5-Aminolevulinic acid alleviates herbicide-induced physiological and ultrastructural changes in Brassica napus

XU Ling, Faisal Islam, ZHANG Wen-fang, Muhammad A Ghani, Basharat Ali,

1 Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang/College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, P.R.China

2 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China

3 Jiading District Agro-Technology Extension Service Center, Shanghai 201800, P.R.China

4 Institute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, Pakistan

5 Institute of Crop Science and Resource Conservation, University of Bonn, Bonn 53115, Germany

1.Introduction

Oilseed rape (Brassica napus L.) is one of the main oilseed crops in the word and is the major source of edible oil in China (Zhou 2001; Momoh et al.2002).However, the cruciferous weeds are hard to control and have become a serious threat to winter oilseed rape production in the field(Diepenbrock 2000).They compete with crop plants for light,water, nutrients and space (Song et al.2005).Chemical weed management is the mostly used practice to control weeds.Among these herbicides, a new effective herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino)benzoate (ZJ0273) is being widely used in the oilseed rape fields with the advantages of low dosage, low mammalian toxicity, broad weeding spectrum and environmental compatibility (Tang et al.2005).This original herbicide is a derivative of 2-pyrimidinyloxy-N-aryl benzoate with novel structure and efficient biological activity (Wu et al.2003),and it also was supposed to be one of those herbicides which inhibit biosynthesis of amino acids, protein and so on in the plants (Zhang et al.2009; Jin et al.2010).Moreover,ZJ0273 has similar phytotoxic symptoms and biological activities as the acetolactate synthase (ALS, EC 4.1.3.18)inhibiting herbicides (Zhou et al.2007; Xu et al.2015).More precisely, this new herbicide ZJ0273 shows pro-herbicide characters as it has shown only a weak inhibiting effect on ALS in vitro (Zhang et al.2009).

However, application of herbicide produces stress responses in crop plants, which results in cell damage because of peroxidation of membrane lipids, protein oxidation,enzyme inhibition, DNA and RNA damage (Islam et al.2016;Kaya and Doganlar 2016).In order to adapt the stress conditions, plants have developed different molecular and bio-physiological responses through hormones dependent signaling pathways (Yang et al.2012; Xia et al.2015, 2016).Previous studies revealed that 5-aminolevulinic acid (ALA)application can enhance chlorophyll biosynthesis and photosynthesis, and improve antioxidant capacity as well as reduce membrane lipid peroxidation damage of plants(Hotta et al.1997).Similarly, ALA is considered as one of most important plant growth regulators, which is known as essential precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll (Akram and Ashraf 2013).Recently, Ali et al.(2014b) reported that ALA improved the plant biomass, uptake of nutrients in the leaves and roots of B.napus plants, and enhanced the performance of antioxidant and some non-antioxidant enzyme activities due to its ameliorative potential under Pb stress conditions.ALA has been demonstrated to alleviate the water stress in B.napus (Liu et al.2011).Moreover, ALA has been found to promote the plant growth through alleviation of antioxidant systems under cadmium stress in B.napus (Ali et al.2013a, b, 2014b).Thus, wealth of knowledge indicated that exogenous application of ALA could modulate antioxidant defense, and thereby increasing the resistance of plants to the abiotic stresses (Naeem et al.2010, 2011, 2012; Akram et al.2012).Although, the previous study showed that ALA alleviated toxicity of new herbicide ZJ0273 (Zhang et al.2008a, b), the information regarding the optimal treatment interval, ultrastructural and proteomic changes induced by exogenous ALA under herbicide stress is poorly understood.Keeping in view the importance of oilseed rape and the alleviating effects of ALA under abiotic stress, the present study was conducted to prove the hypothesis that ALA has the ameliorating capacity to improve the plant growth under herbicide ZJ0273 stress by recovering antioxidant enzyme activities, mitigating ultrastructural and proteomic attributes.

2.Materials and methods

2.1.Chemicals

The ALA was purchased from Cosmo Oil Co., Ltd.(Japan)and the herbicide propyl ZJ0273 (10%, emulsion) was provided by the Shanghai Institute of Organic Chemistry,Chinese Academy of Sciences.

2.2.Seed germination experiments

Seeds of oilseed rape (B.napus cv.ZS758, popular commercial cultivars in China) were selected for the experiments.Seeds were washed with distilled water and air-dried before use.The concentrations of ALA and herbicide ZJ0273 treated on the seeds were determined according to the results of our previous study (Zhang et al.2008b).The details of the experiments were conducted as the following procedures:

1) ALA pre-treatment: 6 mL 0 and 1 mg L-1ALA solutions were added to two pieces of filter paper in Petri dishes previously, respectively, and then 50 seeds were sown for different intervals (48 or 72 h).Later the excess solutions were removed and 6 mL 0, 100, 200, and 500 mg L-1ZJ0273 solutions were added to the Petri dishes, respectivley, for another 48 or 72 h, respectively.

