Low temperature exposure decreases Cry1Ac insecticidal endotoxin content in cotton seeds

Low temperature exposure decreases Cry1Ac insecticidal endotoxin content in cotton seeds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Low temperature exposure decreases Cry1Ac insecticidal endotoxin content in cotton seeds Yuan Chen, Shu Dong, Yuyang Dai, Xiang Zhang, Dehua Chen, Yinglong Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4450426/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Aug, 2024 Read the published version in BMC Plant Biology → Version 1 posted 15 You are reading this latest preprint version Abstract Background Sudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020–2021, two Bt transgenic cotton cultivars, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20°C) with different durations (12h, 24h and 48h) were applied during the peak boll-setting period. Results As the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the temperature causing a significant change in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17°C to 19°C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content. Conclusion In summary, when the daily average temperature was ≤ 19°C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production. low-temperature cotton seed Cry1Ac endotoxin protein metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bacillus thuringiensis (Bt) transgenic cotton, featuring the expression of the Cry1Ac endotoxin (Bt protein), represents a revolutionary cotton variety achieved by introducing the Bt gene into cotton tissue cells. It stands as one of the most extensively cultivated and widely distributed genetically modified crops globally [ 1 ]. Bacillus thuringiensis produces a parasporal crystal named δ-endotoxin during its metabolic process, exhibiting toxic effects on various pests, particularly lepidopteran pests like cotton bollworm [ 2 – 4 ]. The introduction of the first genetically modified cotton variety with Bt traits in Xinjiang at the end of the last century brought about significant economic and ecological benefits through large-scale cultivation. The expression of insect-resistant proteins notably inhibits the growth of lepidopteran pests, such as cotton bollworm, reducing their impact on cotton and consequently increasing yield. Simultaneously, it curtails the use of chemical pesticides, alleviating environmental pressures [ 5 – 8 ]. Despite the widespread cultivation of Bt cotton in China, the instability in the expression of Bt cotton's insecticidal protein has emerged as a pressing concern in the current landscape of transgenic cotton in China [ 9 , 10 ]. Cry1Ac endotoxin content gradually decreases throughout the growing season, reaching its lowest concentration during the boll-setting stage [ 11 – 13 ]. Furthermore, Cry1Ac endotoxin content varies across different cotton organs, with higher concentrations in the leaves and lower levels in reproductive organs like squares, flowers, and bolls [ 14 – 16 ]. Adverse environmental conditions, including extreme temperatures, especially low temperatures, can significantly decrease the expression of insecticidal proteins [ 17 – 23 ]. Among these adverse environments, prolonged exposure to low temperatures has been shown to decrease the Cry1Ac endotoxin content in leaves throughout the growth period, with a more pronounced decrease during the boll-setting period [ 24 ]. Field experiments have demonstrated that continuous low temperatures lead to a decrease in Cry1Ac endotoxin content, exacerbating cotton bollworm damage [ 25 ]. Additionally, under low-temperature conditions, the insecticidal protein content in leaves changes early and significantly decreases throughout the entire stress period [ 26 ]. With the shift of China's cotton production regions to Xinjiang, over 80% of cotton production in China now originates from Xinjiang, constituting nearly one-fifth of global cotton production [ 27 ]. Specifically, in 2020, Xinjiang's cotton planting area and output accounted for 79.0% and 87.3% of China's respective values [ 28 ]. Consequently, low-temperature stress occurs more frequently in Chinese cotton production due to the high latitude of Xinjiang, with an increasing trend in the frequency and intensity of extreme low temperatures [ 29 ]. Temperatures below 15°C can impede the growth and development of cotton, and temperatures below 20°C in later stages are detrimental to cotton maturation [ 30 ]. In July and August, the critical boll development stage, Xinjiang often experiences nightly temperatures around 15°C, even consecutive days with average temperatures below 20°C [ 31 ]. On July 9, 2022, Kashgar experienced temperatures as low as 9°C, while in mid-August 2022, Altay encountered a week-long period