Reduced square Bacillus thuringiensis insecticidal protein content of transgenic cotton under N deficit

CHEN Yuan,LIU Zhen-yu,Leila I.M.TAMBEL,ZHANG Xiang,CHEN Yuan,CHEN De-hua

1 Jiangsu Key Laboratory of Crop Genetics and Physiology/Jiangsu Key Laboratory of Crop Cultivation and Physiology/Agricultural College,Yangzhou University/Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops,Yangzhou University,Yangzhou 225009,P.R.China

2 Agricultural Research Cooperation,Biotechnology and Biosafety Research Center,Khartoum 999129,Sudan

Abstract To clarify the effect of the N deficit on the amount of square Bt insecticidal protein,different N application rates (0,75,150,225,and 300 kg ha-1) were imposed on the conventional cultivar Sikang 1 (SK-1) and hybrid cultivar Sikang 3 (SK-3)during 2015-2016 cotton growth seasons. Under different N application rates,the square number per plant,square volume and square dry weight reduced when the N rates decreased from conventional rate (300 kg ha-1) to 0 kg ha-1. And the square Bt protein content decreased accordingly. The analysis of N metabolism showed that soluble protein content,GPT and GOT activities decreased,free amino acid,peptidase and protease activities increased under N deficit. Correlation analysis indicated that the reduced Bt protein content under N deficit was related to altered N metabolism. In conclusion,square development and the amount of square Bt toxin both decreased under N deficit,indicating that promoting the square development under appropriate N application rate would also promote the insect resistance during squaring stage.

Keywords:Bt cotton,reproductive organ,insecticidal protein,N deficit,N metabolism

1.Introduction

TheBacillus thuringiensis(Bt) transgenic cotton which produces the Cry1Ac endotoxin could suppress lepidopteran pests (James 2013). Transgenic Bt cotton is currently cultivated on a large scale across the world,but its benefits have been reduced due to the variable insecticidal efficiency under field conditions (Dong and Li 2007; Huanget al.2010; Stone 2011; Qiaoet al.2017). It was reported that environmental factors such as soil salinity,extreme air humidity,water deficit,and extreme temperature all resulted in the reduced amount of the insecticidal protein (Chenet al.2005b,2012,2014; Olsenet al.2005; Shenet al.2010; Zhanget al.2012,2014,2017; Wanget al.2018).Besides the environmental factors,the spatial and temporal differential expression of the endotoxin was also observed in Bt transgenic cotton plants. The quantity of Bt endotoxin was not stable across the growth season and reduced during the growth season,especially from flowering period onwards,and the lowest toxin efficacy was recorded during boll development period (Liet al.2006; Zhanget al.2011).Transgenic cotton leaves exhibited much higher insecticidal protein level related to the reproductive organs,particularly the bolls (Shenet al.2010; Stone 2011). To realize the full transgenic genetic potential,it is important to guarantee the stable expression of Bt endotoxin.

China has established and implemented a light and simplified system (LSC) of sustainable cotton production.LSC refers to the replacement of manual labor with agricultural machinery and equipment,and reduction of cultivation management frequency. Thus,by combining technologies such as precision sowing,simplified pruning,light and simplified fertilization,and plant population control,LSC could reduce labor intensity and promote the sustainable cotton production in China. Light and simplified fertilization,as the key technology used in LSC,requires less N application during cotton production (Donget al.2017). For the Yangtze River Valley,China,the suitable N application for LSC is 225-270 kg ha-1for light and simplified fertilization (Linet al.2013). And based on recent studies,reducing the application rate of nitrogen fertilizer to 180 kg N ha-1would not reduce the final yield (data not published). The N application level was reported to affect the insecticidal efficacy to boll worms (Chenet al.2018,2019). Pettigrew and Adamczyk (2006) found that different N application strategies could influence the level of Bt endotoxin,with 112/56 kg N ha-1split application treatment exhibited considerably higher leaf Bt concentration than 112 kg N ha-1anhydrous,112 kg N ha-1liquid and 56/56 kg N ha-1split N treatments. The Bt toxin levels in plant tissues were associated with the amount of total soluble protein and overall N (Chenet al.2005a; Dong and Li 2007; Wanget al.2018). The varied quantity of Bt insecticidal protein was reported to be related with the altered N metabolism caused by environmental stress,developmental stages and cultural practices (Chenet al.2005b,2012,2017). Thus,it is highly possible that reduced N application rate in the light and simplified cotton cropping system would influence the insect resistance in Bt transgenic cotton production. In our previous study,cotton boll shell and seeds,the primary target of bollworm in boll development stage was studied.Under N deficiency,seed Bt protein content decreased,but shell Bt protein content increased with decreasing N application rates (Chenet al.2018,2019). However,as one of the main targets for bollworm in square development stage,the square was not studied for its insecticidal protein content under N deficiency. Thus,the significant effect of N metabolism on Bt transgenic cotton underscore the importance to better understand the variation in toxin efficiency and the resulting mechanism under N deficit.