2) ALA post-treatment: 6 mL 0, 100, 200, and 500 mg L-1ZJ0273 solutions were added firstly, then 6 mL 0 and 1 mg L-1ALA solutions was added, respectively, other procedures were the same as the ALA pre-treatment procedure 1).

3) Culture conditions: The excess solutions were discarded; seeds and filter paper were transferred onto a sponge floating in a high-wall white porcelain plate filled with half-strength Hoagland solution.Seeds were cultured in the incubator at a temperature of 20°C under a 12-h photoperiod (light intensity of 140 μmol m-2s-1) and high relative humidity (85-90%).

2.3.Determination of biomass and physiological characters

Fresh weights of shoot (10 plants) and root (50 plants), and shoot lengths of B.napus seedlings were measured after seven days of ALA pre- or post-treatment.Root oxidizability was determined according to the triphenyl tetrazolium chloride(TTC) reduction method (Zhou and Ye 1996).A total of 0.5 g fresh root samples were placed in a 25-mL test tube before adding 5 mL of 0.4% TTC solution and 5 mL of 1/15 mol L-1phosphate buffer (pH 7.0).After incubation for 1 h at 37°C in an oven, the reaction was terminated by adding 2 mL of 1 mol L-1H2SO4immediately.The roots were decolored with 10 mL of methyl alcohol and the extraction was read at 485 nm.

The oxidative damage to lipids was determined according to Zhou and Leul (1998) as lipid peroxidation in terms of malondialdehyde (MDA) production.Fresh samples(0.2 g) were homogenized and extracted in 10 mL of 0.25%thiobarbituric acid (TBA) made in 10% trichloroacetic acid(TCA), then the extract was heated at 95°C for 30 min and cooled on ice quickly.The samples were centrifuged at 5 000×g for 10 min.The absorbance was measured at 532 nm.Correction of nonspecific turbidity was made by subtracting the absorbance value taken at 600 nm.The level of MDA was expressed as μmol g-1fresh weight by using extinction coefficient of 155 mmol L-1cm-1.

For enzymes activities, samples were homogenized under ice cold conditions.Homogenate was centrifuged at 10 000×g for 20 min at 4°C and the supernatant was used for the determination of the following enzyme activities.Total superoxide dismutase (SOD, EC 1.15.1.1) activity was determined with the method of Zhang et al.(2008b)using the photochemical NBT method.Reaction mixture of 3 mL contained 50 mmol L-1phosphate buffer (pH 7.8),26 mmol L-1L-methionine, 750 μmol L-1NBT, 1 μmmol L-1EDTA, and 20 μmmol L-1riboflavin.One unit of SOD activity was measured as the amount of enzyme required to cause 50% inhibition of the NBT reduction measured at 560 nm.

Peroxidase (POD, EC1.11.1.7) activity was assayed by Zhou and Leul (1999) with some modifications.Reaction mixture of 3 mL contained 50 mmol L-1potassium phosphate buffer (pH 6.1), 1% guaiacol, 0.4% H2O2and 100 μL enzyme extract.Variation due to guaiacol in absorbance was measured at 470 nm.

The assay for ascorbate peroxidase (APX, EC 1.11.1.11)activity was measured in a reaction mixture of 3 mL containing 100 mmol L-1phosphate (pH 7.0), 0.1 mmol L-1EDTA-Na2, 0.3 mmol L-1ascorbic acid, 0.06 mmol L-1H2O2and 100 μL enzyme extract.The change in absorption was taken at 290 nm after addition of H2O2for 30 s (Nakano and Asada 1981).

2.4.Transmission electron microscopy

The ultrastructural and proteomic materials were selected according to the following process: 45-day-old B.napus seedlings (four leaf stage in the greenhouse,25°C at day/10°C at night, the highest light intensity of 1 250 μmol m-2s-1at noon) were selected for the ultrastructural and proteomic experiments.The concentrations of ALA and ZJ0273 were determined according to Zhang et al.(2008b).And 100 mg L-1ALA was sprayed firstly, 24 h later,500 mg L-1ZJ0273 was sprayed.Seven days later, seedlings were selected for the transmission electron microscopy and two-dimensional gel electrophoresis (2-DE) analysis.