with average temperatures below 15°C. Due to the occurrence of low temperatures in the reproductive growth stage and the relatively lower expression of insecticidal protein in reproductive organs, it is crucial to explore the changes in insect resistance of Bt cotton in reproductive organs under low-temperature stress. However, prior studies have predominantly focused on leaves under low-temperature stress, and the impact of low temperatures on different components of cotton bolls, such as boll shells, fibers, and seeds, has been scarcely explored. Therefore, this study aimed to simulate low-temperature conditions that may occur in Xinjiang, compare the changes in the expression of Cry1Ac endotoxin in Bt cotton boll components under different degrees and durations of low temperatures, and investigate the associated physiological mechanisms. Results Low temperature occurrence during boll development stage in Xinjiang Summarizing the daily average temperatures from July 1 to August 31 in the past decade (2014–2023), the East Xinjiang Cotton Region generally experienced higher temperatures with a lower threat of low temperatures. In recent years, both the North Xinjiang Cotton Region and the South Xinjiang Cotton Region have faced greater challenges from low temperatures during the boll-setting period, with particular attention needed in the North Xinjiang Cotton Region (Fig. 1 ). Compared to the previous five years (2014–2018), in the last five years (2019–2023), the total number of days with a daily average temperature lower than 20°C increased by 72.73% in Changji Hui Autonomous Prefecture, by 35.56% in Tacheng Prefecture, and by 54.84% in Bortala Mongol Autonomous Prefecture. It is evident that, in recent years, with climate change, the cotton-growing areas of Xinjiang, especially in Northern Xinjiang, have experienced more frequent occurrences of cold weather during the peak boll-setting period of Bt cotton. Consequently, investigating the impact of low temperatures on the Cry1Ac endotoxin content in cotton bolls holds significant importance for cotton security in China and globally. Cry1Ac endotoxin content According to the low-temperature levels and frequency in Xinjiang, we set the low-temperature range from 16 to 20°C and tested the effect of different levels and durations of low temperature on Cry1Ac endotoxin contents in cotton bolls. Under low temperatures, the Cry1Ac endotoxin content in boll shell, fiber, and seed decreased, and with the decrease in temperature, their content showed a decreasing trend (Table 1 ). In 2020, compared with CK (27°C), after 12 hours of stress, the Cry1Ac endotoxin content in SK1 and SK3 boll shell significantly decreased at 16°C-17°C, fiber at 16°C, while the seeds showed no significant difference from CK. After 24 hours of stress, the Cry1Ac endotoxin content in SK1 boll shell, fiber, and seeds significantly decreased at 16°C-18°C, and the Cry1Ac endotoxin content in SK3 boll shell significantly decreased at 16°C-18°C, fiber at 16°C-17°C, and seeds at 16°C-18°C. After 48 hours of stress, the Cry1Ac endotoxin content in SK1 boll shell significantly decreased at 16°C-20°C, fiber at 16°C-18°C, seeds at 16°C-19°C, the Cry1Ac endotoxin content in SK3 boll shell significantly decreased at 16°C-20°C, fiber at 16°C-17°C, and seeds at 16°C-18°C. From the above results, it can be concluded that the temperatures causing a significant decrease in Cry1Ac endotoxin content in boll shell, fiber, and seed increased with the prolonged duration of low-temperature stress. Additionally, with the prolonged duration of stress, the Cry1Ac endotoxin content in boll shell, fiber, and seeds under each low-temperature treatment showed a gradual decrease. Table 1 Effects of varying temperatures and stress durations on the Cry1Ac endotoxin content in cotton boll shell, fiber, and seed (ng g − 1 FW) Years Varieties T Boll shell Fiber Seed 12h 24h 48h 12h 24h 48h 12h 24h 48h 2020 SK-1 27℃ 129.68a 130.08a 130.01a 66.30a 66.89a 66.04a 233.35a 233.2a 232.74a 20℃ 128.53a 124.68a 119.97b 65.26a 63.47ab 63.91a 230.75a 220.96a 213.51a 19℃ 128.76a 124.21a 118.88b 65.18a 62.57ab 63.75a 228.99a 212.33ab 177.17b 18℃ 128.25a 111.41b 107.16c 65.49a 61.11b 54.05b 228.99a 197.89b 161.16bc 17℃ 115.18b 109.90b 95.30d 64.03a 55.77c 53.44b 228.03a 176.70c 143.99cd 16℃ 116.08b 99.08c 94.05d 57.26b 50.10d 47.70c 225.49a 154.64d 124.66d SK-3 27℃ 134.60a 134.10a 133.87a 69.58a 70.03a 69.69a 322.85a 302.19a 305.34a 20℃ 133.96a 129.25a 122.73b 69.16a 67.99a 68.21a 321.58a 291.91a 287.77a 19℃ 133.54a 126.55a 122.87b 69.45a 67.76a 67.28a 321.30a 283.91ab 274.91ab 18℃ 129.79ab 112.71b 107.30c 68.91a 67.33a 61.46ab 320.53a 267.08b 246.54bc 17℃ 125.12b 111.46bc 97.42d 67.41ab 62.09b 55.53bc 318.74a 244.17c 228.19cd 16℃ 125.37b 