Introduction

Bt transgenic cotton have been planted widely in China and other cotton production areas in the world (Clive 2012; Huang et al. 2010). The production of Bacillus thuringiensis (Bt) transgenic cotton decreased environmental pollution, increased worker safety by reduced chemical use, and enhanced grower income (Gould 1988; Gasser and Fraley 1989; Huang et al. 2010). The Bt cotton can encode the CryIAc protein to control the harm of Helicoverpa amigera larvae. However, the insecticidal activity is unstable, variation of insect efficiency due to altered CryIAc expression has been related to the extreme environmental factors, the silence or switch off of introduced gene, and/or developmental stage (Xia and Guo 2004; Wang et al. 2009; Chen et al. 2012a, b). However, the insect resistance expression was different in various organs and at different growth stages during a cotton growth season (Greenplate et al. 2000; Glenn 2011). The square, flower and boll usually had lower Bt toxin content than the leaf (Adamczyk and Meredith 2004; Shen et al. 2010), and the lowest Bt insect resistance was observed during flowering and boll formation stage in cotton growth season (Chen et al. 2005a, b; Chen et al. 2012a, b). Our previous studies found that cultivars and leaf-square regulation affected boll size, which contributed to changed Bt toxin protein content (Wang et al. 2009). Our previous studies also observed that the Bt insecticidal efficacies of square and boll were associated with nitrogen metabolism, and the Bt toxin content was impacted by protein synthesis and degradation process (Zhang et al. 2007; Chen et al. 2017). These results suggested that nitrogen and amino acid can influence Bt toxin content in Bt cotton, and exterior application of nitrogen fertilizer proved that nitrogen could increase insecticidal efficacy of Bt cotton. But little is known about the effect of amino acids application on the Bt content in Bt cotton, especially for the Bt protein content of reproductive organ. The flower is one of the first chosen reproductive organ harmed by boll worm, in order to uncover the mechanism of the impact of amino acids on insect resistance of flowers, it is necessary to study the effect of amino acids application on Bt toxin content of the flowers and the related mechanism. The current study tested the effect of amino acids application on the leaf insecticidal protein concentration during flowering period.

Materials and methods

Materials and experimental design

Field experiments were carried out at Yangzhou University Farm, Jiangsu Province, China (32°30′N, 119°25′E) in 2016–2017. S1 and S3, which are two widely grown Bt cotton cultivars in China, were used in this study with the planting density of 27 000 (S3) and 37 500 (S1) plants per hectare. Seeds were sown on April 3rd (2016) and April 7th (2017) in a plastic cover lilliputian greenhouse. Seedlings were transplanted to the field on May 15th (2016) and May 19th (2017). The soil [sandy loam texture (Typical fluvaquents, Entisols (U.S. taxonomy))] contained 22.5 and 22.1 g·kg− 1 organic matter and 110.5 and 113.7, 21.6 and 20.9, 85.6 and 86.8 mg·kg− 1 available N-P-K in 2016 and 2017, respectively. Cultivation practices, including application of fertilizers and insecticides, chemical plant growth retardant DPC (1,1-dimethyl piperidinium chloride, C7H16CIN) spray, and irrigation, were carried out following local recommendations.

Before planting, K (120 kg·hm− 2 as KCl) and P (300 kg·hm− 2 as single superphosphate) were applied. At early flowering, K (120 kg·hm− 2 as KCl) and P (300 kg·hm− 2 as single superphosphate) were top-dressed. N (urea) was applied before transplanting (25%), at early flowering (18%), and at peak flowering (57%). Three hundred kg·hm− 2 is the nitrogen fertilization dose in the experiments.

The experiment was arranged with split plot designs. The main plot treatment was cultivars (S1 and S3), and the subplot treatment consisted of three amino acids treatments, which consisted of 0 (CK), 5 (A1), and 21 (A2) types of amino acids, respectively; the applied amino acid concentration was 20 mg·kg− 1. The selected five kinds of amino acids were aspartic acid, glutamic acid, proline, methionine, arginin, which affected Bt protein content remarkably based on the previous studied results (Abidallha et al. 2017). The selected 21 kinds of amino acids were aspartic acid, glutamic acid, proline, methionine, arginin, glycine, tyrosine, phenylalanine, histidine, serine, threonine, alanine, cysteine, valine, isoleucine, leucine, lysine, tryptophan, asparagine, ornithine, and glutamine. The solutions of the treatment were sprayed on the flower at 8 days before opening. And the flowers were sampled for analysis on the same day as they opened. Three replications were used in the field. Each plot consisted of 6 m length with rows spaced 0.9 m apart.

Preparation of plant material

Sampling

Five flowers were harvested from the first position of the fourth to sixth fruiting branches. The flowers were mixed thoroughly before subsampling. Three subsamples of flower (0.2 g FW) per each plot were used to determine the following parameters.

