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Photosynthesis and growth responses of grapevine to acetochlor and fluoroglycofen
Pesticide Biochemistry and Physiology 103 (2012) 210–218
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Photosynthesis and growth responses of grapevine to acetochlor and fluoroglycofen Wei Tan a, Qingliang Li b, Heng Zhai a,⇑ a b
College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China College of Plant Protection, Shandong Agricultural University, Taian 271018, China
a r t i c l e
i n f o
Article history: Received 9 February 2012 Accepted 17 May 2012 Available online 15 June 2012 Keywords: Acetochlor Fluoroglycofen Grapevine Photosynthesis Antioxidant enzymes Chlorophyll florescence
a b s t r a c t Acetochlor and fluoroglycofen are herbicides used in vineyards to eradicate weeds. This present study characterized the effects of these chemicals on photosynthetic characteristics and the antioxidant enzyme system in non-target grape leaves. The results showed that acetochlor and fluoroglycofen reduced net photosynthetic rate in a dose-dependent manner, but also reduced or increased pigment contents, respectively. According to chlorophyll fluorescence measurements, acetochlor and fluoroglycofen decreased the photochemical efficiency of photosystem II in the light and increased non-photochemical quenching. These herbicides enhanced malondialdehyde contents and accelerated the superoxide anion production rate in dose-dependent manners, which might be associated with lower antioxidant enzyme activities, especially at higher concentrations of the herbicides. Acetochlor and fluoroglycofen inhibited grapevine growth in the growth season one-year after herbicide treatment, and stem height was inhibited by up to 55.4% and 88.0%, respectively. Taken together, these results suggest that both herbicides are detrimental for grape photosynthesis and this might be associated with increased oxidative stress in the first year, while growth inhibition in the second year might be due to after effects of herbicide treatment. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Herbicides are widely used to control weeds in agriculture. However, in recent years, consumers have become increasingly aware of the impact of farming practices on the environment and food quality, especially any deleterious effects. Thus, herbicide toxicity against non-target crop species continues to warrant thorough investigation. Various strains and cultivars within the same crop species show marked variability in tolerance to different herbicides and other environmental stresses such as drought and low temperature [1–6]. Various herbicides have different targets and modes of action, which results in variations in the performances of different plants [7,8]. Moreover, the old and young leaves from an individual plant can show differential resistances to herbicides and environmental stresses. Some reports have indicated that older leaves are more tolerant to herbicides and environmental stresses [9]; however, in some species, such as pea, cucumber and squash, Abbreviations: APX, ascorbate peroxidase; CAT, catalase; Fv/Fm, efficiency of photosystem II; MDA, malondialdehyde; Pn, net photosynthetic rate; NPQ, nonphotochemical quenching coefficient; POD, peroxidase; QP, photochemical quenching coefficient; PS, photosystem; UPSII, quantum yield of PSII electron transport; SOD, superoxide dismutase; Gs, stomatal conductance. ⇑ Corresponding author. Fax: +86 5388246017. E-mail address: [email protected] (H. Zhai). 0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.05.010
younger leaves are often more tolerant to chemical and environmental stresses [10–13]. Many studies have demonstrated that herbicides can inhibit plant growth [5,14,15], reduce chlorophyll contents and photosynthesis [16–24]. Application of herbicides can also lead to decreases in the maximal photochemical efficiency of photosystem (PS) II (Fv/ Fm), the photochemical quenching coefficient (QP) and the quantum yield of PSII electron transport (UPSII) [14,16,19]. In addition, long-term exposure to herbicides may cause imbalances in reactive oxygen species that could ultimately damage the plant. Nevertheless, plants have evolved various protective strategies to minimize herbicide toxicity. One of these protective mechanisms is the antioxidant system that operates via the sequential and simultaneous actions of various enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT). The activities of some enzymes increase at low herbicide concentrations but decrease at higher exposures [25,26]. The effects of herbicides on enzyme activities may differ due to the length of exposure time and the time at which enzyme activity is measured. Acetochlor (a chloroacetanilide) and fluoroglycofen (a diphenylether) can both be absorbed by grapevine roots. Diphenylether herbicides inhibit protoporphyrinogenoxidase, while acetochlor can disturb and inhibit the photosynthetic electron transport [24]. In China, both of these herbicides are used commonly on farmland largely due to their low cost. But in Qufu, Shandong,
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China, we observed in two of vineyards which was applied with paraquat, acetochlor and fluoroglycofen perennially, the grape leaves grown dark and round, however the photosynthesis rate decreased compared with the control, which used artificial weeding. In this present study, we studied the effects of acetochlor and fluoroglycofen on the photosynthesis and antioxidant enzyme activities in non-target grape leaves. Moreover, the growth of the grape plants was examined in the second year in order to understand further the mechanisms of any detrimental effects caused by these chemicals.
2. Materials and methods 2.1. Plant and growth conditions Experiment was conducted in a greenhouse at the Shandong Agriculture University, China. One-year old grapevines (Vitis vinifera Vitis labrusca cv. Kyoho) were grown in plastic pots (25 cm in diameter) containing garden earth, sand and matrix soil (2:1:1) in a greenhouse operating at photosynthetic photon flux density of 300 lmol m2 s1, relative humidity of 75–80% and a photoperiod of 14/10 h light/dark at 25 °C. When the shoots had ten leaves, acetochlor or fluoroglycofen were sprayed on the soil as follows: (1) T1, 2,246 g ai ha1 acetochlor (according to the area of pots, 0.014 g ai per pot); (2) T2, 11,230 g ai ha1 acetochlor (0.070 g ai per pot); (3) T3, 22,460 g ai ha1 acetochlor (0.140 g ai per pot); (4) T4, 37.5 g ai ha1 fluoroglycofen (0.00023 g ai per pot); (5) T5, 187.5 g ai ha1 fluoroglycofen (0.00115 g ai per pot); (6) T6, 375 g ai ha1 fluoroglycofen (0.00230 g ai per pot). Simultaneously, control (CK) soil was sprayed with water. The experiment was performed twice with three plants each treatment. Once grapes had been exposed to herbicide for 30 d, physiological indices were analyzed on the leaves at the upper-node (13–14), middle-node (8–9) and bottom-node (3–4) in grape seedlings. During the experiment, the plants were irrigated with equal volume of water (guarantee the amount of water not flowing out of the pot) once every two days. 2.2. Gas exchange measurements Measurements of the net photosynthetic rate (Pn) and the stomatal conductance (Gs) were made on the upper-node (13–14), middle-node (8–9) and bottom-node (3–4) leaves of grape seedlings using an open system (Ciras-2, PP Systems, Hitchin, UK).
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NPQ, non-photochemical quenching of chlorophyll fluorescence. NPQ = (Fm Fm0 )/Fm0 . 2.4. Measurements of pigment content The chlorophyll and carotenoid content was determined spectrophotometrically in 80% acetone with a double beam spectrophotometer Unicam UV 550 (ThermoSpectronic, Cambridge, UK) according to Lichtenthaler [28]. 2.5. Measurements of lipid peroxidation and superoxide radical The comparative rates of lipid peroxidation were assayed by determining the levels of malondialdehyde (MDA) in 0.5 g aliquots of leaves. Malondialdehyde is a product of lipid peroxidation and was assayed by the thiobarbituric acid (TBA) reaction [29]. The production rate of O2 was determined according to Elstner and Heupel [30] by monitoring the nitrite formation from hydroxylamine in the presence of O2. 2.6. Assays of antioxidant enzyme activities After herbicide treatment for 30 d, the different node leaves were harvested and immediately frozen in liquid N2, and then stored at 80 °C until experimental analyses. Frozen leaves (0.5 g) were crushed into fine powder in a mortar and pestle under liquid N2. Cell-free homogenates for antioxidant enzyme assays were prepared essentially by the method described by Jiang and Zhang [31]. Soluble proteins were extracted by homogenizing the powder in 10 ml of 50 mM natrium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP), with addition of 1 mM ascorbate in the case of ascorbate peroxidase (APX) assay. The homogenate was centrifuged at 15,000g for 30 min at 4 °C and the supernatant was used for the following enzyme assays. Total superoxide dismutase (SOD) activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) according to the method of Giannopolitis and Ries [32]. Catalase (CAT) activity was determined by following the consumption of H2O2 (extinction coefficient 39.4 mM1 cm1) at 240 nm for 3 min [33]. Ascorbate peroxidase (APX) activity was measured by monitoring the decrease in absorbance at 290 nm (extinction coefficient 2.8 mM1 cm1) [34]. Guaiacol peroxidase (POD) activity was assayed following the method described by Cakmak and Marschner [35]. Protein content was determined according to the method of Bradford [36].