102.46c 96.14d 62.68b 56.51c 49.00c 319.39a 221.26d 211.74d 2021 SK-1 27℃ 120.43a 120.82a 117.93a 62.12a 61.52a 60.63a 282.68a 281.96a 277.95a 19℃ 118.88a 116.66a 107.88b 61.23a 58.34ab 57.18a 281.67a 270.76a 250.43b 18℃ 118.00a 106.90b 95.74c 61.65a 57.08b 52.00b 278.04a 263.36a 233.06c 17℃ 111.76b 102.46b 84.20d 60.11a 51.99c 50.49b 276.90a 216.74b 200.99d 16℃ 111.35b 90.72c 83.55d 55.99b 47.23d 46.26c 273.06a 202.09b 180.42e SK-3 27℃ 133.10a 132.82a 132.12a 65.12a 65.95a 64.76a 413.65a 404.90a 386.83a 19℃ 132.52a 128.69a 121.17a 64.33a 63.88a 62.42ab 405.66a 385.21ab 370.04a 18℃ 130.73ab 119.00b 107.83b 63.61a 62.99a 56.40bc 406.19a 359.75b 330.47b 17℃ 124.68b 113.90b 95.31c 63.34ab 58.85b 55.44bc 403.82a 320.57c 294.26c 16℃ 124.44b 101.08c 94.73c 59.50b 55.07c 50.57c 400.2a 310.14c 269.08d Note: FW represents fresh weight. Different lowercase letters indicate significant differences between treatments of each variety at P<0.05 in the same year. SK-1 and SK-3 represent cultivars Sikang 1 and Sikang 3, respectively. In 2020, after 12–48 hours of stress at 20°C, Cry1Ac endotoxin content decreased by 0.89%-7.72% in the SK1 boll shell, 1.57%-3.23% in fiber, and 1.11%-8.26% in seeds (Fig. 2 ). Similarly, after 12–48 hours of stress at 19°C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 0.71%-8.56%, 1.69%-6.46%, and 1.87%-23.88%, respectively. This trend continued with 12–48 hours of stress at 18°C, where Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 1.10%-17.58%, 1.22%-18.16%, and 1.87%-30.76%, respectively. The pattern persisted with further decreases in temperature; after 12–48 hours of stress at 17°C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 11.18%-26.70%, 3.42%-19.08%, 2.28%-38.13%, respectively. Similarly, after 12–48 hours of stress at 16°C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 10.49%-27.66%, 13.63%-27.77%, and 3.37%-46.44%, respectively. The comparison of the decrease in Cry1Ac endotoxin content in the cotton boll, fiber, and seeds after the same duration of stress revealed that in 2020, Cry1Ac endotoxin content in SK1 boll shell, fiber, seeds decreased by 0.89% − 10.49%, 1.57% − 13.63%, and 1.11% − 3.67% after 12h of stress at 16–20℃. After 24h of stress, Cry1Ac endotoxin content decreased by 4.15% − 23.83%, 5.11% − 25.10%, and 5.25% − 33.69% in the cotton boll shell, fiber and seeds, respectively. Similar trend was observed with longer stress duration, after 48h of stress, Cry1Ac endotoxin content in the boll shell, fiber and seeds decreased by 7.22% − 27.66%, 3.23% − 27.77%, and 8.26% − 46.44%, respectively. The trend of change in SK3 was similar to SK1. The experimental results in 2021 were similar to those in 2020. Consequently, with decreasing temperature and prolonged stress duration, the Cry1Ac endotoxin content in seeds within cotton bolls exhibited the greatest decline, rendering it the most unstable compared to boll shell and fiber, particularly susceptible to low temperatures. Therefore, this study focused on the seed insecticidal protein level under low temperatures and the nitrogen metabolism to uncover the underlying mechanism. In summary, compared to bolls and fibers, low temperature exhibited a greater impact on the Cry1Ac endotoxin content in cotton seed. After 24h and 48h of stress, the temperatures causing significant changes in Cry1Ac endotoxin content in SK1 seed were 18℃ and 19℃, and for SK3 were 18℃ and 18℃ in 2020; in 2021, for SK1, they were 17℃ and 19℃ respectively, and for SK3 were 18℃ and 18℃ respectively. Therefore, extending the duration of low-temperature stress not only increased the temperature at which Cry1Ac endotoxin content significantly decreased but also further reduced the Cry1Ac endotoxin content. Nitrogen metabolism Soluble protein and amino acid content Under low-temperature treatment, the soluble protein content in seeds decreased, and with the decrease in temperature and extension of stress duration, its content showed a declining trend (Fig. 3 A, Fig. 3 B). In 2020, after 12 hours of stress, the soluble protein content in seeds under low-temperature showed no significant difference compared to CK; after 24 hours of stress, low-temperature 3 significantly decreased the soluble protein content in seeds at 16℃-17℃, with reductions of 23.10%-30.69% and 26.48%-29.60%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly decreased the SK1 soluble protein content in seeds at 16℃-18℃, and the SK3 soluble protein content in seeds at 16℃-19℃, with reductions of 16.32%-33.68% and 14.78%-37.11%, respectively, compared to CK. The experimental results in 2021 were similar to those in 2020. In conclusion, the temperature leading to a significant decrease in soluble protein content in seeds increased with the prolongation of low-temperature