Leaf fragments without veins (approximately 1 mm×3 mm)were selected and then fixed for 24 h in 4% glutaraldehyde(v/v) in 0.1 mol L-1sodium phosphate buffer (PBS), pH 7.0) and rinsed three times with the same PBS.Samples were post-fixed in 1% OsO4for 1 h, washed three times in 0.1 mol L-1PBS (pH 7.0).After 15-20 min interval, the samples were dried in a graded series of ethanol (50, 70, 80,95, 95 and 100%) for 15 min each time.The samples were then infiltrated and embedded in Spurr’s resin overnight.After heating at 70°C for 16 h, ultra-thin sections (80 nm)of specimens were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOL TEM-1230EX, Japan) at an accelerating voltage of 60.0 kV(Najeeb et al.2011).

2.5.Soluble protein extraction and SDS-PAGE

The 0.2 g fresh rape leaves were homogenized in 3 mL buffer solution (30 mmol L-1Tris-HCl, pH 8.0, 1 mmol L-1ethylene diamine tetraacetic acid (EDTA), 5 mmol L-1MgCl2,6 mmol L-1ascorbic acid, 1% PVP, 5% glycerol, and 1%DL-dithiothreitol (DTT)).The homogenate was centrifuged at 15 000×g for 15 min under the temperature of 4°C, the soluble protein was contained in supernatant fluid.Protein concentrations in suspensions were determined according to Bradford’s method (1976).SDS-PAGE was performed using a 4% stacking and a 12% separating gel according to the Guo’s (2005) protocol.Sample volume was controlled to 20 μg proteins in each sample with 5 μL of 0.002% bromophenol blue.Electrophoresis was conducted at a constant current of 80 V through the stacking gel and 120 V through the resolving gel.The gel was scanned by the machine of Bio-Rad GS-800 (USA).

2.6.2-DE and image analysis

2-DE with immobilized pH gradient (IPG) strips was utilized to analyze the proteins changes induced by ALA under herbicide stress in oilseed leaves.The SDS-PAGE was carried out at 20°C and 10 mA for 15 min, and then 20 mA for approximately 5 h using an SE600 apparatus (Bio-Rad,USA).Spot intensities were obtained in pixel units, normalized to the total absorbance of the gel, and calculated as relative volumes.The relative volume of each spot was assumed to represent its expression level (Xu et al.2015).

2.7.Statistical analysis

The data were analyzed using the statistical program SAS and the analysis of variance (ANOVA) was followed by Fisher’s protected LSD test to identify homogeneous groups within the means.All data presented are mean values.All treatments were replicated three times.Significant differences among treatments were considered at the P≤0.05 level.

3.Results

3.1.Effects of ALA and ZJ0273 on physiological changes in B.napus seedlings

The data regarding the biomass of B.napus in response to pre/post treatment of ALA with different doses of herbicide (ZJ0273) for different treatment durations have been shown in Tables 1-3.The fresh weight of shoots was significantly lower under ZJ0273 stress conditions.Decline rate of shoot weight was consistently higher with each successive increase of herbicide concentration.Both preand post-treatment with 1 mg L-1ALA for 48 h significantly increased the shoot fresh weight of oilseed rape seedlings under the different concentrations of ZJ0273 (Table 1).As expected, the highest shoot fresh weight was investigated in the pretreatment of 1 mg L-1ALA for 48 h alone.Nevertheless, seeds pretreated with the highest ZJ0273 (500 mg L-1) for 72 h could not germinate, which led to the biomass was undetectable.Similar results were found in the 500 mg L-1ZJ0273 post-treatment for 72 h.

In the 48 h pre-treatment experiments, the values of shoot length declined dramatically with the successive increase of herbicide concentrations from 0 to 500 mg L-1,and the higher the concentration of herbicide ZJ0273, the lower the shoot length is (Table 2).Addition of exogenous ALA (1 mg L-1) significantly enhanced the shoot length as compared with the relative control.The longest shoot(8.8 cm) was observed when the seedlings were pretreated by 1 mg L-1ALA for 48 h alone.However, with the prolongingof the treatment time from 48 to 72 h, the shoot elongation was suppressed obviously under herbicide conditions.And the lowest shoot length (3.17 cm) was found after 72 h pretreated with 200 mg L-1ZJ0273.Nevertheless, the shoot length was un-detectable after the seeds pretreated with the 500 mg L-1ZJ0273 for 72 h due to the un-germinated seeds.Moreover,ALA could not recover the negative effects induced by 72 h pre-treatment with 500 mg L-1ZJ0273, indicating that high concentration of ZJ0273 treated for long time was lethal to the seeds.The similar results were found in the post-treatment tests.Thus, exogenous addition of 1 mg L-1ALA significantly promoted the shoot growth as reflected by the increase of shoot length both in the pre- and post-treatment experiments.