2.3. Analysis of chlorophyll fluorescence Chlorophyll florescence was measured with a FMS-2 pulse modulated fluorometer (Hansatech, UK). The minimal fluorescence (Fo) was determined by a weak modulated light which was low enough not to induce any significant variable fluorecence. A 0.8 s saturating light of 8000 lmol m2 s1 was used on dark-adapted leaves to determine the maximal fluorescence (Fm). Then the leaf was illuminated by an actinic light of 500 lmol m2 s1. When the leaf reached steady-state photosynthesis, the steady-state fluorescence (Fs) was recorded and a second 0.8s saturating light 8000 lmol m2 s1of was given to determine the maximal fluorescence (Fm0 ) in the light-adapted state. The actinic light was then turned off; the minimal fluorescence in the light-adapted state (Fo0 ) was determined by the illumination of the 3 s far red light. The following parameters were then calculated: (1) QP, the photochemical quenching coefficient, QP = (Fm0 Fs)/(Fm0 Fo0 ); (2) Fv/ Fm, maximal photochemical efficiency of PSII; (3) UPSII, quantum yield of PSII electron transport, UPSII = (Fm0 Fs)/Fm0 , [27]; (4)
2.7. Growth analysis in 2011 The treated grape seedlings were germinated in 2011, when the shoots of control had eight leaves, the grape seedlings treated with acetochlor and fluoroglycofen were photographed, respectively; the length of stems were measured, the leaves and roots of each plantlet were weighed (roots were first rinsed with distilled water) to calculate the ratio of root to shoot (FW). 2.8. Statistical analysis Each reported data are the mean ± standard error (SE) of six replicates combined in the two experimental repeats. Statistical analyses were performed by analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Chicago, USA) and comparisons between the mean values were made by least significant difference (LSD) at a 0.05 probability level.
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3. Results 3.1. Effects of acetochlor and fluoroglycofen on CO2 assimilation and stomatal conductance in grape leaves Acetochlor and fluoroglycofen reduced the net photosynthetic rate (Pn) as concentrations of these herbicides increased (Figs. 1A and B). Lower concentrations of acetochlor caused a significant decrease in Pn in upper- and middle-node leaves, but lower concentrations of fluoroglycofen reduced Pn significantly only in middle-node leaves. In other higher concentration treatments, Pn was reduced significantly. Compared with the control (CK), acetochlor reduced Pn in upper-, middle- and bottom-node leaves by up to 38.0%, 53.2% and 48.9%, respectively; fluoroglycofen reduced Pn by up to 55.7%, 32.9%, 40.2% in upper-, middle- and bottom-node leaves, respectively. Acetochlor and fluoroglycofen treatments had little effect on stomatal conductance (Gs) (Fig. 1C and D), and Gs values were significantly lower than controls only for upper-node leaves in T2 and T3 groups and bottom-node leaves in the T3 group, with reductions of 48.8%, 37.4% and 31.2%, respectively. 3.2. Effects of acetochlor and fluoroglycofen on chlorophyll and carotenoid contents in grape leaves As shown in Fig. 2A and C, lower concentrations of acetochlor enhanced carotenoid contents in upper-node leaves, but when concentrations increased further, carotenoid and chlorophyll contents decreased in a dose-dependent manner. Acetochlor had no effects on the pigment contents of middle-node leaves. Lower concentrations of acetochlor significantly enhanced pigment contents
in bottom-node leaves, but at the maximum concentration of acetochlor used there was a significant reduction in the chlorophyll content of these leaves. The effects of fluoroglycofen on pigment contents differed from the effects of acetochlor (Fig. 2B and D). In upper-node leaves, pigment contents were significantly greater in the T5 group, but in the T6 group pigment contents were lower than observed in the control group. Fluoroglycofen caused an increase in pigment contents in middle-node leaves, but this was only significant at 187.5 g ai ha1. In bottom-node leaves, fluoroglycofen caused a dose-dependent increase in chlorophyll contents, while carotenoid contents increased in a dose-dependent manner at lower concentrations only, as they reduced at the greatest concentration of fluoroglycofen tested.
3.3. Effects of acetochlor and fluoroglycofen on chlorophyll fluorescence in grape leaves As shown in Fig. 3, acetochlor did not affect Fv/Fm and QP of upper- and bottom-node leaves or the UPSII of bottom-node leaves. The UPSII of upper-node leaves in the T1 group was 12.4% greater than the control, while UPSII was markedly lower in the T2 and T3 groups than in the control group (Fig. 3B). Acetochlor significantly reduced UPSII in middle-node leaves, but no significant differences were observed between these values at the three concentrations of acetochlor used. QP values of middle-node leaves in the T2 and T3 groups were significantly lower than in the control group (Fig. 3C). The non-photochemical quenching coefficient (NPQ) in middle- and bottom-node leaves increased greatly after treatment with acetochlor, and the greatest increase was observed in the T3
Fig. 1. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on net photosynthetic rate (Pn; A and B) and stomatal conductance (Gs; C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
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Table 1 Effects of acetochlor and fluoroglycofen on stem height, number of nodes and root/shoot ratio observed in grapes in 2011. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Herbicide
Stem height (cm) Number of nodes Root/shoot
Acetochlor Fluoroglycofen Acetochlor Fluoroglycofen Acetochlor Fluoroglycofen
Treatment CK
T1/T4
T2/T5
T3/T6
54.88 ± 7.55a 54.88 ± 7.55a 10.5 ± 0.6a 10.5 ± 0.6a 1.551 ± 0.214b 1.551 ± 0.214c
49.33 ± 7.82a 40.00 ± 1.41b 10.3 ± 0.6a 9.0 ± 0.0b 1.534 ± 0.151b 2.318 ± 0.092b
43.33 ± 3.55a 35.00 ± 1.41b 8.7 ± 0.6b 7.5 ± 0.7c 2.017 ± 0.067b 2.481 ± 0.110b
24.50 ± 4.92b 6.60 ± 2.69c 6.7 ± 0.6c 4.5 ± 0.7d 2.341 ± 0.531a 3.755 ± 0.442a
Different lower-case letters in the same row indicate a significant difference between treatments (P < 0.05).
Fig. 2. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on chlorophyll (A and B) and carotenoid contents (C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
group (Fig. 3D). In upper-node leaves, the NPQ in the T2 and T3 groups were greater than in the T1 and control groups. Fluoroglycofen had no significant effects on Fv/Fm in upper-, middle- and bottom-leaves, UPSII in bottom-node leaves, QP in upper- and middle-node leaves and NPQ in upper-node leaves (Fig. 4). Application of fluoroglycofen significantly reduced UPSII in the upper-node leaves, while UPSII in middle-node leaves of the T5 and T6 groups were significantly lower than the control (Fig. 4B). QP in bottom-node leaves of T4 and T5 groups were significantly lower than the control (Fig. 4C). NPQ in the middle- and bottom-node leaves of the T5 and T6 groups were much greater than in the control group, but no significant differences was observed between these values at the three concentrations of fluoroglycofen used (Fig. 4D).
