formula Y_NA = (P′_m − P_m)/P′_m. Y_I, the photochemical quantum yield of PSI, was calculated according to the formula Y_I = (P′_m− P)/P_m. Y_ND, the nonphotochemical quantum yield of PSI, is a measure of the donor side limitation and calculated as Y_ND = (P − P₀)/P_m. The sum of these three quantum yields is one, i.e.: Y_I + Y_ND + Y_NA = 1. The electron transfer efficiency of PSI, ETR_I was assessed by the Dual PAM software.

Measurement of endogenous hormones

Fresh foxtail millet leaves (0.5 g) were homogenized with four mL of 80% methanol (one mmol/L 2, 6-di-tert-butyl-4-methylphenol) in an ice bath. The homogenate mixture was refrigerated at 4 °C for 4 h, and centrifuged at 3,500 rpm for 8 min. The precipitate was added to one mL of 80% methanol, refrigerated at 4 °C for 1 h, and centrifuged. The supernatant passed through a C₁₈ solid phase extraction column, and then dried with nitrogen. The hormone extract was obtained by mixing with one mL of the sample diluent (PBS containing 0.1% Tween-20, gelatin one µg/L) and determined using enzyme-linked immunosorbent assay. The hormone assay kit was supplied by China Agricultural University, and the content of auxin (IAA), abscisic acid (ABA), zeatin (ZR), and gibberellin (GA) contents were measured by using the Thermo Multiskan FC enzyme-labeled instrument.

Statistical analysis

Statistical analyses were performed using Statistical Product and Service Solutions 19.0 (SPSS Inc., Chicago, IL, USA). Quantitative data were expressed as the mean ± standard error, and multiple comparisons were analyzed by Duncan’s multiple range test. P < 0.05 was considered significantly different. In this study, Fisher discriminant and principal component analyses were calibrated by using the selected indices with the highest difference between the different herbicide treatments and control, to simplify the indicators.

Effects of fluroxypyr on growth parameters

After exposing the seedlings to 0.5–4.0 L ai ha⁻¹ fluroxypyr for 5 days, the plant height of Zhangza 5 significantly differed between the control and herbicide treatments. At 10 days post-fluroxypyr treatment, the plant height increased by 10.16%, 12.62%, and 5.73% after exposure to 0.5, 1.0, and 2.0 L ai ha⁻¹, respectively. As shown in Table 1, the leaf area of Zhangza 5 significantly differed between the recommended dose (one L ai ha⁻¹) and other treatments after 5 days. A similar response of the leaf area was recorded when Zhangza 5 was exposed to fluroxypyr for 10 days, thereby showing significant difference among treatments compared with the recommended dosage.

Effects of fluroxypyr on stress parameters

Fluroxypyr treatment at doses ranging from zero to four L ai ha⁻¹ increased H₂O₂ and O₂⁻ accumulation in plants, with the maximum expression levels obtained after treatment with a dosage of four L ai ha⁻¹ for 5 and 10 days. As shown in Table 2, maximum H₂O₂ and O₂⁻ accumulations significantly increased by treatment with four L ai ha⁻¹ fluroxypyr for 5 and 10 days compared with the control. Similarly, treatment with fluroxypyr at the recommended dose (one L ai ha⁻¹) increased H₂O₂ and O₂⁻ contents by 1.01- and 1.02- fold relative to the control after 5 days and by 2.02- and 1.05-fold relative to the control after 10 days, respectively. In our studies, we observed a time-dependent decrease in reactive oxygen species (ROS) in response to fluroxypyr exposure.

Effects of fluroxypyr on enzyme activities

As shown in Fig. 1, the SOD, POD, CAT, APX, and GR activities significantly increased. The exposure of Zhangza 5 to increasing levels of fluroxypyr led to increases in SOD, CAT, APX, and GR activities. However, further increases in fluroxypyr concentration beyond two L ai ha⁻¹ failed to promote SOD, CAT, APX, and GR accumulation. The activities of SOD, CAT, APX, and GR significantly increased after treatment with two L ai ha⁻¹ fluroxypyr relative to the control. POD activities were stimulated after exposure to different fluroxypyr concentrations, and its effect was concentration-dependent; the highest POD activity was observed at four L ai ha⁻¹ fluroxypyr. POD activities significantly increased by 2.91- and 3.53-fold relative to the control after treatment with four L ai ha⁻¹ fluroxypyr for 5 and 10 days, respectively.