Table 1 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on shoot fresh weight (g fresh weight (FW) 10 plants-1) of Brassica napus cv.ZS 758

Table 2 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on shoot length (cm) of Brassica napus cv.ZS 758

Results showed that root fresh weight was declined with the excessive increase of ZJ0273 concentrations from 0 to 500 mg L-1in both pre- and post-treatment experiments(Table 3).Thus, the herbicide ZJ0273 imposed negative effects on root fresh weight.Exogenously applied ALA (1 mg L-1) increased the root fresh weight as compared to relative controls.The pre-treatment with 1 mg L-1ALA alone for 48 h produced the highest root fresh weight (0.980, 0.507,0.383, and 0.297 g FW 50 plants-1) when the seeds were subsequently treated with different dosages of ZJ0273 (0,100, 200, and 500 mg L-1), respectively.

The root oxidizability declined with the successive increase of herbicide dosages from 0 to 500 mg L-1(Table 4).Pretreatment of seeds with 1 mg L-1ALA alone for 48 and 72 h significantly increased the root oxidizability by 10 and 7%, respectively, whereas recovery effect of ALA was much more obvious under the herbicide stress.Our study found that the pre-treatment with 1 mg L-1ALA for 48 h dramatically improved root oxidizability by 14, 23 and 30% when the seeds were subsequently treated with different dosages of ZJ0273 (100, 200, and 500 mg L-1), respectively (Table 4).The similar results were also found in the post-treatment testing.However, with the prolonging of treatment duration to 72 h, the root oxidizability could not be detected under the highest dosage of herbicide (500 mg L-1) due to the death of seeds.Therefore, the herbicide significantly decreased the root oxidizability of rape seedlings, and the exogenous ALA could promote the growth of rape seedlings as reflected by the enhancing of root oxidizability under the ZJ0273 stress.

Table 3 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on root fresh weight (g fresh weight (FW) 50 plants-1) of Brassica napus cv.ZS 758

Table 4 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on root oxidizability (2,3,5-triphenyltetrazoliumchloride (TTC) mg g-1 fresh weight (FW) h-1) of Brassica napus cv.ZS 758

A positive effect of ALA (both pre- and post-treatments)was observed as reflected by the decrease of MDA accumulation after addition of 1 mg L-1ALA (Table 5).During the pre-treatment for 48 h, the application of 1 mg L-1ALA significantly decreased the MDA accumulation by 10, 15,14 and 12% when the seeds were subsequently treated with different dosages of ZJ0273 (0, 100, 200, and 500 mg L-1), respectively (Table 5).Moreover, with the prolonging treatment time from 48 to 72 h, the MDA accumulation was much more obvious.However, we could not detect the MDA content after treated with the highest dosage of ZJ0273(500 mg L-1) for 72 h due to the death of pants.Similar results were also observed in the post-treatment experiments.

A significant decrease in SOD activity was observed with the successive increase of ZJ0273 concentrations(Table 6).Both pre- and post-treatment with 1 mg L-1ALA significantly enhanced SOD activities as compared with the relative controls, and the highest value (381.9 U g-1FW)was achieved after 48 h pre-treatment with 1 mg L-1ALA.Additionally, the SOD activity could not be detected under the highest dosage of ZJ0273 (500 mg L-1) for 72 h in both pre- and post-treatment experiments (Table 6).A decrease in POD activity with the successive increase of ZJ0273 concentrations in both pre- and post-treatment and the least POD activity (5.918 OD470g-1FW min-1) was detected in response to the post-treatment with 500 mg L-1of ZJ0273 for 48 h (Table 7).Exogenous ALA obviously alleviated the negative effects induced by the herbicide ZJ0273 (0-500 mg L-1) as reflected by the increase of POD activity.The plants pre- and post-treatment only with 1 mg L-1ALA for 48 h had a significantly higher POD activity, 44 and 43% over the control, respectively.Similarly, pre- and post-treatment with 1 mg L-1ALA for 72 h also significantly increased (20 and 18%, respectively) POD activity as compared to the control.Therefore, with the prolonging of treatment durations from 48 to 72 h, the recovery effects of ALA decreased.