3.4. Effects of acetochlor and fluoroglycofen on lipid peroxidation and superoxide production rate in grape leaves Malondialdehyde (MDA) contents increased in middle- and bottom-node leaves after treatment with acetochlor, but there were no significant differences between the three acetochlor treatments of middle-node leaves, or between the T2 and T3 groups in MDA contents of bottom-node leaves (Fig. 5A). Lower concentrations of acetochlor (T1) had no effect on MDA contents in upper-node leaves, while higher concentrations enhanced MDA contents significantly. Acetochlor caused a significant acceleration in the O2 production rate, which increased in dose-dependent manner in uppernode leaves (Fig. 5C); in middle-node leaves, the O2 production
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Fig. 3. Effects of acetochlor and fluoroglycofen on maximal photochemical efficiency of PSII in darkness (Fv/Fm; A), actual photochemical efficiency of PSII in the light (UPSII; B) photochemical quenching (QP; C) and non-photochemical quenching (NPQ; D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
rate was 3.4% lower in the T1 group compared with the control, but this rate was 21.8% and 38.2% higher in the T2 and T3 groups compared with the control, respectively. In bottom-node leaves, O2 production rate was greater at lower acetochlor concentrations, but lower in the T3 group (though the rate was still greater than in the T1 group). When fluoroglycofen concentration was greater than 37.5 g ai ha1, MDA contents were significantly higher (Fig. 5B). Moreover, the O2 production rate accelerated in a dose-dependent manner (Fig. 5D), with increases of up to 323%, 57% and 78% in upper-, middle- and bottom-node leaves, respectively. 3.5. Effects of acetochlor and fluoroglycofen on antioxidant enzyme activities in grape leaves The herbicide treatments induced significant changes in the activities of various antioxidant enzymes. At lower concentrations, acetochlor improved SOD activity in upper- and middle-node leaves, but activity was reduced at the greatest concentration of acetochlor tested; however, in bottom-node leaves, SOD activity decreased in a dose-dependent way (Fig. 6A). CAT activities in upper- and middle-node leaves were higher in the T1 group (compared to the controls) but decreased to control levels in the T3 group. Acetochlor had no significant effects on CAT activity in bottom-node leaves (Fig. 6B). POD activity was reduced significantly by acetochlor in upper- and middle-node leaves, but no significant differences were observed between the T1 and T2 groups; however, compared with other treatments, POD activity was significantly greater in the bottom-node leaves in the T2 group (Fig. 6C). Acetochlor caused dose-dependent decreases in APX activity in
upper-node leaves, while in middle-node leaves APX activity was 8.0% greater in the T1 group, but 47.3% and 75.0% lower in the T2 and T3 groups, respectively. In bottom-node leaves, APX activity was reduced compared to the control by 33.8% and 53.8% in the T2 and T3 groups, respectively (Fig. 6D). In upper-node leaves, SOD and APX activities decreased significantly and gradually when fluoroglycofen concentrations were greater than 37.5 g ai ha1 (Fig. 7A and D). Meanwhile, CAT and POD activities were greater in the T4 group but decreased dramatically as fluoroglycofen concentrations increased further (Fig. 7B and C). In the middle-node leaves, the changes in SOD activity were similar to changes observed in POD activity in upper-node leaves (Fig. 7A), whereas CAT and POD activities decreased significantly only when fluoroglycofen concentrations reached 387.5 or 187.5 g ai ha1, respectively (Fig. 7B and C). APX activity was greater in the T4 and T5 treatments compared to the control, but then it decreased in the T6 group to a level 36.2% lower than the control (Fig. 7D). In bottom-node leaves, fluoroglycofen reduced SOD and POD activities in a dose-dependent manner (Fig. 7A and C), while only the greatest concentration of fluoroglycofen reduced CAT activity (Fig. 7B). Finally, APX activity in bottom-node leaves in the T5 and T6 groups were lower than the control and T4 groups (Fig. 7D). 3.6. Acetochlor and fluoroglycofen inhibited the growth of grapes in 2011 Acetochlor and fluoroglycofen treatments in 2010 inhibited the growth of grapevines in 2011 (Table 1). Stem heights were reduced by up to 55.4% and 88.0% after treatment with acetochlor and
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Fig. 4. Effects of acetochlor and fluoroglycofen on maximal photochemical efficiency of PSII in darkness (Fv/Fm; A), actual photochemical efficiency of PSII in the light (UPSII; B) photochemical quenching (QP; C) and non-photochemical quenching (NPQ; D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
fluoroglycofen, respectively; however, the ratio of root and shoot was enhanced in these treatment groups by up to 50.9% and 142.1%, respectively. Inhibition of grape growth was more serious with fluoroglycofen than acetochlor, especially at the maxium concentrations of fluoroglycofen which even caused deformation of the leaves.
4. Discussion Many herbicides are photosystem inhibitors [37], including atrazine that binds to the D1 protein of PSII, blocking electron transfer to the plastoquinone pool [38,39]; clomazone that greatly reduces electron transport in thylakoid membranes of chloroplasts [18]; and flumioxazin that acts as an protoporphyrinogenoxidase inhibitor, causing decreases in Pn and Gs in grape leaves [19]. Acetochlor and fluoroglycofen are also absorbed by roots, and both herbicides reduced photosynthesis in grape leaves. Herbicides can enter the plant through the apoplast, symplast and apoplast-symplast systems in roots, and then be transported via the xylem and catheter action due to transpiration. Differences in penetration rate, the in vivo metabolic situation and the toxicity of the herbicide can each affect the herbicide transport mechanism in different plant species. Furthermore, environmental conditions, such as temperature, can moderate and influence herbicide transport. The responses to herbicides in different node leaves might be attributed to these diverse transport pathways. Acetochlor caused greater decreases in Pn in middle- and bottom-node leaves compared to upper-node leaves. Conversely, fluoroglycofen treatment decreased Pn in upper-node leaves to a greater extent than in
middle- and bottom-node leaves. Old and young leaves showed opposing tolerances to the two herbicides examined herein, which might be related with the plants requiring distinct resistant mechanisms to counter these different herbicides. Acetochlor caused decreases in pigment contents. Conversely, fluoroglycofen treatment resulted in increases in pigment contents at lower concentrations, which differs from observations with a similar herbicide, flumioxazin, that caused pigment contents to decrease [19]. In 2009, three vineyards were observed that applied different concentrations of herbicides. One vineyard was perennially exposed to higher levels of paraquat, acetochlor and fluoroglycofen, and in this vineyard the grape leaves grew dark and round, but the photosynthetic rate and the grape yields were lower than obtained with artificial weeding. The mechanisms behind this phenomenon required further study and this formed the focus of the present study. The fluorescence arising from chlorophyll is almost exclusively associated with PSII [40]. Since PSII functioning is sensitive to a wide range of environmental conditions, chlorophyll fluorescence provides a wealth of information on the effects of particular stresses on plants [40]. Paraquat and norflurazon (each at 100 lg L1) caused significant decreases in Fv/Fm, UPSII and QP, while significantly increasing NPQ [1]; however, flumioxazin and terbutryn reduced NPQ in grape and Vicia faba, respectively [17,19]. A decrease in QP indicates a higher proportion of closed PSII reaction centers, i.e., an increase in the proportion of the reduced state of QA [27], which probably leads to a decrease in the proportion of available excitation energy used for photochemistry [41]. Moreover, the value of NPQ is linearly related to energy dissipation in leaf tissues and is viewed as essential in protecting the leaf from light-induced
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Fig. 5. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on malondialdehyde (MDA) contents (A and B) and O2 production rates (C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
Fig. 6. Effects of acetochlor on superoxide dismutase (SOD; A), catalase (CAT; B), peroxidase (POD; C) and ascorbate peroxidase (APX; D) activities in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
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Fig. 7. Effects of fluoroglycofen on superoxide dismutase (SOD; A), catalase (CAT; B), peroxidase (POD; C) and ascorbate peroxidase (APX; D) activities in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
damage [42]. Acetochlor and fluoroglycofen caused increases in NPQ and decreases in UPSII, especially at higher concentrations. Thus, the two herbicides reduced PSII activity and were involved in non-radiative energy dissipation. Decreases in CO2 accumulation and the photochemical efficiency of PSII might be associated with higher levels of reactive oxygen species. Different classes of herbicides are direct or indirect sources of oxidative damage in plants [43,44]. While plants have a suite of protective antioxidant enzymes and substances, the induction of these enzymes’ activities often accompanies a disturbance in the redox homeostasis and these can be used to indicate oxidative stress [45]. MDA is the final product of membrane lipid peroxidation and its accumulation can result from toxicity caused by reactive oxygen species. Many studies have reported that many herbicides induce the accumulation of reactive oxygen species and MDA [46–49]. Meanwhile, the responses of antioxidant enzymes to herbicide treatments in various plants may differ due to differences in the treatment times and the point at which measurements are made, and there are also concentration and time effects [13,14,16,47]. This present study shows that both acetochlor and fluoroglycofen accelerated O2 production and caused exacerbation of membrane lipid peroxidation, especially higher concentrations, which might be relevant to lowest antioxidant enzyme activities. Nevertheless, in agreement with other reports, lower concentrations of acetochlor enhanced the activities of SOD and CAT in the upper- and middle-node leaves and APX in the middle-node leaves. Furthermore, CAT and POD activities in the upper-node leaves and APX activities in middle- and bottom-node leaves were increased after treatment with lower concentrations of fluoroglycofen. Herbicides can affect plant growth and reduce biomass [8,15,24]. In an earlier study, vines treated with flazasulfuron were
found to exhibit yellow leaves and alterations in photosynthetic activity, but these changes disappeared in the following year, indicating that vines have the potential to recover after one season from the stress caused by this particular herbicide [50]. However, the data in this present report suggest that acetochlor and fluoroglycofen not only resulted in decreases in photosynthesis in 2010, but also led to inhibition of growth in 2011, especially at higher concentrations of fluoroglycofen, which caused leaf deformities and seriously inhibited growth. The reasons behind these observations require further research. According to the above results, both of higher levels of acetochlor and fluoroglycofen exert detrimental effects on grapevines not only in the first year but also in the second year. Furthermore, another postgraduate in our lab measured the grape quality in grapevines after application of combination of 2246 g ai ha1 acetochlor and 22.5 g ai ha1 fluoroglycofen, the results showed that the grape acidity, anthocyanin, vitamin C content and soluble solid decreased significantly. We also measured the acetochlor residues in grapes, but no fluoroglycofen residues. However, many authors have already reported that vineyard cover crops could reduce must acidity and increase berry sugar accumulation, tartaric acid/malic acid ratio and berry skin total phenols and anthocyanin content [51,52], we suggested that the orchardman should reduce the use number and dosage of herbicide, or instead of it, better to cover crops.