Effects of fluroxypyr on net photosynthetic rate and pigments

As shown in Table 3, P_N, E, G_s, and Chl showed a progressive decrease with increasing fluroxypyr concentration. After one L ai ha⁻¹ (recommended dose) fluroxypyr treatment for 5 days, P_N and E significantly decreased by 23.72% and 22.56% compared with the control, respectively. However, one L ai ha⁻¹ fluroxypyr treatment for 5 days inhibited G_s by 3.53%. Moreover, after with one L ai ha⁻¹ fluroxypyr for 10 days, P_N, E, and G_s of the treated plants were reduced by 18.78%, 15.12%, and 18.79% compared with the control, respectively. According to our results, significant differences were recorded between the treatments and control. Chlorophyll content was also significantly affected following fluroxypyr treatment; however, no significant differences were observed between the control and treatment using the recommended dose after 5 and 10 days of exposure.

Effects of fluroxypyr on chlorophyll fluorescence and P₇₀₀ parameters

After 5 days of exposure to fluroxypyr, the F_v/F_m of treated plants decreased to 0.13%, 0.32%, 1.71%, and 3.29% compared with the control, and the differences between treatments were insignificant. Moreover, after 10 days, the F_v/F_m of the treated plants recovered to control levels. As shown in Fig. 2, the changes in Y(II) and ETR(II) were consistent under fluroxypyr treatment, and the values were reduced in a dose-dependent manner. After 5 and 10 days of treatment, Y(II) decreased from 12.44% to 22.96%, and from 18.25% to 32.14%, respectively, and ETR(II) decreased from 12.37% to 22.69%, and from 14.91% to 32.31%, respectively. Additionally, Y(NPQ) increased with increasing fluroxypyr doses, and the maximum accumulation was observed at two L ai ha⁻¹. During treatment with two L ai ha⁻¹ fluroxypyr, the Y(NPQ) values were 36.39%, and 50.75%, compared with the control.

PSI activities were substantially affected by fluroxypyr treatment. As shown in Fig. 2, P_m, Y(I), and ETR(I) were evidently decreased with increasing fluroxypyr dosage; however, no significant differences between the control and herbicide-treated plants were observed after 5 days of exposure. Treatment with increasing fluroxypyr concentration increased the Y(ND) of Zhangza 5. The maximum Y(ND) level was observed at fluroxypyr treatment. However, further increasing the dosages of fluroxypyr beyond two L ai ha⁻¹ no longer increased Y(ND).

Effects of fluroxypyr on endogenous hormones

Fluroxypyr treatment at doses ranging from 0.5 to 4 L ai ha⁻¹ increased IAA and ABA accumulation in plants, with the maximum accumulation observed at four L ai ha⁻¹ (Fig. 3). The application of four L ai ha⁻¹ fluroxypyr significantly increased IAA and ABA by 26.69% and 23.36% relative to that of the control after 5 days of treatment and 5.37% and 35.53% relative to that of the control after 10 days of treatment, respectively. A similar response was observed between GA and ZR accumulation in Zhangza 5 exposure to fluroxypyr; GA and ZR decreased with increasing fluroxypyr concentration, and the difference observed between the control and treated plants was significant. After treatment with one L ai ha⁻¹ (recommended dose) fluroxypyr for 5 and 10 days, the GA and ZR values significantly decreased by 10.46%, 18.72%, and by 13.50%, 23.59% relatively to the control treatment, respectively.

Comprehensive evaluation of fluroxypyr on various foxtail millet indices

To further comprehensively evaluate the effects of fluroxypyr on various foxtail millet indices, Fisher discriminant analysis was used. Herbicide treatment was used as the grouping variable Y, and the physiological parameters were used as the independent variable X.