stress duration. Under low-temperature treatment, the content of free amino acids in seeds increased, and with the decrease in temperature and prolongation of stress duration, its content showed an upward trend. In 2020, after 12 hours of stress, the free amino acid content in seeds for SK1 and SK3 under 16℃-20℃ treatment showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly increased the free amino acid content in SK1 seeds at 16℃-18℃, and significantly increased the free amino acid content in SK3 seeds at 16℃-17℃, with increases of 9.25%-22.45% and 21.54%-23.70%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly increased the free amino acid content in SK1 seeds at 16℃-19℃, and significantly increased the free amino acid content in SK3 seeds at 16℃-18℃, with increases of 8.08%-29.66% and 11.73%-31.92%, respectively, compared to CK. The experimental results in 2021 were similar to those in 2020. In conclusion, the temperature leading to a significant increase in free amino acid content in seeds increased with the prolongation of low-temperature stress duration. GOT and GPT activity Under low-temperature treatment, the activities of seed GOT and GPT decreased, and with decreasing temperature and prolongation of stress duration, their activities showed a declining trend (Fig. 3 C, Fig. 3 D). In 2020, after 12 hours of stress, the seed GOT activity of SK1 and SK3 under treatment at 16°C-20°C showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly decreased seed GOT activity at 16°C-17°C, with decreases of 25.50%-26.85% and 25.83%-28.13% compared to CK; after 48 hours of stress, low-temperature significantly decreased the SK1 seed GOT activity at 16°C-19°C, and significantly decreased the SK3 seed GOT activity at 16°C-18°C, with decreases of 7.45%-31.45% and 14.75%-32.65% compared to CK. In 2020, after 12 hours of stress, under treatment at 16°C-20°C, the seed GPT activity of SK1 showed no significant difference compared to CK, while low-temperature significantly decreased the SK3 seed GPT activity by 8.96% at 16°C; after 24 hours of stress, low-temperature significantly decreased seed GPT activity at 16°C-18°C, with decreases of 8.31%-23.71% and 11.17%-24.69% in SK1 and SK3 respectively compared to CK; after 48 hours of stress, low-temperature significantly decreased seed GPT activity at 16°C-18°C, with decreases of 11.56%-28.44% and 11.28%-36.34% in SK1 and SK3 compared to CK. The results of the 2021 experiment were similar to those of 2020. In summary, the temperatures leading to a significant decrease in seed GOT and GPT activities increased with the prolonged duration of low-temperature stress. Protease and peptidase activity Under low-temperature treatment, the activities of seed protease and peptidase increased, and with decreasing temperature and prolonged stress duration, their activities showed an upward trend (Fig. 3 E, Fig. 3 F). In 2020, after 12 hours of stress, the seed protease activity of SK1 and SK3 at 16°C-20°C showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly increased seed protease activity at 16°C-17°C, with increases of 24.43%-27.79% and 19.92%-21.85% respectively in SK1 and SK3, compared to CK; after 48 hours of stress, low-temperature significantly increased seed protease activity at 16°C-19°C, with increases of 15.54%-40.00% and 14.40%-32.78% compared to CK. In 2020, after 12 hours of stress, there was no significant difference in cotton seed peptidase activity at 16°C-20°C compared to the control (CK); after 24 hours of stress, low-temperature significantly increased the SK1 seed peptidase activity at 16°C-18°C, and significantly increased the SK3 seed peptidase activity at 16°C-19°C, with increases of 8.63%-16.88% and 4.13%-28.00%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly increased the SK1 seed peptidase activity at 16°C-19°C, and significantly increased the SK3 seed peptidase activity at 16°C-18°C, with increases of 7.61%-31.52% and 11.68%-39.21%, respectively. The results of the 2021 experiment were similar to those of 2020. In summary, the temperatures leading to a significant increase in cotton seed protease and peptidase activity increased with the prolonged duration of low-temperature stress. Correlation analysis and pathway analysis According to Fig. 4 , it can be observed that after 12 hours of low-temperature treatment, Cry1Ac endotoxin content in seeds was not correlated with the soluble protein content and peptidase activity. However, it showed a significant positive correlation with GOT, GPT, and protease activity, and a highly significant negative correlation with free amino acid content. After 24 hours of low-temperature treatment, the Cry1Ac endotoxin content in seeds showed a significant positive correlation with soluble protein