The synergistic effect of ALA and ZJ0273 on APX activity was obvious in both pre- and post-treatment (Table 8).A significant decrease in APX activity was observed with the successive increase of ZJ0273 concentrations from 0 to 500 mg L-1.Exogenous 1 mg L-1ALA application dramatically improved the APX activity of the rape seedlings.Plantspost-treated with 1 mg L-1ALA for 48 h produced the highest APX activity (53.17 μmol g-1FW) without the herbicide stress,and the APX activity also declined with the prolonging of treatment time from 48 to 72 h.Whereas the 1 mg L-1ALA could not recover the APX activity after being pre- or post-treatment with the highest dosage of ZJ0273 (500 mg L-1) for 72 h.

Table 5 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on malondialdehyde (MDA) content (μmol g-1 fresh weight (FW)) of Brassica napus cv.ZS 758

Table 6 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on superoxide dismutase (SOD) activity (U g-1 fresh weight (FW)) of Brassica napus cv.ZS 758

3.2.Effects of ALA and ZJ0273 on ultrastructure’s in B.napus seedlings

The chloroplast of the control mesophyll cells in rape seedlings had normal typical structure with the fusiform shape,containing many compact grana stacks, well-arranged thylakoids, clear thylakoid membranes, and little plastoglobuli(Fig.1-A).Electron microscopic image under 500 mg L-1ZJ0273 stress alone showed the amounts of grana and thylakoid membranes were obviously lower, however, the number of plastoglobuli inside chloroplast was more than control (Fig.1-B).Application of 100 mg L-1ALA under 500 mg L-1ZJ0273 stress, the disintegrated thylakoid was recovered with more compact grana, thicker thylakoid membranes and less plastoglobuli, the number of grana increased with clear lamella and thylakoid arrangement (Fig.1-C).Electron microscopic image at 100 mg L-1ALA alone showed that the thylakoid membrane was intact but the number of grana was more than control.The number of plastoglobuli inside chloroplast was also more than control (Fig.1-D).

Synergistic effects of ALA and ZJ0273 on mitochondria in the mesophyll cells of B.napus seedlings are presented in Fig.2.The mitochondria had normal typical structure with intact and clear bilayer, mitochondrial crista membrane was clear and cristaes were obvious with short vesicular shape(Fig.2-A).When the seedlings exposed to 500 mg L-1herbicide ZJ0273, the abnormal and swollen mitochondria were observed (Fig.2-B).After the application of 100 mg L-1ALA under 500 mg L-1herbicide ZJ0273 stress, the bilayer of mitochondria started to recover with the appearance of clear crista membrane and cristae (Fig.2-C).Moreover, no considerable change in the appearance of mitochondria was observed after treated with 100 mg L-1ALA alone (Fig.2-D).

Electron micrographs of B.napus seedlings demonstrated the synergistic effects of ALA and ZJ0273 on thenucleus structures (Fig.3).Indeed, there were clear nucleus structures with well-developed nuclear membrane and nucleolus except the treatment with 500 mg L-1ZJ0273 alone.Ultrastructural studies of herbicide-toxicity showedundesirable effects on the mesophyll cells of B.napus under 500 mg L-1ZJ0273 stress alone (Fig.3-B).Deformation of nucleus, rupturing of nuclear membrane and turbid nucleoplasm were some of the obvious damages (Fig.3-B).The TEM micrographs of mesophyll cells after the addition of 100 mg L-1ALA under 500 mg L-1ZJ0273 stress showed well developed nucleus in the cell (Fig.3-C).The mesophyll cells possessed well-shaped nucleus, uniform nucleoplasm with intact nuclear membrane, and the nucleolus were clearly detected in nucleus (Fig.3-C).Under the application of ALA (100 mg L-1) alone, the ultra-structure of nucleus was similar to the control (Fig.3-D).Thus, 100 mg L-1ALA could alleviate the herbicide-toxicity in Brassica seedlings as reflected by the mitigating structures of organelles under ZJ0273 stress condition.

Table 7 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on peroxidase (POD) activity (OD470 g-1 fresh weight (FW) min-1) of Brassica napus cv.ZS 758

Table 8 Effects of treatments of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) and 5-aminolevulinic acid (ALA) on peroxidase (APX) activity (μmol g-1 fresh weight (FW)) of Brassica napus cv.ZS 758

Fig.1 Electron micrographs of chloroplast of Brassica napus cv.ZS 758 seedlings under different treatments.A, control.B, 500 mg L-1 propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273) alone.C, 100 mg L-1(5-aminolevulinic acid) ALA+500 mg L-1 ZJ0273.D, 100 mg L-1 ALA alone.G, grana; P, plastoglobuli; T, thylakoid membranes.A-D, 40 000×.