Also,the differential insecticidal protein expression between the squares and different boll tissues were compared to study the varied insecticidal expression in different reproductive growth stages under N deficit.

2.Materials and methods

2.1.Experimental materials and design

Field experiments were conducted at Yangzhou University Farm of Jiangsu Province,China (32°30´N,119°25´E),in the Yangtze River Valley region of China,from 2015 to 2016. The experimental site has a sandy loam soil (Typical fluvaquents,Entisols (U.S.taxonomy)) with 22.3 g kg-1organic matter and 110.5,21.6 and 85.6 mg kg-1available N-P-K in 2015,21.9 g kg-1organic matter and 113.7,20.9 and 86.8 mg kg-1available N-P-K in 2016. In our present study,two widely grown Bt transgenic cotton cultivars,Sikang 1 (SK-1,conventional) and Sikang 3 (SK-3,hybrid),were used. The seeds were planted on 3 April in 2015 and 7 April in 2016 in the greenhouse,and seedlings were transplanted to field with the planting density of 27 000 (SK-3) and 37 500 (SK-1) plants ha-1on 15 May in 2015 and 19 May in 2016. A split plot design with three replications was used. The main plot was cultivars (SK-3 and SK-1),while the N rates (0,75,150,225,and 300 kg ha-1) constituted the subplots. Each subplot contained six rows,6 m long spaced 0.9 m apart.

K (120 kg ha-1as KCl) and P (300 kg ha-1as single superphosphate) were used before planting,followed by K (120 kg ha-1as KCl) and P (300 kg ha-1as single superphosphate) at early flowering. N (urea) was used before transplanting,at early flowering and peak flowering with the proportion of 25,18 and 57%,respectively. Other management practices were conducted according to local recommendations unless otherwise indicated.

2.2.Data collection

Data collection was performed in the middle two rows of each subplot. The square Cry1Ac endotoxin contents and N metabolic parameters were assessed at 20,25 and 30 days after square appearance (DASA). Fifteen squares locating at the first and second fruiting positions of the 4th to 6th fruiting branches were collected for further analysis in each subplot. For each parameter,the subsamples per subplot were used.

As described by Chenet al.(1997),Cry1Ac endotoxin content was analyzed by immunological analysis ELISA. We analyzed the the total free amino acid content by ninhydrin assay according to Yemmet al.(1955). The total soluble protein concentration was determined by the Coomassie blue dye-binding assay of Bradford (1976). Seed samples(0.2 g) were homogenized in 5 mL of 0.05 mmol L-1Tris-HCl butter (pH 7.2) and the homogenate was centrifuged at 26 100×g at 4°C for 10 min. The supernatant was used for the analysis of GOT and GPT activities,following the procedure described by Tonhazyet al.(1950). Seed samples (0.8 g) were homogenized at 4°C in 1 mL of β-mercaptoethanol extraction buffer (a mixture of ethylene glycol,sucrose and phenylmethylsulfonyl fluoride,pH 6.8). The supernatant was collected to estimate the square protease acitivity (Vance and Johnson 1979).For peptidase activity measurement,seed samples (0.5 g)were homogenized in 8 mL Tris-HCl buffer (4 mmol L-1DTT,4 mmol L-1EDTA,1% PVP,pH 7.5) at 4°C and measured for the peptidase activity based on Setlow (1975).

2.3.Statistical analysis

Analysis of variance was performed using PROC ANOVA in SAS 9.4. Means were separated using LSD test at the 5% significant level.