5. Conclusion From our results we conclude the followings: (1) application of acetochlor and fluoroglycofen reduce photosynthesis in grape leaves in the first year and this is associated with higher levels of
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reactive oxygen species, which is, in turn, may be attributed to lower activities of antioxidant enzymes; (2) the growth inhibition in the second year might due to the after effects of herbicide treatment. Acknowledgments We sincerely thank Prof. Xinghong Yang (College of Life Science, Shandong Agriculture University, China) for assistance in editing the manuscript. This research was supported by China Agriculture Research System (CARS-30). References [1] C. Frankart, P. Eullaffroy, G. Vernet, Comparative effects of four herbicides on non-photochemical fluorescence quenching in Lemna minor, Environ. Exp. Bot. 49 (2003) 159–168. [2] J.O. Guh, K. Ishizuka, J.Y. Pyon, Differential absorption and translocation of oxyfluorfen between selected rice cultivars, Korean J. Weed Sci. 8 (1988) 37– 44. [3] S. Kim, K.K. Hatzios, Differential response of two soybean cultivars to paraquat, Verlag der Zeitschrift für Naturforschung 48 (1993) 379–384. [4] J.S. Choi, H.J. Lee, I.T. Hwang, J.Y. Pyon, K.Y. Cho, Differential susceptibilities of wheat and barley to diphenyl ether herbicide oxyfluorfen, Pestic. Biochem. Physiol. 65 (1999) 62–72. [5] J.D.M. Belgers, R.J. Van Lieverloo, L.J.T. Van der Pas, P.J. Van den Brink, Effects of the herbicide 2,4-D on the growth of nine aquatic macrophytes, Aquat. Bot. 86 (2007) 260–268. [6] C.M. Smart, Gene expression during leaf senescence, New Phytol. 126 (1994) 419–448. [7] S. Kaushik, Effect of herbicides with different modes of action on physiological and cellular traits of Anabaena fertilissima, Paddy Water Environ, 8 (2010) 277– 282. [8] Y. Sunohara, S. Shirai, N. Wongkantrakorn, H. Matsumoto, Sensitivity and physiological responses of Eleusine indica and Digitaria adscendens to herbicide quinclorac and 2,4-D, Environ. Exp. Bot. 68 (2010) 157–164. [9] T.D. Sherman, J.M. Becerril, H. Matsumoto, M.V. Duke, J.M. Jacobs, N.J. Jacobs, S.O. Duke, Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides, Plant Physiol. 97 (1991) 280–287. [10] Y.I. Kuk, J.-S. Shin, H.I. Jung, J.O. Guh, S. Jung, N.R. Burgos, Mechanism of paraquat tolerance in cucumber leaves of various ages, Weed Sci. 54 (2006) 6– 15. [11] Y.I. Kuk, J.S. Shin, Mechanisms of low-temperature tolerance in cucumber leaves of various ages, J. Am. Soc. Hortic. Sci. 132 (2007) 294–301. [12] J.L. Donahue, C.M. Okpodu, C.L. Cramer, E.A. Grabau, R.G. Alscher, Responses of antioxidants to paraquat in pea leaves (Relationships to Resistance), Plant Physiol. 113 (1997) 249–257. [13] J.Y. Yoon, J.S. Shin, D.Y. Shin, K.H. Hyun, N.R. Burgos, S. Lee, Y.I. Kuk, Tolerance to paraquat-mediated oxidative and environmental stresses in squash (Cucurbita spp.) leaves of various ages, Pestic. Biochem. Physiol. 99 (2011) 65–76. [14] R.S. Macedo, A.T. Lombardi, C.Y. Omachi, L.R. Rörig, Effects of the herbicide bentazon on growth and photosystem II maximum quantum yield of the marine diatom Skeletonema costatum, Toxicol. In Vitro 22 (2008) 716–722. [15] G. Saladin, C. Magné, C. Clément, Impact of flumioxazin herbicide on growth and carbohydrate physiology in Vitis vinifera L, Plant Cell Rep. 21 (2003) 821– 827. [16] L. Geoffroy, C. Frankart, P. Eullaffroy, Comparison of different physiological parameter responses in Lemna minor and Scenedesmus obliquus exposed to herbicide flumioxazin, Environ. Pollut. 131 (2004) 233–241. [17] R. Piñol, E. Simón, Effect of 24-epibrassinolide on chlorophyll fluorescence and photosynthetic CO2 assimilation in Vicia faba plants treated with the photosynthesis-inhibiting herbicide terbutryn, J. Plant Growth Regul. 28 (2009) 97–105. ˇ a, M. Špundová, P. Ilík, D. Lazár, K. Klem, P. Tomek, J. Nauš, O. Prášil, [18] R. Kan Effect of herbicide clomazone on photosynthetic processes in primary barley (Hordeum vulgare L.) leaves, Pestic. Biochem. Physiol. 78 (2004) 161–170. [19] A. Bigot, F. Fontaine, C. Clément, N. Vaillant-Gaveau, Effect of the herbicide flumioxazin on photosynthetic performance of grapevine (Vitis vinifera L.), Chemosphere 67 (2007) 1243–1251. [20] J. Shen, W. Luo, Effects of monosulfuron on growth, photosynthesis, and nitrogenase activity of three nitrogen-fixing cyanobacteria, Arch. Environ. Contam. Toxicol. 60 (2011) 34–43. [21] D.A. Kopsell, G.R. Armel, K.R. Abney, J.J. Vargas, J.T. Brosnan, D.E. Kopsell, Leaf tissue pigments and chlorophyll fluorescence parameters vary among sweet corn genotypes of differential herbicide sensitivity, Pestic. Biochem. Physiol. 99 (2011) 194–199. [22] M.I. Hussain, L. GonzáLez, M. Reigosa, Phytotoxic effects of allelochemicals and herbicides on photosynthesis, growth and carbon isotope discrimination in Lactuca sativa, Allelopathy J. 26 (2010) 157–174.