3.3.Effects of ALA and ZJ0273 on proteomic changes in B.napus seedlings

To investigate the synergistic effect of ALA and ZJ0273 on the protein patterns of B.napus seedlings, rape leaves under 500 mg L-1ZJ0273 alone, ALA alone and in combination with 500 mg L-1ZJ0273 and 100 mg L-1ALA were selected for total proteins extraction and then analyzed by 2-D SDSPAGE.Results demonstrated significant differences in soluble protein contents under different treatments (Fig.4).Under ZJ0273 stress alone, a significant increase in protein content was observed as compared to the control; while, the combined treatment of ALA and ZJ0273 gently decreased protein content as compared to the herbicide treatment alone, but in both treatments, total protein contents were higher than control.However, the soluble protein contents had no significant differences between plants treated with ALA (100 mg L-1) alone and the control (Fig.4).

Fig.3 Electron micrographs of nucleus of Brassica napus cv.ZS 758 seedlings under different treatments.A, control.B, 500 mg L-1 propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273) alone.C, 100 mg L-1 5-aminolevulinic acid (ALA)+500 mg L-1 ZJ0273.D, 100 mg L-1 ALA alone.N, nucleus.A-D, 25 000×.

The protein band patterns were approximately the same between all the treatments and the control, but there were clear differences among protein relative contents.The SDS-PAGE pattern clearly showed that there were some changes in the components of soluble protein.The protein content was significantly higher at MW 20 kD when treated with herbicide ZJ0273 (500 mg L-1) alone as compared to the control.However, the protein content at MW 20 kD decreased when treated with ALA (100 mg L-1) alone or combined with 500 mg L-1ZJ0273 as compared to the herbicide treatment alone (Fig.5).

The changes in protein components were analyzed by 2-DE for further characterization (Fig.6).Compared to the control, the protein spots were obviously different after treated with herbicide treatment alone, combination with 100 mg L-1ALA or 100 mg L-1ALA alone.We observed from the profiles that the protein spots were abundant at MW 14.4-66.0 kD, and contents of some proteins increased,some protein spots disappeared with the appearance of some new protein spots at the same time.We found one new protein spot appearance after treated with 100 mg L-1ALA alone (Fig.6-D), or combined with 500 mg L-1ZJ0273(Fig.6-C) as compared to the control.But the new protein spot disappeared when treated with herbicide alone(Fig.6-B).And as compared to the control, two protein spots were significantly down-regulated under ZJ0273 stress alone, whereas the expressions were enhanced after combined treatment with 100 mg L-1ALA.Furthermore, another two protein spots were up-regulated under the ZJ0273 treatment alone as compared to the control or ALA treatment alone.However, the addition of ALA under the ZJ0273 stress down-regulated the expression of these two protein spots as compared to the herbicide treatment alone.So, it can be predicted that some stress proteins produced under the herbicide conditions and ALA alleviated the herbicide toxicity through regulating these stress proteins encoded by some special genes.

Fig.4 Soluble protein content in leaves of Brassica napus cv.ZS 758 seedlings under different treatments.A, control.B, 500 mg L-1 propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273) alone.C, 100 mg L-1 5-aminolevulinic acid (ALA)+500 mg L-1 ZJ0273.D, 100 mg L-1 ALA alone.FW, fresh weight.Bars mean SD.Different letters among the treatments are significantly different by the LSD test at P≤0.05.

4.Discussion

The results of this study were in accordance with the previous reports showing that ALA application could promote the plant growth through activating antioxidative defense system in B.napus (Liu et al.2011).Our research indicated that herbicide ZJ0273 inhibited the growth of B.napus as reflected by the decrease of biomass, root oxidizability, and antioxidant enzymes activities.Reactive oxygen species(ROS) under several abiotic stress conditions such as herbicides and heavy metal had been reported (Jin et al.2010; Ali et al.2013b, 2014a).Moreover, biomass was severely depressed under the 500 mg L-1herbicide ZJ0273 stress, leading to the common primary symptoms in ALS-inhibitor-treated plants (Lee and Dwen 2000; Zhang et al.2008a, b).Herbicide ZJ0273 had significantly negative effects on antioxidant enzymes (POD, SOD, APX), the higher the dosages of ZJ0273, the lower activities of the antioxidant enzymes was.Moreover, ZJ0273 stress enhanced the accumulation of MDA.However, exogenous application of ALA could significantly alleviate the negative effects induced by the herbicide as reflected by the increase of plant biomass,root oxidizability and antioxidant enzymes activities.