3.Results

3.1.Effects of N rates on square development

Square volume increased from 20 to 30 DASA,which indicated an increasing trend during the growth season for both cultivars in 2015 and 2016 (Table 1). The square volume was also affected by N rates,with decreased square volume detected when N rates were reduced from the conventional rate (300 kg ha-1) to the untreated control(0 kg ha-1). At 30 DASA,with the decreasing N rates from 300 to 0 kg ha-1,the square size was decreased by 37.6%for SK-1 and 45.2% for SK-3 in 2015,and 57.0% for SK-1 and 54.0% for SK-3 in 2016. The largest difference was observed at 20 DASA,with square volume at high N rate(300 kg ha-1) treatment exhibited 56.5% higher relate to the untreated control for SK-1,and 56.4% for SK-3 in 2015.

Similar trend was detected in square dry weight and square number per plant. Both of the square growth parameters increased during the growth season,and they both reduced when N rates decreased from 300 to 0 kg ha-1. And this trend was observed in both cultivars (Table 2).Multiple comparisons showed that there were significant differences in square dry weight and square number per plant among different N rates. The greatest reduction of square number due to N deficit were detected at 20 DASA.the N rates reached less than 3/4 of the conventional rate,the square dry weight and square number per plant reduced dramatically. At 30 DASA,the 225,150,75,and 0 kg N ha-1decreased the square dry weight by 8.3,12.5,20.8,and 33.3% in SK-1,compared with the conventional rate,and SK-3 was reduced by 11.5,19.2,36.9,and 38.5%,respectively. The 225,150,75,and 0 kg N ha-1decreased the square number by 23.8,32.5,49.7,and 50% in SK-1,compared with the conventional rate,and SK-3 was reduced by 19.5,39.5,52.7,and 53.9%,respectively.

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3.2.Square Bt toxin content under different N rates

Bt protein concentration in the square was significantly affected by N rates,with reduced Bt protein concentration observed under low N rates (Table 3). The Bt protein content in the square was the highest under the conventional N rate,and the lowest under the untreated control. The 225,150,75,and 0 kg N ha-1decreased the square Bt toxin amount by 7.5,13.8,22.2,and 30.0% in SK-1,relate to the conventionalrate,and SK-3 was reduced by 10.1,19.5,26.4,and 30.6%respectively at 30 DASA in 2015. In 2016,the 225,150,75,and 0 kg N ha-1reduced the contents of square Bt protein by 5.5,11.0,16.4,and 20.6% in SK-1,compared with the conventional rate,and hybrid SK-3 was reduced by 5.5,9.9,15.4,and 20.7% respectively at 30 DASA.

Table 1 The effect of the N rates on square volume (cm3) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3) at different square development stages in 2015 and 20161)

Table 2 The effect of the N rates on square dry weight and square number per plant of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3) at different square development stages in 20161)

Table 3 The effect of the N rates on square Bt protein contents (ng g-1 FW) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3(SK-3) at different square development stages in 2015 and 20161)

3.3.Relationship between square Bt insecticidal protein content and boll Bt insecticidal protein content under N deficit

The correlation analysis showed that there was significant positive correlation between seed Bt protein contents and square Bt toxin contents (R=0.903＊＊),but significant negative correlation between shell Bt protein contents and square Bt protein contents (R=-0.843＊＊) (Fig.1). The results indicated that decreased N rates reduced square Bt protein concentration and seed Bt protein content,but increased shell Bt protein content.

Fig.1 Correlations between square Bt protein content at 30 days after square appearance (DASA) vs.seed Bt protein content at 20 DAF (empty circle) and square Bt protein content at 30 DASA vs.shell Bt protein content at 20 DAF (solid circle)for Sikang 3 and Sikang 1 Bt cotton cultivars under different N rates in 2015 and 2016. ＊＊,significance at 0.01 level.

3.4.Effects of N rates on square N metabolism

The amount of square soluble protein reduced considerably with the decrease of the N rates from 300 to 0 kg ha-1(Table 4). The lowest square soluble protein content was observed under the untreated control. In 2015,for SK-1,the 0 kg N ha-1treatment exhibited the 75.0,73.9 and 75.9%decrease at 20,25 and 30 DASA,respectively,compared to 300 kg N ha-1. For SK-3,the soluble protein content was reduced by 73.3,66.7 and 71.6% at 20,25 and 30 DASA,respectively. In 2016,for SK-1,the untreated control treatment reduced the soluble protein content by 51.6,37.4 and 36.2% at 20,25 and 30 DASA,respectively,compared to that of the conventional rate. For SK-3,the decrease of soluble protein content was 46.1,41.6 and 41.4% at 20,25 and 30 DASA,respectively.