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Contents lists available at SciVerse ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Photosynthesis and growth responses of grapevine to acetochlor and fluoroglycofen Wei Tan a, Qingliang Li b, Heng Zhai a,⇑ a b
College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China College of Plant Protection, Shandong Agricultural University, Taian 271018, China
a r t i c l e
i n f o
Article history: Received 9 February 2012 Accepted 17 May 2012 Available online 15 June 2012 Keywords: Acetochlor Fluoroglycofen Grapevine Photosynthesis Antioxidant enzymes Chlorophyll florescence
a b s t r a c t Acetochlor and fluoroglycofen are herbicides used in vineyards to eradicate weeds. This present study characterized the effects of these chemicals on photosynthetic characteristics and the antioxidant enzyme system in non-target grape leaves. The results showed that acetochlor and fluoroglycofen reduced net photosynthetic rate in a dose-dependent manner, but also reduced or increased pigment contents, respectively. According to chlorophyll fluorescence measurements, acetochlor and fluoroglycofen decreased the photochemical efficiency of photosystem II in the light and increased non-photochemical quenching. These herbicides enhanced malondialdehyde contents and accelerated the superoxide anion production rate in dose-dependent manners, which might be associated with lower antioxidant enzyme activities, especially at higher concentrations of the herbicides. Acetochlor and fluoroglycofen inhibited grapevine growth in the growth season one-year after herbicide treatment, and stem height was inhibited by up to 55.4% and 88.0%, respectively. Taken together, these results suggest that both herbicides are detrimental for grape photosynthesis and this might be associated with increased oxidative stress in the first year, while growth inhibition in the second year might be due to after effects of herbicide treatment. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Herbicides are widely used to control weeds in agriculture. However, in recent years, consumers have become increasingly aware of the impact of farming practices on the environment and food quality, especially any deleterious effects. Thus, herbicide toxicity against non-target crop species continues to warrant thorough investigation. Various strains and cultivars within the same crop species show marked variability in tolerance to different herbicides and other environmental stresses such as drought and low temperature [1–6]. Various herbicides have different targets and modes of action, which results in variations in the performances of different plants [7,8]. Moreover, the old and young leaves from an individual plant can show differential resistances to herbicides and environmental stresses. Some reports have indicated that older leaves are more tolerant to herbicides and environmental stresses [9]; however, in some species, such as pea, cucumber and squash, Abbreviations: APX, ascorbate peroxidase; CAT, catalase; Fv/Fm, efficiency of photosystem II; MDA, malondialdehyde; Pn, net photosynthetic rate; NPQ, nonphotochemical quenching coefficient; POD, peroxidase; QP, photochemical quenching coefficient; PS, photosystem; UPSII, quantum yield of PSII electron transport; SOD, superoxide dismutase; Gs, stomatal conductance. ⇑ Corresponding author. Fax: +86 5388246017. E-mail address: [email protected] (H. Zhai). 0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.05.010
younger leaves are often more tolerant to chemical and environmental stresses [10–13]. Many studies have demonstrated that herbicides can inhibit plant growth [5,14,15], reduce chlorophyll contents and photosynthesis [16–24]. Application of herbicides can also lead to decreases in the maximal photochemical efficiency of photosystem (PS) II (Fv/ Fm), the photochemical quenching coefficient (QP) and the quantum yield of PSII electron transport (UPSII) [14,16,19]. In addition, long-term exposure to herbicides may cause imbalances in reactive oxygen species that could ultimately damage the plant. Nevertheless, plants have evolved various protective strategies to minimize herbicide toxicity. One of these protective mechanisms is the antioxidant system that operates via the sequential and simultaneous actions of various enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT). The activities of some enzymes increase at low herbicide concentrations but decrease at higher exposures [25,26]. The effects of herbicides on enzyme activities may differ due to the length of exposure time and the time at which enzyme activity is measured. Acetochlor (a chloroacetanilide) and fluoroglycofen (a diphenylether) can both be absorbed by grapevine roots. Diphenylether herbicides inhibit protoporphyrinogenoxidase, while acetochlor can disturb and inhibit the photosynthetic electron transport [24]. In China, both of these herbicides are used commonly on farmland largely due to their low cost. But in Qufu, Shandong,
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China, we observed in two of vineyards which was applied with paraquat, acetochlor and fluoroglycofen perennially, the grape leaves grown dark and round, however the photosynthesis rate decreased compared with the control, which used artificial weeding. In this present study, we studied the effects of acetochlor and fluoroglycofen on the photosynthesis and antioxidant enzyme activities in non-target grape leaves. Moreover, the growth of the grape plants was examined in the second year in order to understand further the mechanisms of any detrimental effects caused by these chemicals.
2. Materials and methods 2.1. Plant and growth conditions Experiment was conducted in a greenhouse at the Shandong Agriculture University, China. One-year old grapevines (Vitis vinifera Vitis labrusca cv. Kyoho) were grown in plastic pots (25 cm in diameter) containing garden earth, sand and matrix soil (2:1:1) in a greenhouse operating at photosynthetic photon flux density of 300 lmol m2 s1, relative humidity of 75–80% and a photoperiod of 14/10 h light/dark at 25 °C. When the shoots had ten leaves, acetochlor or fluoroglycofen were sprayed on the soil as follows: (1) T1, 2,246 g ai ha1 acetochlor (according to the area of pots, 0.014 g ai per pot); (2) T2, 11,230 g ai ha1 acetochlor (0.070 g ai per pot); (3) T3, 22,460 g ai ha1 acetochlor (0.140 g ai per pot); (4) T4, 37.5 g ai ha1 fluoroglycofen (0.00023 g ai per pot); (5) T5, 187.5 g ai ha1 fluoroglycofen (0.00115 g ai per pot); (6) T6, 375 g ai ha1 fluoroglycofen (0.00230 g ai per pot). Simultaneously, control (CK) soil was sprayed with water. The experiment was performed twice with three plants each treatment. Once grapes had been exposed to herbicide for 30 d, physiological indices were analyzed on the leaves at the upper-node (13–14), middle-node (8–9) and bottom-node (3–4) in grape seedlings. During the experiment, the plants were irrigated with equal volume of water (guarantee the amount of water not flowing out of the pot) once every two days. 2.2. Gas exchange measurements Measurements of the net photosynthetic rate (Pn) and the stomatal conductance (Gs) were made on the upper-node (13–14), middle-node (8–9) and bottom-node (3–4) leaves of grape seedlings using an open system (Ciras-2, PP Systems, Hitchin, UK).
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NPQ, non-photochemical quenching of chlorophyll fluorescence. NPQ = (Fm Fm0 )/Fm0 . 2.4. Measurements of pigment content The chlorophyll and carotenoid content was determined spectrophotometrically in 80% acetone with a double beam spectrophotometer Unicam UV 550 (ThermoSpectronic, Cambridge, UK) according to Lichtenthaler [28]. 2.5. Measurements of lipid peroxidation and superoxide radical The comparative rates of lipid peroxidation were assayed by determining the levels of malondialdehyde (MDA) in 0.5 g aliquots of leaves. Malondialdehyde is a product of lipid peroxidation and was assayed by the thiobarbituric acid (TBA) reaction [29]. The production rate of O2 was determined according to Elstner and Heupel [30] by monitoring the nitrite formation from hydroxylamine in the presence of O2. 2.6. Assays of antioxidant enzyme activities After herbicide treatment for 30 d, the different node leaves were harvested and immediately frozen in liquid N2, and then stored at 80 °C until experimental analyses. Frozen leaves (0.5 g) were crushed into fine powder in a mortar and pestle under liquid N2. Cell-free homogenates for antioxidant enzyme assays were prepared essentially by the method described by Jiang and Zhang [31]. Soluble proteins were extracted by homogenizing the powder in 10 ml of 50 mM natrium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP), with addition of 1 mM ascorbate in the case of ascorbate peroxidase (APX) assay. The homogenate was centrifuged at 15,000g for 30 min at 4 °C and the supernatant was used for the following enzyme assays. Total superoxide dismutase (SOD) activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) according to the method of Giannopolitis and Ries [32]. Catalase (CAT) activity was determined by following the consumption of H2O2 (extinction coefficient 39.4 mM1 cm1) at 240 nm for 3 min [33]. Ascorbate peroxidase (APX) activity was measured by monitoring the decrease in absorbance at 290 nm (extinction coefficient 2.8 mM1 cm1) [34]. Guaiacol peroxidase (POD) activity was assayed following the method described by Cakmak and Marschner [35]. Protein content was determined according to the method of Bradford [36].