The cry IAc protein content

Immunological analysis ELISA was used to test the CryIAc content in the flower extracts as described by Chen et al. (1997).

Free amino acid and soluble protein content

Based on Yemm et al. (1955), the total free amino acid content was measured by ninhydrin assay. The Coomassie Blue dye-binding Assay of Bradford was used for total soluble protein content determination (Bradford 1976).

Glutamic-pyruvic transaminase (GPT) and glutamate oxaloacetate transaminase (GOT)

Activity flowers (0.2 g FW) were homogenized in 0.05 mmol·L− 1 Tris-HCl, pH 7.2 buffer. The supernatant was collected after centrifugation at 26 100 g for 10 min at 4 °C. For GOT activity assay, 0.2 mL of the supernatant was added to a mixture containing 0.5 mL of 0.8 mol·L− 1 alanine in 0.1 mol·L− 1 Tris-HCl (pH 7.5), 0.1 mL of 2 mmol·L− 1 pyriodoxal phosphate solution, and 0.2 mL of 0.1 mol·L− 1 2-oxoglutarate solution. The reaction mixture was incubated at 37 °C for 10 min followed by adding 0.1 mL of a 0.2 mol·L− 1 trichloroacetic acid solution to stop the reaction. The color intensity was read at 520 nm. The GPT activity assay was similar to the GOT assay. In GPT assay, 0.5 mL of a 0.1 mol·L− 1 buffered aspartate solution in the reaction mixture was used instead of 0.5 mL of a 0.8 mol·L− 1 alanine in 0.1 mol·L− 1 Tris-HCl (pH 7.5) (Tonhazy et al. 1950).

Protease and peptidase activity

Flowers (0.8 g) were homogenized at 4 °C in 1 mL of β-mercaptoethanol extraction buffer (a mixture of ethylene glycol, sucrose, and phenyl methyl sulfonyl fluoride, pH 6.8). The supernatant was collected to estimate the square protease. Protease activity was determined spectrophotometrically at 400 nm using azocasein as a substrate (Vance and Johnson 1979) and expressed as mg protein·g− 1 flower fresh weight (FW)·h− 1. Flowers samples (0.5 g) were homogenized at 4 °C in 8 mL of Tris-HCl extraction buffer (a mixture of 4 mmol·L− 1 DTT, 4 mmol·L− 1 EDTA, 1% PVP, pH 7.5). The supernatant (0.4 mL) was collected by centrifugation at 15 000 g for 30 min at 4 °C and added to a mixture [0.4 mL acetate buffer (pH 4.8), 1% bovine hemoglobin compounded with 0.2 mL acetate buffer (pH 4.8)] and incubated at 38 °C for 60 min. One mL of a 10% trichloroacetic acid solution was added to stop the reaction. The supernatant collected by centrifugation (4 000 g for 5 min) was used for amino acid content analysis by ninhydrin assay (Yemm et al. 1955), and peptidase activity was expressed as μmol amino acid·g− 1 flower fresh weight·h− 1.

Results

Flower insecticidal protein concentration under the amino acids application treatments

Similar trends were observed for flower Bt protein content under different amino acids application treatments in both years. In comparison with the control, the flower Bt protein contents increased significantly under the two amino acid treatments in both cultivars (Fig. 1). However, no significant differences were detected between the two treatments of amino acid application. In 2016, the increase caused by treatments A1 and A2 on flowers insecticidal protein contents were 22.7 and 25.3% in S1 and 22.9 and 25.8% in S3. In 2017, amino acids application treatments A1 and A2 increased the flower Bt protein contents by 15.2 and 18.8% in S1 and by 16.4 and 19.1% in S3. Cultivar S3 had higher flower Bt protein content than that of cultivar S1.

Fig. 1
figure 1

The effect of application of 5 amino acids and 21 amino acids on flower Bt protein contents at flowering period in Bt cotton Sikang1(S1) and Sikang3 (S3). A1, A2, CK represented 5 amino acids treatment, 21 amino acids treatment, and the control, respectively. Differences between treatments within the same year labeled by the same letter are statistically not significant (LSD test at 0.05 significance level)

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Flower nitrogen metabolism under the amino acids application treatments

GPT and GOT, the key enzymes in amino acid synthesis, their activities increased remarkably under the amino acid application treatments in both years (Table 1). Compared with the control, the increase caused by amino acids application treatments A1 and A2 on flower GOT activity was 31.1 and 34.6% in Sikang1 and 40.3 and 51.4% in Sikang3 in 2016. In 2017, amino acids application treatments A1 and A2 increased the flower GOT activity by 25.0 and 39.0% in Sikang1 and by 28.0 and 34.7% in Sikang3. Similar results for GPT activity were also detected in both cultivars in 2016 and 2017.