Fig.5 SDS-PAGE pattern and scan of SDS-PAGE pattern of soluble proteins in the leaves of Brassica napus cv.ZS 758 seedlings.Lane 0, broad range protein marker; lane 1,control (distilled water); lane 2, 500 mg L-1 ZJ0273 alone; lane 3, 100 mg L-1 5-aminolevulinic acid (ALA)+500 mg L-1 propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate(ZJ0273); lane 4, 100 mg L-1 ALA alone.

Fig.6 Two-dimensional gel-electrophorisis (2-DE) profiles of proteins in the leaves of Brassica napus cv.ZS 758 seedlings.A, control.B, 500 mg L-1 propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) alone.C, 100 mg L-1 5-aminolevulinic acid (ALA)+500 mg L-1 ZJ0273.D, 100 mg L-1 ALA alone.Circles in the profiles mean different protein spots, and the arrow ididated the spots were special and stressed in the text.

ALA is considered as important plant growth regulator(PGR) and it is a precursor for biosynthesis of chlorophyll (Von Wettstein et al.1995).It acts as biodegradable herbicide at higher concentration, and shows more effective in dicotyledon than monocotyledon (Zhang and Zhou 2000).ALA exerts positive effects on the plants and increases the chlorophyll content and antioxidants under saline stress conditions(Naeem et al.2011, 2012).Recently, it was found that ALA improved the concentration of different nutrients in B.napus under salinity stress conditions (Naeem et al.2010).Similarly,an increase in antioxidant enzymes activities was reported by ALA under heavy metal stress in B.napus (Ali et al.2013b).The present results showed that herbicide ZJ0273 significantly decreased the plant biomass (Tables 1-3).However, plant biomass was obviously improved after treated with 1 mg L-1ALA under herbicide stress (Tables 1-3).Moreover, reduction in root weight might be due to herbicide-induced inhibition of cell division in root cells (Sharma and Dubey 2005).This growth recovery by the exogenous ALA is because ALA could regulate different metabolic processes, thereby improving growth and yield of most plants under abiotic stresses (Akram et al.2012).Our findings are similar to Hotta et al.(1997,1998) who demonstrated that the yields of plants, including kidney bean, barley, potato, and garlic, were improved by 10-60% after treated with ALA at low concentrations.

Previous studies demonstrated that the exogenous plant growth regulators could recover the plant growth through reduction the damage in antioxidant defense system (Farooq et al.2016; Gill et al.2016; Wang et al.2016).Promotive role of 5-aminolevulinic acid on antioxidative defense system under heavy metal abiotic stress is recently evaluated in B.napus by Ali et al.(2014b).Our study also revealed that the activities of POD, SOD and APX were declined with the successive increase of ZJ0273 concentration; and low concentration of ALA (1 mg L-1) improved the antioxidant activities as compared to relative controls.The findings were consistent with the results of Ali et al.(2013b) who indicated that ALA could enhance the antioxidant activities under abiotic stress.The mitigating effects of ALA on antioxidant machinery were also reported in oilseed rape under herbicide stress (Zhang et al.2008a, b).While, 500 mg L-1ZJ0273 treatment for 72 h led to the death of plants that might be due to mal-functioning of antioxidant machinery due to uncontrolled ROS production (Tables 1-8).These findings are consistent with the results of Zhang et al.(2008a, b).Thus, it can be suggested that the activities of antioxidant enzymes could protect thylakoids against the potentially cytotoxic species of activated oxygen under herbicide stress.