2.3. Analysis of chlorophyll fluorescence Chlorophyll florescence was measured with a FMS-2 pulse modulated fluorometer (Hansatech, UK). The minimal fluorescence (Fo) was determined by a weak modulated light which was low enough not to induce any significant variable fluorecence. A 0.8 s saturating light of 8000 lmol m2 s1 was used on dark-adapted leaves to determine the maximal fluorescence (Fm). Then the leaf was illuminated by an actinic light of 500 lmol m2 s1. When the leaf reached steady-state photosynthesis, the steady-state fluorescence (Fs) was recorded and a second 0.8s saturating light 8000 lmol m2 s1of was given to determine the maximal fluorescence (Fm0 ) in the light-adapted state. The actinic light was then turned off; the minimal fluorescence in the light-adapted state (Fo0 ) was determined by the illumination of the 3 s far red light. The following parameters were then calculated: (1) QP, the photochemical quenching coefficient, QP = (Fm0 Fs)/(Fm0 Fo0 ); (2) Fv/ Fm, maximal photochemical efficiency of PSII; (3) UPSII, quantum yield of PSII electron transport, UPSII = (Fm0 Fs)/Fm0 , [27]; (4)
2.7. Growth analysis in 2011 The treated grape seedlings were germinated in 2011, when the shoots of control had eight leaves, the grape seedlings treated with acetochlor and fluoroglycofen were photographed, respectively; the length of stems were measured, the leaves and roots of each plantlet were weighed (roots were first rinsed with distilled water) to calculate the ratio of root to shoot (FW). 2.8. Statistical analysis Each reported data are the mean ± standard error (SE) of six replicates combined in the two experimental repeats. Statistical analyses were performed by analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Chicago, USA) and comparisons between the mean values were made by least significant difference (LSD) at a 0.05 probability level.
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3. Results 3.1. Effects of acetochlor and fluoroglycofen on CO2 assimilation and stomatal conductance in grape leaves Acetochlor and fluoroglycofen reduced the net photosynthetic rate (Pn) as concentrations of these herbicides increased (Figs. 1A and B). Lower concentrations of acetochlor caused a significant decrease in Pn in upper- and middle-node leaves, but lower concentrations of fluoroglycofen reduced Pn significantly only in middle-node leaves. In other higher concentration treatments, Pn was reduced significantly. Compared with the control (CK), acetochlor reduced Pn in upper-, middle- and bottom-node leaves by up to 38.0%, 53.2% and 48.9%, respectively; fluoroglycofen reduced Pn by up to 55.7%, 32.9%, 40.2% in upper-, middle- and bottom-node leaves, respectively. Acetochlor and fluoroglycofen treatments had little effect on stomatal conductance (Gs) (Fig. 1C and D), and Gs values were significantly lower than controls only for upper-node leaves in T2 and T3 groups and bottom-node leaves in the T3 group, with reductions of 48.8%, 37.4% and 31.2%, respectively. 3.2. Effects of acetochlor and fluoroglycofen on chlorophyll and carotenoid contents in grape leaves As shown in Fig. 2A and C, lower concentrations of acetochlor enhanced carotenoid contents in upper-node leaves, but when concentrations increased further, carotenoid and chlorophyll contents decreased in a dose-dependent manner. Acetochlor had no effects on the pigment contents of middle-node leaves. Lower concentrations of acetochlor significantly enhanced pigment contents
in bottom-node leaves, but at the maximum concentration of acetochlor used there was a significant reduction in the chlorophyll content of these leaves. The effects of fluoroglycofen on pigment contents differed from the effects of acetochlor (Fig. 2B and D). In upper-node leaves, pigment contents were significantly greater in the T5 group, but in the T6 group pigment contents were lower than observed in the control group. Fluoroglycofen caused an increase in pigment contents in middle-node leaves, but this was only significant at 187.5 g ai ha1. In bottom-node leaves, fluoroglycofen caused a dose-dependent increase in chlorophyll contents, while carotenoid contents increased in a dose-dependent manner at lower concentrations only, as they reduced at the greatest concentration of fluoroglycofen tested.
3.3. Effects of acetochlor and fluoroglycofen on chlorophyll fluorescence in grape leaves As shown in Fig. 3, acetochlor did not affect Fv/Fm and QP of upper- and bottom-node leaves or the UPSII of bottom-node leaves. The UPSII of upper-node leaves in the T1 group was 12.4% greater than the control, while UPSII was markedly lower in the T2 and T3 groups than in the control group (Fig. 3B). Acetochlor significantly reduced UPSII in middle-node leaves, but no significant differences were observed between these values at the three concentrations of acetochlor used. QP values of middle-node leaves in the T2 and T3 groups were significantly lower than in the control group (Fig. 3C). The non-photochemical quenching coefficient (NPQ) in middle- and bottom-node leaves increased greatly after treatment with acetochlor, and the greatest increase was observed in the T3
Fig. 1. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on net photosynthetic rate (Pn; A and B) and stomatal conductance (Gs; C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
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Table 1 Effects of acetochlor and fluoroglycofen on stem height, number of nodes and root/shoot ratio observed in grapes in 2011. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Herbicide
Stem height (cm) Number of nodes Root/shoot
Acetochlor Fluoroglycofen Acetochlor Fluoroglycofen Acetochlor Fluoroglycofen
Treatment CK
T1/T4
T2/T5
T3/T6
54.88 ± 7.55a 54.88 ± 7.55a 10.5 ± 0.6a 10.5 ± 0.6a 1.551 ± 0.214b 1.551 ± 0.214c
49.33 ± 7.82a 40.00 ± 1.41b 10.3 ± 0.6a 9.0 ± 0.0b 1.534 ± 0.151b 2.318 ± 0.092b
43.33 ± 3.55a 35.00 ± 1.41b 8.7 ± 0.6b 7.5 ± 0.7c 2.017 ± 0.067b 2.481 ± 0.110b
24.50 ± 4.92b 6.60 ± 2.69c 6.7 ± 0.6c 4.5 ± 0.7d 2.341 ± 0.531a 3.755 ± 0.442a
Different lower-case letters in the same row indicate a significant difference between treatments (P < 0.05).
Fig. 2. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on chlorophyll (A and B) and carotenoid contents (C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
group (Fig. 3D). In upper-node leaves, the NPQ in the T2 and T3 groups were greater than in the T1 and control groups. Fluoroglycofen had no significant effects on Fv/Fm in upper-, middle- and bottom-leaves, UPSII in bottom-node leaves, QP in upper- and middle-node leaves and NPQ in upper-node leaves (Fig. 4). Application of fluoroglycofen significantly reduced UPSII in the upper-node leaves, while UPSII in middle-node leaves of the T5 and T6 groups were significantly lower than the control (Fig. 4B). QP in bottom-node leaves of T4 and T5 groups were significantly lower than the control (Fig. 4C). NPQ in the middle- and bottom-node leaves of the T5 and T6 groups were much greater than in the control group, but no significant differences was observed between these values at the three concentrations of fluoroglycofen used (Fig. 4D).