content, GOT, and GPT activity, a highly significant negative correlation with free amino acid content, and a negative correlation with protease and peptidase activity, although not significant. After 48 hours of low-temperature treatment, the Cry1Ac endotoxin content in seeds exhibited a highly significant positive correlation with soluble protein content, GOT, and GPT activity, and a significant negative correlation with free amino acid content, as well as protease and peptidase activity. The above results indicated that low temperature primarily reduced protein synthesis capacity, and enhanced protein degradation capability, thereby affecting the Cry1Ac endotoxin content in seeds. Moreover, with the prolonged duration of stress, a more significant correlation was found between Cry1Ac endotoxin content in seeds and the content of key nitrogen metabolism substances and key enzyme activities. Using Cry1Ac endotoxin content (Y) as the dependent variable and SP (X1), AA (X2), GOT (X3), GPT (X4), protease (X5), and peptidase activity (X6) as independent variables, stepwise regression analysis was conducted, and the coefficient of determination of the regression equation was R2 = 0.682 (Table 2 ). This indicated that the selected indicators reflected the main factors influencing the content of Cry1Ac endotoxin in seeds. To further clarify the effects of the indicators determined by stepwise regression on Cry1Ac endotoxin content, pathway analysis was performed on the selected indicators with Cry1Ac endotoxin content (Y) as the dependent variable. The results showed that AA (X2) and peptidase (X6) both had significant negative effects on Cry1Ac endotoxin content. Although peptidase had a direct positive effect on Cry1Ac endotoxin content, its indirect effect was − 1.049 (Fig. 5 ). In summary, the increase in amino acid content and peptidase activity in cotton seeds under low-temperature stress mainly contributed to the decrease in Cry1Ac endotoxin content. Table 2 Results of stepwise regression analysis Dependent variable Significant level The order of stepwise Regression equation Cry1Ac endotoxin content 0.05 X 2 , X 6 Y = 655.956-75.095X 2 + 21.308X 6 Note: X2 represents AA content. X6 represents peptidase activity. Y represents seed Cry1Ac endotoxin content. Discussion The increasingly frequent low-temperature stress reduced the Cry1Ac endotoxin content in cotton bolls, with the greatest impact observed on cotton seeds. Due to the distribution of soil and topographical influences in China, regions like Xinjiang and Inner Mongolia, characterized by vast land and sparse population, have formed fertile and expansive plains suitable for mechanized cultivation of crops. However, the higher latitudes in regions like Xinjiang result in lower temperatures throughout the year. Previous studies have shown that temperatures below 15℃ seriously affect the growth and development of cotton [ 32 ]. Maho et al. found that the survival rate of cotton bollworm larvae was higher under low temperatures (14 ~ 22℃) [ 33 ]. Zhou et al. studied the impact of temperature on the insecticidal activity of expressed insecticidal proteins in cotton plants, finding that at 16℃, the insecticidal activity of Bt cotton was significantly reduced [ 34 ]. Zhang et al. demonstrated that the entire growth period of Bt cotton was affected by low-temperature and high-humidity conditions. The decline in insecticidal protein content in Bt cotton leaves varied with the degree and duration of stress, with a faster decline during the boll development period compared to the flowering period [ 24 ]. Chen et al. demonstrated that low-temperature stress reduced the insecticidal protein content in cotton bolls, and prolonging the duration of low temperatures increased the temperature at which significant changes occur [ 35 ]. Therefore, it can be concluded that low-temperature stress during the cotton growth stages, especially during the boll development period, leads to a decrease in the insect resistance of Bt cotton. Our current study indicated that in recent years, both Southern Xinjiang and Northern Xinjiang cotton regions frequently experience low temperatures during the Bt cotton boll development period. Moreover, low temperatures reduced the Cry1Ac endotoxin content in boll shell, fiber, and seeds of cotton bolls. As the temperature decreased, the Cry1Ac endotoxin content in boll shell, fiber, and seeds all showed a declining trend. Additionally, the temperature at which the Cry1Ac endotoxin content underwent significant changes increased with the prolonged duration of low-temperature stress. Moreover, as the most affected part of bolls by low-temperature stress, after 24h and 48h of low-temperature stress, the Cry1Ac endotoxin content in cotton seeds