With the decrease of N rates from the conventional rate to the untreated control,the square free amino acid contents increased,and the greatest incline of square free amino acid content was detected at 20 DASA (Table 5).In 2015,for SK-1,the free amino acid content under the untreated control was elevated by 234.7,63.3 and 73.1%at 20,25 and 30 DASA,respectively,compared to that of conventional rate. For SK-3,the free amino acid content was improved by 134.4,87.1 and 84.7% at 20,25 and 30 DASA respectively. In 2016,for SK-1,the untreated control elevated the free amino acid content by 245.3,94.4 and 67.2% at 20,25 and 30 DASA,respectively,compared to that of conventional rate. For SK-3,the enhancement of free amino acid content was 174.1,78.8 and 58.1%at 20,25 and 30 DASA,respectively. Greater elevation of free amino acid under N deficit was detected in SK-1 related to SK-3.

Similar trend of the square GPT and GOT activities was observed with decreasing N level in both cultivars(Tables 6 and 7). The square GPT and GOT activities both reduced significantly when the N rates decreased from 300 to 0 kg ha-1,exhibiting the lowest activities under the untreated control. In 2015,for SK-1,the GOT activity was decreased by 64.7,67.9 and 64.5% at 20,25 and 30 DASA,respectively,compared to that of conventional rate.For SK-3,the GOT activity exhibited 42.1,60.4 and 62.2%reduction at 20,25 and 30 DASA,respectively. In 2016,for SK-1,the GOT activity was decreased by 19.2,14.8 and 19.0% at 20,25 and 30 DASA,respectively,compared to that of conventional rate. For SK-3,the reduction of GOT activity was 19.0,23.1 and 15.8% at 20,25 and 30 DASA,respectively. Similar trend were observed in GPT activity.

Table 4 The effect of the N rates on square soluble protein content (mg g-1 FW) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3(SK-3) at different square development stages in 2015 and 20161)

Table 6 The effect of the N rates on square glutamic-pyruvic transaminase (GPT) activities (μmol g-1 FW h-1) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3) at different square development stages in 2015 and 20161)

Table 7 The effect of the N rates on square glutamic-oxalacetic transaminase (GOT) activities (μmol g-1 FW h-1) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3) at different square development stages in 2015 and 20161)

The square protease and peptidase activities both increased with decreasing N rates for both cultivars in 2015 and 2016 (Tables 8 and 9). In 2015,for SK-1,the square protease activity under conventional rate was elevated by 86.4,53.2 and 85.1% at 20,25 and 30 DASA,respectively,compared to that of the untreated control. For SK-3,the square protease at the highest N rate treatment exhibited 92.5,92.7 and 77.6% increase at 20,25 and 30 DASA,respectively. In 2016,for SK-1,the conventional rate treatment increased the square protease activity by 50.0,60.0 and 77.7% at 20,25 and 30 DASA,respectively,compared to that of the untreated control. For SK-3,the square protease at highest N rate was increased by 106.8,103.3 and 101.2% at 20,25 and 30 DASA,respectively.Similar trend was observed in GPT activity.

3.5.The relationship between square Bt protein content with N metabolism parameters

The correlation analysis showed that Bt square protein content was positively correlated with soluble protein content,GPT and GOT activities,but negatively correlated with free amino acid content,protease and peptidase activities (Table 10). The results suggested that square Bt protein content was affected by N metabolism under different N application rates.

4.Discussion

4.1.N deficit decreased square development and the amount of Bt toxin

In our present study,decreased N application reduced square development and square Bt protein content. This result was in agreement with previous research which indicated positive correlation of square development and square Bt protein content (Wanget al.2009). Squares fromleaf cut treated plants,with reduced square size,had lower quantity of insecticidal protein relate to untreated control plants. In addition,square removal resulted in bigger square and higher insecticidal protein amount (Zhouet al.2019).Moreover,high planting density led to smaller square and lower insecticidal protein content. In contrast,square from plants with lower planting density were bigger and exhibited higher insecticidal protein content (Chenet al.2017). This result suggested that the square development parameters and square Bt protein content was positively correlated,with larger square exhibited higher amount of insecticidal protein. The study of the relationships between the square toxin level and square size provides fundamental information for the development of an efficient pest management strategy. Data concerning toxin levels in the different square sizes in conjunction with varieties,the square number and physiological data can be used to determine the toxin thresholds of different square sizes under different conditions (variety,cultural and pest management),which provided an accurate picture for the development of an efficient pest management strategy.