3.4. Effects of acetochlor and fluoroglycofen on lipid peroxidation and superoxide production rate in grape leaves Malondialdehyde (MDA) contents increased in middle- and bottom-node leaves after treatment with acetochlor, but there were no significant differences between the three acetochlor treatments of middle-node leaves, or between the T2 and T3 groups in MDA contents of bottom-node leaves (Fig. 5A). Lower concentrations of acetochlor (T1) had no effect on MDA contents in upper-node leaves, while higher concentrations enhanced MDA contents significantly. Acetochlor caused a significant acceleration in the O2 production rate, which increased in dose-dependent manner in uppernode leaves (Fig. 5C); in middle-node leaves, the O2 production
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Fig. 3. Effects of acetochlor and fluoroglycofen on maximal photochemical efficiency of PSII in darkness (Fv/Fm; A), actual photochemical efficiency of PSII in the light (UPSII; B) photochemical quenching (QP; C) and non-photochemical quenching (NPQ; D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
rate was 3.4% lower in the T1 group compared with the control, but this rate was 21.8% and 38.2% higher in the T2 and T3 groups compared with the control, respectively. In bottom-node leaves, O2 production rate was greater at lower acetochlor concentrations, but lower in the T3 group (though the rate was still greater than in the T1 group). When fluoroglycofen concentration was greater than 37.5 g ai ha1, MDA contents were significantly higher (Fig. 5B). Moreover, the O2 production rate accelerated in a dose-dependent manner (Fig. 5D), with increases of up to 323%, 57% and 78% in upper-, middle- and bottom-node leaves, respectively. 3.5. Effects of acetochlor and fluoroglycofen on antioxidant enzyme activities in grape leaves The herbicide treatments induced significant changes in the activities of various antioxidant enzymes. At lower concentrations, acetochlor improved SOD activity in upper- and middle-node leaves, but activity was reduced at the greatest concentration of acetochlor tested; however, in bottom-node leaves, SOD activity decreased in a dose-dependent way (Fig. 6A). CAT activities in upper- and middle-node leaves were higher in the T1 group (compared to the controls) but decreased to control levels in the T3 group. Acetochlor had no significant effects on CAT activity in bottom-node leaves (Fig. 6B). POD activity was reduced significantly by acetochlor in upper- and middle-node leaves, but no significant differences were observed between the T1 and T2 groups; however, compared with other treatments, POD activity was significantly greater in the bottom-node leaves in the T2 group (Fig. 6C). Acetochlor caused dose-dependent decreases in APX activity in
upper-node leaves, while in middle-node leaves APX activity was 8.0% greater in the T1 group, but 47.3% and 75.0% lower in the T2 and T3 groups, respectively. In bottom-node leaves, APX activity was reduced compared to the control by 33.8% and 53.8% in the T2 and T3 groups, respectively (Fig. 6D). In upper-node leaves, SOD and APX activities decreased significantly and gradually when fluoroglycofen concentrations were greater than 37.5 g ai ha1 (Fig. 7A and D). Meanwhile, CAT and POD activities were greater in the T4 group but decreased dramatically as fluoroglycofen concentrations increased further (Fig. 7B and C). In the middle-node leaves, the changes in SOD activity were similar to changes observed in POD activity in upper-node leaves (Fig. 7A), whereas CAT and POD activities decreased significantly only when fluoroglycofen concentrations reached 387.5 or 187.5 g ai ha1, respectively (Fig. 7B and C). APX activity was greater in the T4 and T5 treatments compared to the control, but then it decreased in the T6 group to a level 36.2% lower than the control (Fig. 7D). In bottom-node leaves, fluoroglycofen reduced SOD and POD activities in a dose-dependent manner (Fig. 7A and C), while only the greatest concentration of fluoroglycofen reduced CAT activity (Fig. 7B). Finally, APX activity in bottom-node leaves in the T5 and T6 groups were lower than the control and T4 groups (Fig. 7D). 3.6. Acetochlor and fluoroglycofen inhibited the growth of grapes in 2011 Acetochlor and fluoroglycofen treatments in 2010 inhibited the growth of grapevines in 2011 (Table 1). Stem heights were reduced by up to 55.4% and 88.0% after treatment with acetochlor and
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Fig. 4. Effects of acetochlor and fluoroglycofen on maximal photochemical efficiency of PSII in darkness (Fv/Fm; A), actual photochemical efficiency of PSII in the light (UPSII; B) photochemical quenching (QP; C) and non-photochemical quenching (NPQ; D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
fluoroglycofen, respectively; however, the ratio of root and shoot was enhanced in these treatment groups by up to 50.9% and 142.1%, respectively. Inhibition of grape growth was more serious with fluoroglycofen than acetochlor, especially at the maxium concentrations of fluoroglycofen which even caused deformation of the leaves.
4. Discussion Many herbicides are photosystem inhibitors [37], including atrazine that binds to the D1 protein of PSII, blocking electron transfer to the plastoquinone pool [38,39]; clomazone that greatly reduces electron transport in thylakoid membranes of chloroplasts [18]; and flumioxazin that acts as an protoporphyrinogenoxidase inhibitor, causing decreases in Pn and Gs in grape leaves [19]. Acetochlor and fluoroglycofen are also absorbed by roots, and both herbicides reduced photosynthesis in grape leaves. Herbicides can enter the plant through the apoplast, symplast and apoplast-symplast systems in roots, and then be transported via the xylem and catheter action due to transpiration. Differences in penetration rate, the in vivo metabolic situation and the toxicity of the herbicide can each affect the herbicide transport mechanism in different plant species. Furthermore, environmental conditions, such as temperature, can moderate and influence herbicide transport. The responses to herbicides in different node leaves might be attributed to these diverse transport pathways. Acetochlor caused greater decreases in Pn in middle- and bottom-node leaves compared to upper-node leaves. Conversely, fluoroglycofen treatment decreased Pn in upper-node leaves to a greater extent than in
middle- and bottom-node leaves. Old and young leaves showed opposing tolerances to the two herbicides examined herein, which might be related with the plants requiring distinct resistant mechanisms to counter these different herbicides. Acetochlor caused decreases in pigment contents. Conversely, fluoroglycofen treatment resulted in increases in pigment contents at lower concentrations, which differs from observations with a similar herbicide, flumioxazin, that caused pigment contents to decrease [19]. In 2009, three vineyards were observed that applied different concentrations of herbicides. One vineyard was perennially exposed to higher levels of paraquat, acetochlor and fluoroglycofen, and in this vineyard the grape leaves grew dark and round, but the photosynthetic rate and the grape yields were lower than obtained with artificial weeding. The mechanisms behind this phenomenon required further study and this formed the focus of the present study. The fluorescence arising from chlorophyll is almost exclusively associated with PSII [40]. Since PSII functioning is sensitive to a wide range of environmental conditions, chlorophyll fluorescence provides a wealth of information on the effects of particular stresses on plants [40]. Paraquat and norflurazon (each at 100 lg L1) caused significant decreases in Fv/Fm, UPSII and QP, while significantly increasing NPQ [1]; however, flumioxazin and terbutryn reduced NPQ in grape and Vicia faba, respectively [17,19]. A decrease in QP indicates a higher proportion of closed PSII reaction centers, i.e., an increase in the proportion of the reduced state of QA [27], which probably leads to a decrease in the proportion of available excitation energy used for photochemistry [41]. Moreover, the value of NPQ is linearly related to energy dissipation in leaf tissues and is viewed as essential in protecting the leaf from light-induced
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Fig. 5. Effects of acetochlor (A and C) and fluoroglycofen (B and D) on malondialdehyde (MDA) contents (A and B) and O2 production rates (C and D) in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
Fig. 6. Effects of acetochlor on superoxide dismutase (SOD; A), catalase (CAT; B), peroxidase (POD; C) and ascorbate peroxidase (APX; D) activities in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
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Fig. 7. Effects of fluoroglycofen on superoxide dismutase (SOD; A), catalase (CAT; B), peroxidase (POD; C) and ascorbate peroxidase (APX; D) activities in upper-, middle- and bottom-node leaves of grapevines. Different concentrations of acetochlor and fluoroglycofen were sprayed in a single treatment onto soil containing one-year old grapevines (>10 leaves). CK: control; T1, T2, T3: three gradual increases in acetochlor concentration; T4, T5, T6: three gradual increases in fluoroglycofen concentration. Values are means ± standard deviation (n = 6). Columns marked with different lower-case letters indicate a significant difference between treatments (P < 0.05).