experienced the greatest decline, reaching up to 33.69% and 46.44% in SK1 and SK3, respectively, with a significant decrease occurring at temperatures between 17℃ and 19℃. The decrease in Cry1Ac endotoxin content would endanger the safety of cotton production in Xinjiang, and thus special attention should be given to low-temperature hazards, and appropriate practices should be applied when the temperature drops below 19℃ during the boll development period. The hybrid variety SK-3 had a smaller decrease compared to the conventional variety SK-1, possibly due to the better stress resistance of the hybrid variety. However, this conclusion needs further verification. Low-temperature stress reduced the protein synthesis capacity and enhanced the protein degradation capability, leading to decreased Cry1Ac endotoxin content in cotton seeds. Protein metabolism in living organisms is in dynamic balance. While new proteins are synthesized, there is also degradation of old proteins, and the amino acids produced from degradation can be utilized for protein synthesis [ 36 ]. Multiple studies have shown that the concentration of Cry1Ac endotoxin protein content is closely related to protein turnover in plants [ 37 – 39 ]. Chen et al. [ 40 ] proved the altered square nitrogen metabolic intensity was main physiological cause of changed square growth and Cry1Ac endotoxin content. The changes in seed Cry1Ac endotoxin content under nitrogen increasing and nitrogen deficient conditions have also been shown to be the result of protein turnover [ 13 , 41 ]. In terms of environment, the protein synthesis in the alternating high temperature cotton boll shell decreases and degradation increases, thereby reducing the Cry1Ac endotoxin content [ 42 ]. Under high-temperature conditions, the insecticidal protein content in Bt cotton leaves decreased, possibly due to the greater degradation of soluble proteins in leaves by high temperatures [ 18 ]. When the temperature was above 38℃, and diurnal temperature variations persisted for more than 7 days, the ability of protein degradation in squares and bolls exceeded the synthetic capacity, resulting in a significant decrease in insecticidal protein content [ 43 ]. Therefore, high-temperature stress accelerates protein degradation, slows down protein synthesis, and ultimately leads to a decrease in insecticidal protein content. This study yielded consistent findings in low-temperature conditions. Specifically, we observed peptidase and protease activities increased under low temperatures, leading to increased protein degradation and a subsequent rise in free amino acids. Concurrently, diminished activities of enzymes such as GOT and GPT directly contributed to reduced protein synthesis and a decline in soluble protein content, consequently leading to a decrease in Cry1Ac endotoxin content. Cry1Ac endotoxin content exhibited a significant positive correlation with soluble protein content, GOT activity, and GPT activity, while displaying a significant negative correlation with free amino acid content, protease activity, and peptidase activity. In summary, low temperatures impaired protein synthesis capacity while augmenting protein degradation capacity, ultimately resulting in a reduction in Cry1Ac endotoxin content. The increase of free amino acid content and peptidase activity in cotton seeds mainly contributed to the deduction in Cry1Ac endotoxin content While previous studies have highlighted the relationship between Cry1Ac endotoxin content and protein metabolism, few have delved into a detailed analysis. Our study utilized stepwise regression and pathway analysis to elucidate this relationship further. We found that the rise in free amino acid content exerted a significant direct negative effect on Cry1Ac endotoxin content, while the indirect negative effect of peptidase activity was even more pronounced. In summary, the elevation of free amino acid content and the increase in of peptidase activity in seeds under low-temperature stress primarily contributed to the decline in Cry1Ac endotoxin content. These findings offer novel insights and a theoretical foundation for mitigating the detrimental effects of low-temperature stress on the insect resistance of Bt cotton. Conclusion In recent years, both the Northern and Southern Xinjiang cotton regions in China have experienced a growing incidence of low-temperature stress during the boll development period. These low temperatures have had a significant impact on the Cry1Ac endotoxin content in cotton bolls. Moreover, as the duration of stress persisted, the threshold temperature leading to a significant decrease in Cry1Ac endotoxin content had risen. Notably, cotton seeds were susceptible to low-temperatures, highlighting the need for practices