Table 8 The effect of the N rates on square protease (mg g-1 FW h-1) of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3)at different square development stages in 2015 and 20161)

Table 9 The effect of the N rates on square peptidase (μmol g-1 FW h-1) activities of Bt transgenic cotton Sikang 1 (SK-1) and Sikang 3 (SK-3) at different square development stages in 2015 and 2016

Table 10 The correlation of square Bt protein content and N metabolism parameters1)

Light and simplified fertilization,as the key technology used in LSC,requires less N application during cotton production. For the Yangtze River Valley,the suitable N application for LSC is 225-270 kg ha-1in light and simplified fertilization system (Linet al.2013). And based on recent studies,reducing the application rate of nitrogen fertilizer to 180 kg N ha-1would not reduce the final yield (data not published). Thus,decreased N application rate in light and simplified fertilization system would affect the Bt protein content in squares of Bt transgenic cotton,and it is important to adopt other cultural practice and technologies to increase the insect resistance under reduced N application rate in light and simplified fertilization system.

To further investigate the corresponding mechanism of decreased square Bt protein content under N deficit,the N metabolism which was closely related to protein content in plant tissues was studied in this process. Our results showed decreased soluble protein content,GPT and GOT activities,but elevated free amino acid,protease and peptidase activities under N deficit,indicating increased protein degradation and reduced protein synthesis in this process. Thus,the reduced Bt protein content was related with the altered N metabolism under N deficit.Previous researches also showed that the insecticidal protein content was affected by N metabolism (Chenet al.2005a,2012,2017; Zhanget al.2017; Wanget al.2018).

4.2.Big square would increase insect resistance

The finding that big square cultivars exhibited higher Bt toxin content has important implications in Bt technology utilization in cotton production. Big-square Bt cotton cultivars are preferred by farmers in China due to low abscission and larger bolls,since big-square cultivars can be planted in lower density without yield reduction(Donget al.2006; Daiet al.2015). Cultural practices affected both square development and insect resistance(Chenet al.2005b; Wanget al.2009; Zhouet al.2019).The cultural practices that enhanced square development(square removal) increased Bt toxin content,but cultural practice that decreased square development (leaf cut)decreased Bt toxin content (Chenet al.2017). The plant growth retardant DPC could increase square size and the content of insecticidal protein in square development period (Zhanget al.2010). Thus,cultural practices that could promote square development should be developed and adopted,which would promote the square insectresistance.

Square Bt protein content in square was higher than that in boll shell and seed,which suggested that Bt protein content in reproductive organs decreased as the cotton plants mature. This result was consistent with previous study (Liet al.2006; Zhanget al.2011,2014). Since boll development period is critical for yield formation,the decreased insect resistance and thus more vulnerable bolls to pests in later reproductive growth stage would affect the final yield. Thus,it is important to increase the insect resistance of reproductive organs in boll development period in cotton production.

5.Conclusion

Under different N application rates,the square number per plant,square volume and square dry weight reduced when the N rates decreased from conventional rate (300 kg ha-1) to the untreated control (0 kg ha-1). And the square Bt protein content decreased accordingly. In addition,the square Bt protein content was positively correlated with seed Bt protein content,but was negatively correlated with shell Bt protein content. The analysis of N metabolism showed that soluble protein content,GPT and GOT activities decreased,free amino acid,peptidase and protease activities increased under N deficit. The reduced Bt protein content under N deficit was related to altered N metabolism. These results suggested that promoting the square development under appropriate N application rate would promote the insect resistance during squaring stage.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31901462 and 31671613),the National Key Research and Development Program of China(2018YFD0100406 and 2018YFD1000907),the Natural Science Foundation of the Jiangsu Higher Education Institutions,China (18KJB210013 and 17KJA210003),and the Natural Science Foundation of Jiangsu Province,China(BK20191439).