damage [42]. Acetochlor and fluoroglycofen caused increases in NPQ and decreases in UPSII, especially at higher concentrations. Thus, the two herbicides reduced PSII activity and were involved in non-radiative energy dissipation. Decreases in CO2 accumulation and the photochemical efficiency of PSII might be associated with higher levels of reactive oxygen species. Different classes of herbicides are direct or indirect sources of oxidative damage in plants [43,44]. While plants have a suite of protective antioxidant enzymes and substances, the induction of these enzymes’ activities often accompanies a disturbance in the redox homeostasis and these can be used to indicate oxidative stress [45]. MDA is the final product of membrane lipid peroxidation and its accumulation can result from toxicity caused by reactive oxygen species. Many studies have reported that many herbicides induce the accumulation of reactive oxygen species and MDA [46–49]. Meanwhile, the responses of antioxidant enzymes to herbicide treatments in various plants may differ due to differences in the treatment times and the point at which measurements are made, and there are also concentration and time effects [13,14,16,47]. This present study shows that both acetochlor and fluoroglycofen accelerated O2 production and caused exacerbation of membrane lipid peroxidation, especially higher concentrations, which might be relevant to lowest antioxidant enzyme activities. Nevertheless, in agreement with other reports, lower concentrations of acetochlor enhanced the activities of SOD and CAT in the upper- and middle-node leaves and APX in the middle-node leaves. Furthermore, CAT and POD activities in the upper-node leaves and APX activities in middle- and bottom-node leaves were increased after treatment with lower concentrations of fluoroglycofen. Herbicides can affect plant growth and reduce biomass [8,15,24]. In an earlier study, vines treated with flazasulfuron were
found to exhibit yellow leaves and alterations in photosynthetic activity, but these changes disappeared in the following year, indicating that vines have the potential to recover after one season from the stress caused by this particular herbicide [50]. However, the data in this present report suggest that acetochlor and fluoroglycofen not only resulted in decreases in photosynthesis in 2010, but also led to inhibition of growth in 2011, especially at higher concentrations of fluoroglycofen, which caused leaf deformities and seriously inhibited growth. The reasons behind these observations require further research. According to the above results, both of higher levels of acetochlor and fluoroglycofen exert detrimental effects on grapevines not only in the first year but also in the second year. Furthermore, another postgraduate in our lab measured the grape quality in grapevines after application of combination of 2246 g ai ha1 acetochlor and 22.5 g ai ha1 fluoroglycofen, the results showed that the grape acidity, anthocyanin, vitamin C content and soluble solid decreased significantly. We also measured the acetochlor residues in grapes, but no fluoroglycofen residues. However, many authors have already reported that vineyard cover crops could reduce must acidity and increase berry sugar accumulation, tartaric acid/malic acid ratio and berry skin total phenols and anthocyanin content [51,52], we suggested that the orchardman should reduce the use number and dosage of herbicide, or instead of it, better to cover crops.
5. Conclusion From our results we conclude the followings: (1) application of acetochlor and fluoroglycofen reduce photosynthesis in grape leaves in the first year and this is associated with higher levels of
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reactive oxygen species, which is, in turn, may be attributed to lower activities of antioxidant enzymes; (2) the growth inhibition in the second year might due to the after effects of herbicide treatment. Acknowledgments We sincerely thank Prof. Xinghong Yang (College of Life Science, Shandong Agriculture University, China) for assistance in editing the manuscript. This research was supported by China Agriculture Research System (CARS-30). References [1] C. Frankart, P. Eullaffroy, G. Vernet, Comparative effects of four herbicides on non-photochemical fluorescence quenching in Lemna minor, Environ. Exp. Bot. 49 (2003) 159–168. [2] J.O. Guh, K. Ishizuka, J.Y. Pyon, Differential absorption and translocation of oxyfluorfen between selected rice cultivars, Korean J. Weed Sci. 8 (1988) 37– 44. [3] S. Kim, K.K. Hatzios, Differential response of two soybean cultivars to paraquat, Verlag der Zeitschrift für Naturforschung 48 (1993) 379–384. [4] J.S. Choi, H.J. Lee, I.T. Hwang, J.Y. Pyon, K.Y. Cho, Differential susceptibilities of wheat and barley to diphenyl ether herbicide oxyfluorfen, Pestic. Biochem. Physiol. 65 (1999) 62–72. [5] J.D.M. Belgers, R.J. Van Lieverloo, L.J.T. Van der Pas, P.J. Van den Brink, Effects of the herbicide 2,4-D on the growth of nine aquatic macrophytes, Aquat. Bot. 86 (2007) 260–268. [6] C.M. Smart, Gene expression during leaf senescence, New Phytol. 126 (1994) 419–448. [7] S. Kaushik, Effect of herbicides with different modes of action on physiological and cellular traits of Anabaena fertilissima, Paddy Water Environ, 8 (2010) 277– 282. [8] Y. Sunohara, S. Shirai, N. Wongkantrakorn, H. Matsumoto, Sensitivity and physiological responses of Eleusine indica and Digitaria adscendens to herbicide quinclorac and 2,4-D, Environ. Exp. Bot. 68 (2010) 157–164. [9] T.D. Sherman, J.M. Becerril, H. Matsumoto, M.V. Duke, J.M. Jacobs, N.J. Jacobs, S.O. Duke, Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides, Plant Physiol. 97 (1991) 280–287. [10] Y.I. Kuk, J.-S. Shin, H.I. Jung, J.O. Guh, S. Jung, N.R. Burgos, Mechanism of paraquat tolerance in cucumber leaves of various ages, Weed Sci. 54 (2006) 6– 15. [11] Y.I. Kuk, J.S. Shin, Mechanisms of low-temperature tolerance in cucumber leaves of various ages, J. Am. Soc. Hortic. Sci. 132 (2007) 294–301. [12] J.L. Donahue, C.M. Okpodu, C.L. Cramer, E.A. Grabau, R.G. Alscher, Responses of antioxidants to paraquat in pea leaves (Relationships to Resistance), Plant Physiol. 113 (1997) 249–257. [13] J.Y. Yoon, J.S. Shin, D.Y. Shin, K.H. Hyun, N.R. Burgos, S. Lee, Y.I. Kuk, Tolerance to paraquat-mediated oxidative and environmental stresses in squash (Cucurbita spp.) leaves of various ages, Pestic. Biochem. Physiol. 99 (2011) 65–76. [14] R.S. Macedo, A.T. Lombardi, C.Y. Omachi, L.R. Rörig, Effects of the herbicide bentazon on growth and photosystem II maximum quantum yield of the marine diatom Skeletonema costatum, Toxicol. In Vitro 22 (2008) 716–722. [15] G. Saladin, C. Magné, C. Clément, Impact of flumioxazin herbicide on growth and carbohydrate physiology in Vitis vinifera L, Plant Cell Rep. 21 (2003) 821– 827. [16] L. Geoffroy, C. Frankart, P. Eullaffroy, Comparison of different physiological parameter responses in Lemna minor and Scenedesmus obliquus exposed to herbicide flumioxazin, Environ. Pollut. 131 (2004) 233–241. [17] R. Piñol, E. Simón, Effect of 24-epibrassinolide on chlorophyll fluorescence and photosynthetic CO2 assimilation in Vicia faba plants treated with the photosynthesis-inhibiting herbicide terbutryn, J. Plant Growth Regul. 28 (2009) 97–105. ˇ a, M. Špundová, P. Ilík, D. Lazár, K. Klem, P. Tomek, J. Nauš, O. Prášil, [18] R. Kan Effect of herbicide clomazone on photosynthetic processes in primary barley (Hordeum vulgare L.) leaves, Pestic. Biochem. Physiol. 78 (2004) 161–170. [19] A. Bigot, F. Fontaine, C. Clément, N. Vaillant-Gaveau, Effect of the herbicide flumioxazin on photosynthetic performance of grapevine (Vitis vinifera L.), Chemosphere 67 (2007) 1243–1251. [20] J. Shen, W. Luo, Effects of monosulfuron on growth, photosynthesis, and nitrogenase activity of three nitrogen-fixing cyanobacteria, Arch. Environ. Contam. Toxicol. 60 (2011) 34–43. [21] D.A. Kopsell, G.R. Armel, K.R. Abney, J.J. Vargas, J.T. Brosnan, D.E. Kopsell, Leaf tissue pigments and chlorophyll fluorescence parameters vary among sweet corn genotypes of differential herbicide sensitivity, Pestic. Biochem. Physiol. 99 (2011) 194–199. [22] M.I. Hussain, L. GonzáLez, M. Reigosa, Phytotoxic effects of allelochemicals and herbicides on photosynthesis, growth and carbon isotope discrimination in Lactuca sativa, Allelopathy J. 26 (2010) 157–174.
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