to bolster the resistance of Bt cotton to temperatures ≤ 19℃. Protein metabolism analysis revealed that under low-temperature conditions, the decrease in Cry1Ac endotoxin content was primarily related to enhanced protein degradation and diminished synthesis ability. Notably, the levels of free amino acids and peptidase activity had played a key role in this process, underscoring their significant contribution to decreased Cry1Ac endotoxin content. Materials and methods Plant materials and experimental design The study was conducted using two Bt transgenic cotton cultivars, ‘Sikang1’ (conventional, SK1) and ‘Sikang3’ (hybrid, SK3), at Yangzhou University, Yangzhou, China (32°30’N, 119°25’E) during the 2020–2021 cotton growing season. Seeds were sowed in the greenhouse on April 15th, 2020, and April 18th, 2021, and the seedlings were transplanted to pots (50 cm height, 40 cm diameter, 62.8 L volume) at 35 days after sowing. The pots were filled with 20 kg sandy loam soil (Typic fluvaquents, Entisols), containing 18.8 g kg − 1 organic matter and available N-P-K at 135.2, 22.8, and 80.9 mg kg − 1 , respectively. Plants were watered thoroughly on a daily basis. On the day of transplanting (May 17th), 1.5g N as urea, 0.7g P as single superphosphate, and 2.6g as KCl were mixed into the soil of each pot, and one seedling was transplanted in each pot. At 46 days after transplanting, 1.6g N as urea, 0.7g P as single superphosphate, and 2.6g as KCl were top-dressed into each pot. At 68 days after transplanting, 2.0g N as urea was top-dressed into each pot. The experiments were performed using a completely randomized design with six replications, consisting of six temperature regimes (optimum temperature: 27°C and low temperatures: 16°C, 17°C, 18°C, 19°C, 20°C) in 2020 and five temperature regimes (optimum temperature: 27°C and low temperatures: 16°C, 17°C, 18°C, 19°C) in 2021. The temperature was set based on low-temperature variations in Xinjiang and the previous study by Chen et al. [ 26 ]. At the peak flowering stage (July 18th), flowers on the seventh to eighth fruiting branches were tagged, and temperature treatments were initiated fifteen days after flower appearance by transporting pots to environmentally controlled rooms with different temperature settings (14h d − 1 photoperiod at a photon flux density of 200 mmol m − 2 s − 1 ; and 70% relative humidity). Five labeled bolls of each treatment were collected at 12h, 24h, and 48h after treatments and stored at -80°C for later measurements. Each cotton boll was separated into three parts: shell, fiber, and seed. The boll shell, fiber, and seed were thoroughly mixed respectively before subsampling. Meteorological data sources Xinjiang (73.66°-96.38°E, 34.42°-49.17°N) is located in the border area of Northwest China. Xinjiang is the largest arid and semiarid region in China, characterized by a typical continental climate with a dry climate, little rainfall, sufficient sunshine time, and a large diurnal temperature range (12.9–15℃) [ 44 ]. Xinjiang cotton holds a significant position in global cotton production. Currently, based on different climate characteristics and vegetation types, Xinjiang's cotton-growing areas are divided into three cotton regions: East Xinjiang Cotton Region (Hami City, Turpan City), North Xinjiang Cotton Region (Changji Hui Autonomous Prefecture, Tacheng Prefecture, Bortala Mongol Autonomous Prefecture), and South Xinjiang Region (Aksu Prefecture, Kizilsu Kyrgyz Autonomous Prefecture, Kashgar Prefecture, Bayingol Mongol Autonomous Prefecture) (Fig. 6 ). This data is sourced from the Xinjiang Statistical Yearbook [ 45 ]. Due to the cotton boll formation period being concentrated from July to August each year, we selected the daily average temperature data (T) from July 1st to August 31st in the past decade. The meteorological data is sourced from the National Oceanic and Atmospheric Administration (NOAA) of the United States ( www.noaa.gov ) and the National Center for Environmental Information (NCEI) (ncei.noaa.gov). We used the basic GAODE map data compiled from the DISHU Platform ( https://dycharts.com ), which includes the boundary of Xinjiang, provincial boundaries, and national boundaries. Physiological measurements Determination of Cry1Ac endotoxin content The concentrations of the Cry1Ac endotoxin in the boll shell, fiber, and seed extracts were determined by immunological analysis using enzyme-linked immunosorbent assay (ELISA) [ 22 ]. Tissue extracts were harvested by homogenizing the frozen tissue (1.5 g) in 2 mL of extraction buffer (Na 2 CO 3 1.33 g, DTT 0.192 g, NaCl 1.461 g, and Vc 0.5 g dissolved in 250 mL of distilled water). The extracts were then transferred to 10-mL centrifuge tubes. The tubes were shaken by hand and stored