• (1)

  Fisher discrimination was used to evaluate the seven indicators of foxtail millet leaf, namely, H₂O₂(X₁), O₂⁻ (X₂), SOD(X₃), POD(X₄), CAT(X₅), APX(X₆), and GR(X₇). The following discriminant functions were obtained:

  • Y₁ = −3.850 + 4.868X₃ + 3.588X₆ − 48.734X₇

  • Y₂ = −0.344 − 1.408X₃ + 2.886X₆ − 7.736X₇

  • Y₃ = −5.062 − 0.107X₃ + 0.020X₆ + 12.487X₇

  The cumulative contribution rate of the first-class functions, which is the main function, reached 98.8%. Thus, SOD(X₃), POD(X₄), and GR(X₇) were used as indices to evaluate the peroxidation characteristics of foxtail millet.

• (2)

  Fisher discrimination was used to evaluate the fourteen indicators of foxtail millet leaf, namely, P_N(X₁), E(X₂), G_s(X₃), Chl(X₄), F_v/F_m(X₅), Y(II)(X₆), ETR(I)(X₇), Y(NPQ)(X₈), Y(NO)(X₉), P_m(X₁₀), Y(I)(X₁₁), ETR(I)(X₁₂), Y(ND)(X₁₃), and Y(NA)(X₁₄). The following discriminant functions were obtained:

  • Y₁ = −252.994 + 427.640X₆ − 66.364X₈ + 143.154X₁₀ + 1.396X₁₂

  • Y₂ = −69.158 + 151.904X₆ + 96.548X₈ + 22.743X₁₀ − 0.036X₁₂

  • Y₃ = −5.251 − 91.400X₆ − 1.385X₈ + 7.042X₁₀ + 0.184X₁₂

  • Y₄ = 1.257 + 2.323X₆ − 1.378X₈ + 3.767X₁₀ − 0.018X₁₂

  The cumulative contribution rate of the first-class functions, which is the main function, reached 94.4%. Thus, Y(II)(X₆), Y(NPQ)(X₈), P_m(X₁₀), and ETR(I)(X₁₂) were used as indices to evaluate the photosynthetic characteristics of foxtail millet.

• (3)

  Fisher discrimination was used to evaluate the four indicators of foxtail millet leaf, namely, IAA(X₁), GA(X₂), ZR(X₃), and ABA(X₄). The following discriminant functions were obtained:

  • Y₁ = −78.315 + 8.217X₂ + 0.229X₄

  • Y₂ = −13.841 + 1.139X₂ + 0.073X₄

  The cumulative contribution rate of the first-class functions, which is the main function, reached 94.4%. Thus, GA(X₂) and ABA(X₄) were used as indices to evaluate the endogenous hormone characteristics of foxtail millet.

As shown in Table 4, the cumulative contribution rate of the first two principal components reached 93.72%. Thus, the first two principal components were further analyzed. The first principal component accounted for 59.23% of the variance observed, whereas the second principal component accounted for 34.49% of this variance. The load values of ERT (I), Y(II), and GA, which were 0.978, 0.975 and 0.963, respectively, were the largest in the first principal component. ABA also showed high absolute loading value in the first principal component, but exhibited a negative effect. Therefore, the first principal component could explain the photosynthetic characteristics and endogenous hormones of foxtail millet. In the second principal component, the loading values of plant height and leaf area were relatively high and could represent the growth parameters of foxtail millet.

According to the principal component analysis, the model was set up as follows: F = 0.592Z₁ + 0.345Z₂. The comprehensive evaluation scores of the two and four L ai ha⁻¹ fluroxypyr treatments were relatively low, which indicated that high fluroxypyr concentrations negatively affected the photosynthetic characteristics and endogenous hormones of foxtail millet. Furthermore, the comprehensive evaluation scores of the 0.5 and 1 L ai ha⁻¹ fluroxypyr treatments were 0.941 and 2.007, respectively, and the scores of first two principal components were positive, which indicated that the moderate concentrations of fluroxypyr positively affected the peroxidation characteristics, photosynthetic characteristics, and endogenous hormones of foxtail millet (Table 5). Consequently, the scores of the two factors in one L ai ha⁻¹ fluroxypyr treatments were both relatively high and represented the comprehensive effect of herbicide in foxtail millet (Fig. 4).