Table 1 The effect of application of 5 amino acids and 21 amino acids on flower GOT and GPT activities of the two Bt cotton cultivars at flowering period
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Flower protease activities were increased significantly with increasing amino acids application composition in both years (Table 2). Greater increase was observed at A2 treatment than A1 for both enzyme activities in both years. In 2016, the increase caused by amino acids application treatments A1 and A2 on flowers protease activity were 36.3 and 39.7% in S1 and 38.3 and 56.8% in S3. In 2017, amino acids application treatments A1 and A2 increased the flower protease activity by 58.1 and 29.1% in S1 and by 62.2 and 69.0% in S3. Similar characteristics were observed for flower peptidase activities.

Table 2 The effect of application of 5 amino acids composition and 21 amino acids composition on flower protease and peptidase activities of the two Bt cotton cultivars at flowering period
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Enhanced flowers amino acid and soluble protein content were observed for both years (Table 3). Compared with the control, greater increase for soluble protein content of flower was detected at A2 treatment, and less increase was observed at A1 treatment. The increase caused by amino acids application treatments A1 and A2 on flowers soluble protein content were 68.4 and 73.6% in S1 and 58.5 and 69.9% in S3 in 2016. In 2017, amino acids application treatments A1 and A2 increased the flowers soluble protein content by 37.0 and 64.0% in S1 and by 22.0 and 31.9% in S3. Similar results for flower amino acids were also detected in both cultivars in 2016 and 2017.

Table 3 The effect of application of 5 amino acids and 21 amino acids on flower soluble protein and amino acid content of the two Bt cotton cultivars at flowering period
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Relationship between nitrogen metabolic enzyme activity, chemicals and Bt protein concentration in Bt cotton flowers

There was a significant positive correlation between flower Bt insecticidal protein content with protein metabolism related enzyme activities (Table 4). In addition, flower Bt protein content exhibited a significant positive correlation with amino acid content in 2016 (r = 0.849*) and 2017 (r = 0.874*), and a significant positive correlation with soluble protein content in 2016 (r = 0.839) and 2017 (r = 0.997**). The correlation was highest between Bt contents with protease, followed by Bt contents with soluble protein, and lowest between Bt contents and GOT. Higher correlation was observed in 2017, but no differences were noted between cultivars S1 and S3.

Table 4 Relationship (R value) between nitrogen metabolic enzyme activity, chemicals and Bt protein concentration in Bt cotton flowers
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Discussion

Amino acid application enhanced flower Bt protein concentration in Bt cotton

The extreme environmental conditions, such as high/low temperature, high/low humidity, water deficit, soil salinity, reduced the Bt toxin content, which was related to altered nitrogen metabolism (Chen et al. 2005a, b, 2013, 2012a, b). In these processes, the content of free amino acid and soluble protein content changed, and they were closely correlated with the Bt toxin content. These studied results suggest that nitrogen and amino acid can influence Bt toxin content in Bt cotton, and the application of nitrogen fertilizer proved that nitrogen could increase insecticidal efficacy of Bt cotton leaves (Yang et al. 2005; Pettigrew and Adamczyk 2006; Dong et al. 2000; Zhang and Wen 2011; Dai et al. 2012; Manjunatha 2015). Improvement of boll shell insecticidal protein by decreasing nitrogen fertilizer rates was reported in Bt cotton (Chen et al. 2018). Since nitrogen fertilizer plays an important role in regulating toxin content in Bt transgenic cotton, amino acid, as the basic components of protein, might impact Bt protein content. In our present study, compared with the control, the flower Bt protein contents increased significantly under both amino acid treatments in both cultivars. However, no significant differences were detected between the two amino acid treatments. These results suggested that amino acid application could enhance flower Bt protein concentration in Bt cotton.

Increased protein synthesis and protein degradation by exterior amino acid application caused elevated Bt toxin content in flower

The amino acid application enhanced soluble protein content, amino acid content, protease and peptidase activities, GPT and GOT activities. It is evident that protein degradation and synthesis were increased remarkably in flower under amino acid application, as reflected by enhanced protease and peptidase activities, and GPT and GOT activities. Thus, the enhanced protein metabolism contributed to the increased protein concentration. As a part of the total soluble protein, Bt protein in flower also increased under amino acid application. In our present study, flower Bt protein content had a significant positive correlation with amino acid content and soluble protein content. Our results were consistent with previous studies. The reduced insecticidal protein concentration under extreme environmental conditions, such as high/low temperature, high/low humidity, water deficit, soil salinity, was all related to altered nitrogen metabolism (Chen et al. 2005a, b, 2013, 2012a, b). Therefore, GPT and GOT activity, and the activity of protease and peptidase in nitrogen metabolism were associated with the variation of Bt protein concentration in response to amino acid application in Bt transgenic cotton.

Conclusions

This study showed that exterior application of the amino acids, especially the 21 amino acids application, could bolster the flower insect resistance, which was a result of increased protein metabolism.