Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

Enhancing boll protein synthesis and carbohydrate conversion by the application of exogenous amino acids at the peak flowering stage increased the boll Bt toxin concentration and lint yield in cotton

In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments(i.e., CK, the untreated control;LA1, five amino acids;LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum(i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3%in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase(GPT)activity, glutamate oxaloacetate transaminase(GOT) activity, glucose content, fructose content and soluble acid invertase(SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering(DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.

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

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.

Effects of plant density and mepiquat chloride application on cotton boll setting in wheat–cotton double cropping system

Sowing cotton directly after harvesting wheat in the Yangtze River Valley of China requires early mature of cotton without yield reduction.Boll-setting period synchronisation and more yield bolls distributed at the upper and middle canopy layers are also required for harvesting.The objective of this study is to quantify the individual and interaction effects of plant density and plant growth regulator mepiquat chloride(MC)on temporal and spatial distributions of yield bolls,as well as yield and yield components.During the 2013–2016 cotton growing seasons,the experiments were conducted on a shortseason cotton cultivar CRRI50 at Yangzhou University,China.Various combinations of plant density(12.0,13.5 and 15.0 plants m^(–2))and MC dose(180,270 and 360 g ha^(–1))were applied on cotton plants.The combination of 13.5 plants m^(–2)and 270 g ha^(–1)MC resulted in the greatest boll number per unit area,the highest daily boll setting number and more than 90%of bolls positioned within 45–80 cm above the ground.In conclusion,appropriate MC dose in combination of high plant density could synchronize boll-setting period and retain more bolls at the upper and middle canopy layers without yield reduction in the system of direct-seeded cotton after wheat harvest,and thus overcome the labor-intensive problem in current transplanting cropping system.

Amino acids application enhances flowers insecticidal protein content in Bt cotton

Background:Low insecticidal protein expression at reproductive organs affect insect resistance in Bt transgenic cotton.In order to enhance flower insecticidal protein expression,the conventional cultivar Sikang1(S1)and the hybrid cultivar Sikang3(S3)were used as experimental materials;the applications of selected 5 types of amino acids and 21 types of amino acids were sprayed on the flowers in 2016 and 2017 cotton growing seasons.Results:The flower Bt protein contents increased significantly under the two amino acid treatments in both cultivars,the Bt protein concentration increased by 15.2 to 25.8%compared with the control.However,no significant differences were detected between the two treatments of amino acid application.Increased amino acid and soluble protein contents,enhanced GPT,GOT,protease,and peptidase activities were observed under the amino acid application at the flowering stage.Conclusions:These results suggest that exterior application of the amino acids treatments could bolster the flower insecticidal protein expression.

High plant density increases seed Bt endotoxin content in Bt transgenic cotton

Plant density is the cultivation practice usually employed to manipulate boll distribution, boll setting and yield in cotton production. In order to determine the effect of plant density on the insecticidal protein content of Bacillus thuringiensis(Bt) cotton plants, a study was conducted in Yangzhou University of China in 2015 and 2016. Five plant densities(PD1–PD5, representing 15 000, 30 000, 45 000, 60 000, and 75 000 plants ha^(–1)) were imposed on two Bt cotton cultivars, Sikang 1(the conventional cultivar, SK-1) and Sikang 3(the hybrid cultivar, SK-3). The boll number per plant, boll weight and boll volume all decreased as plant density increased. As plant density increased from 15 000 to 75 000 plants ha–1, seed Bt protein content increased, with increases of 66.5% in SK-1 and 53.4% in SK-3 at 40 days after flowering(DAF) in 2015, and 36.8% in SK-1 and 38.6% in SK-3 in 2016. Nitrogen(N) metabolism was investigated to uncover the potential mechanism. The analysis of N metabolism showed enhanced soluble protein content, glutamic-pyruvic transaminase(GPT) and glutamate oxaloacetate transaminase(GOT) activities, but reduced free amino acid content, and protease and peptidase activities with increasing plant density. At 20 DAF, the seed Bt toxin amount was positively correlated with soluble protein level, with correlation coefficients of 0.825** in SK-1 and 0.926** in SK-3 in 2015, and 0.955** in SK-1 and 0.965** in SK-3 in 2016. In contrast, the seed Bt protein level was negatively correlated with free amino acid content, with correlation coefficients of –0.983** in SK-1 and –0.974** in SK-3 in 2015, and –0.996** in SK-1 and –0.986** in SK-3 in 2016. To further confirm the relationship of Bt protein content and N metabolism, the Bt protein content was found to be positively correlated with the activities of GPT and GOT, but negatively correlated with the activities of protease and peptidase. In conclusion, our present study indicated that high plant density elevated the amount of seed Bt protein, and this increase was associated with decreased boll number per plant, boll weight and boll volume. In addition, altered N metabolism also contributed to the increased Bt protein content under high plant density.

Increasing plant density increases Bt toxin concentration of boll wall in cotton by decreasing boll setting speed

Background: In order to uncover the mechanism of significantly reduced insect resistance at the late developmental stage in cotton(Gossypium hirsutum L.),the relationship between boll setting rate under different planting densities and Bacillus thuringiensis(Bt)insecticidal concentrations in the boll wall were investigated in the present study.Two studies were arranged at Yangzhou,China during the 2017–2018 cotton growth seasons.Five planting densities(15000,25000,45000,60000 and 75000 plants per hectare)and the flower-removal treatment were imposed separately on Bt cotton cultivar Sikang3 to arrange different boll setting rates,and the boll setting rates and Bt toxin content were compared.Results: Higher boll setting rate together with lower Bt toxin contents in boll wall was observed under low planting density,whereas lower boll setting rate and higher Bt toxin contents were found under high planting density.Also,higher Bt protein concentration was associated with higher soluble protein content,glutamic-pyruvic transaminase(GPT),and glutamic oxaloacetate transaminase(GOT)activities,but lower amino acid content,and protease and peptidase activities.It was further confirmed that a higher boll setting rate with lower Bt protein content under flower-removal.Conclusions: This study demonstrated that the insecticidal efficacy of boll walls was significantly impacted by boll formation.Reduced protein synthesis and enhanced protein degradation were related to the reduced Bt toxin concentration.