Due to complementary relation between the structure and function of plants, the effect of ALA on cellular organization is important for understanding the physiological alterations.Electron microscopy helps us to assess the damage at the tissue and ultrastructural levels providing basis for macroscopic examination (Najeeb et al.2011; Ali et al.2013a, b).In the present investigation, the ultrastructural observations showed that the structures of chloroplast, mitochondria and nucleus had significant differences at combined treatment of 100 mg L-1ALA and 500 mg L-1ZJ0273 as compared with the control or singly applied with 100 mg L-1ALA or 500 mg L-1ZJ0273 treatments (Figs.1-3).Chloroplast was highly susceptible to oxidative stress caused by elevated oxygen levels, electron flux, and the presence of metal ions in their micro environment (Zhang et al.2003; Najeeb et al.2011).Thylakoid swelling along with lipid droplets is a general symptom of different stresses (Holopainen et al.1992).Formation of plastoglobuli is linked with the break-down of thylakoids (Inada et al.1998).Damage to chloroplast was the result of herbicide-induced oxidative stress.Herbicide stress was reported to change the morphology of chloroplast, increase the number and size of starch grains as well as plastoglobuli in plants (Zhang et al.2003; Hajri et al.2016).These findings are similar to our results that the increased plastoglobuli in chloroplast is due to the herbicide stress.A reduction in the number of plastoglobuli in the chloroplast of plants treated with ALA and ZJ0273 indicated that ALA might play a role in alleviating the toxic effects of ZJ0273 in B.napus.These results are in agreement with the findings of Ali et al.(2013a, b) who found that ALA ameliorated ultrastructural changes under cadmium stress in B.napus.Moreover, we found significantly improved mitochondria and nucleus structures with the application of ALA alone or in combination with herbicide ZJ0273.The improved cell structure might be due to the application of ALA which reduced lipid peroxidation of thylakoids and cell membranes induced by the antioxidant system (Zhang et al.2008a, b; Ali et al.2013b).Mitigated chloroplast, mitochondria and nucleus structures in seedling treated with ALA alone or in combination with ZJ0273 could be the indication of less oxidative stress.The results were in consistent with Naeem et al.(2012) who demonstrated ALA could recover the chloroplast structure in B.napus under salinity stress.Thus, the alleviation of ALA under the ZJ0273 stress was reflected by mitigating structures of chloroplast, mitochondria and nucleus.

The protein degradation and free amino acid accumulation are important indexes of plant tissue and cell senescence (Xu et al.2015).Proteins in mature tissues are relatively constant turn around, so the steady state of cellular components is due to the balance system of synthesis and decomposition (Huffaker and Peterson 1974; Bray 1988).Our present study showed that soluble protein contents were significantly increased after treated with 500 mg L-1ZJ0273 alone, combined application of 100 mg L-1ALA and 500 mg L-1ZJ0273 declined the content gently as compared to the ZJ0273 treatment alone.Therefore, the proteins contents and components were changed as compared to the control.Recently, Ali et al.(2015) also investigated the regulation of cadmium-induced proteomic changes by ALA in the leaves of B.napus.Previously, Li et al.(1998) reported that the syntheses of normal protein often inhibited under various stresses such as herbicide, drought, salt, pollutant,germ infection and so on.The increased protein content in plants under stress could be used to evaluate plant relative resistance (Ren et al.2000).

Under the stress of herbicide, some degradation protein would be detected in the soluble proteins (Xu et al.2015).Our research also investigated that the soluble proteins of B.napus seedlings increased obviously when exposed to 500 mg L-1ZJ0273 alone as compared to the control.However, the extra application of ALA under herbicide stress could alleviate the soluble protein content gently and the main location changed was focused at the molecular weight of 20 kD.Changes in protein profiles induced by ALA and herbicide application might be due to that translation of the mRNAs was inhibited or stimulated by herbicide induced toxicity or it was due to regulation of mRNA transcription(Zhang et al.2013), or may be related to the reactive oxygen scavenging system or systemic acquired resistance.

Moreover, the investigation of metabolic pathways of plant growth regulator was required for better understanding the effects of ALA on B.napus seedlings under stress conditions (Yang et al.2012; Ali et al.2013b).Overall, in our study we investigated the potential benefits of exogenous ALA for reducing the adverse effects of herbicide stress in B.napus plants.Thus, we can assume that extra ALA could promote the growth of plants under herbicide stress,leading to a reduction of damage to antioxidant systems and cell ultrastructure.

5.Conclusion

In the present study, we found negative effects induced by ZJ0273 on the growth of oilseed seedlings.Plant growth and antioxidant attributes were inhibited obviously under ZJ0273 stress and the rate of decrease was consistently enhanced with the increase of ZJ0273 concentration.Further, application of ZJ0273 (500 mg L-1) produced significant ultrastructural disorders in the leaves of B.napus and increased soluble protein contents in the leaves.However, exogenous application of ALA dramatically improved B.napus seedlings growth through mitigating all the physiological, ultrastructural and proteomic changes under herbicide stress.This study provides new information on physiological and biochemical bases of ALA alleviated the herbicide toxicity on B.napus.And also shed light on molecular mechanisms involved in ALA-induced herbicide tolerance in B.napus leaves and suggested a more active involvement of ALA in plant physiological and proteomic processes.

Acknowledgements

This work was supported by the Science and Technology Department of Zhejiang Province, China (2016C02050-8,2016C32089), the Special Fund for Agro-scientific Research in the Public Interest, China (201303022), the Jiangsu Collaborative Innovation Center for Modern Crop Production,China, the Zhejiang Provincial Top Key Discipline of Biology,China, and the Zhejiang Provincial Open Foundation, China(2014C03, 2016D11).

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