at 4°C for 4 h. After centrifugation at 10,000×g, the extracts were collected, and the filtered supernatants were collected for determination. Microtitration plates were coated with the standard Cry1Ac insecticidal endotoxin and samples, then incubated at 37°C for 4 h. Antibodies against the Cry1Ac insecticidal endotoxin were added. After that, horseradish peroxidase-labelled goat anti-rabbit immunoglobulin was added, and the samples were incubated for 30 min at 37°C. Finally, the buffered enzyme substrate (1,2-phenylenediamine) was added. Fifteen minutes later, the reaction was terminated using 50 µL of H 2 SO 4 (3 mol L –1 ). The results from absorbance measurements at 490 nm were recorded. Assay of soluble protein (SP) and free amino acid (AA) content Fresh seed samples were stirred at 4°C in 3 mL of cold water (Milli-Q reagent grade) and centrifuged. The supernatant was stored on ice. The total free AA content was determined by the ninhydrin assay according to Yemm et al. [ 46 ]. The total soluble protein content was determined by the Coomassie Blue dye-binding assay of Zou [ 47 ]. The absorbance readings were converted into protein concentrations using bovine serum albumin in the standard curve. Activities of glutamic-pyruvic transaminase (GPT) and glutamic oxaloacetate transaminase (GOT) assay Fresh seed samples were homogenized in 0.05 mmol L − 1 Tris-HCl (pH 7.2), the homogenate was centrifuged, and the supernatant was analyzed for GOT activity. A mixture of 0.5 mL of 0.8 mol·L − 1 alanine in 0.1 mol·L − 1 Tris-HCl (pH 7.5) together with 0.1 mL of 2 mmol·L − 1 pyridoxal phosphate solution was used, and 0.2 mL of 0.1 mol·L − 1 2-oxoglutarate solution and 0.2 mL of the prepared enzyme were added to this mixture. The reaction mixture was incubated at 37°C for 10 min followed by termination of the reaction with 0.1 mL of 0.2 mol·L − 1 trichloroacetic acid solution, following which the pyruvate with chromogen was converted to pyruvate hydrazone. The color intensity of the hydrazone in saturated water toluene was measured at 520 nm. GOT activity was calculated simultaneously from authentic pyruvate standards. The procedure used for assaying the activity of GPT was identical to the GOT assay, except that in the GPT assay, 0.5 mL of 0.8 mol alanine in 0.1 mol·L − 1 Tris-HCl (pH 7.5) was substituted for 0.5 mL of 0.1 mol·L − 1 buffered aspartate solution in the reaction mixture, and aniline citrate addition was omitted [ 48 ]. Activities of protease and peptidase assay Fresh seed samples were homogenized at 4°C in 1 mL of β-mercaptoethanol extraction buffer (pH = 6.8). Cell debris was removed by centrifugation, and the supernatant was placed on ice, and the protease activity determined spectrophotometrically at 400 nm using azocasein as a substrate. Peptidase activity was determined as described by Hu et al. [ 49 ]. A total of 0.1 mL extract was added to 1-mL buffer containing 50 mM Tris-HCl (pH 8.0), 1 m Mol L − 1 MnCl2, and 5 m Mol L − 1 peptide, and then 25 mL of the reaction mixture was incubated at 37°C for 30 min in 1 mL of a 1% ninhydrin solution containing 100 mg of cadmium acetate, 85 mL of ethanol, and 15 mL of acetic acid in a total volume of 100 mL. The optical density was measured at 505 nm. Data processing and statistics Experimental data were analyzed using SPSS 25 (IBM Corp., Armonk, NY, USA). The significance of the differences between different treatments was tested with the least significant difference (P ≤ 0.05), and the Pearson correlation coefficient was used to measure their correlations. The tables and figures were processed and plotted with Excel 2016 (Microsoft Corp., Redmond, USA) and Origin 2023 (OriginLab, Northampton, Massachusetts, USA). Path analysis can decompose the direct correlation between the independent variables and the dependent variables, study their direct and indirect importance, and provide a theoretical basis for accurate statistical decisions [ 50 , 51 ]. In this paper, SPSS software, Pearson method, physiological indicators related to nitrogen metabolism (soluble protein, SP content, AA content, GOT activity, GPT activity, protease activity, and peptidase activity) as the independent variable (X), Bt protein content as the dependent variable (Y), and the direct and indirect effect of physiology related to nitrogen metabolism on Bt protein content. Pyi = bisi/sy; Pyij = rij×Pyi Where Pyi and Pyij represent the direct diameter coefficient and the indirect diameter coefficient rij are the correlation coefficient between independent variable i and dependent variable j; bi is the partial regression coefficient of dependent variable y on independent variable i; si and sy represent the standard deviation of i and y respectively. Declarations Acknowledgements We are grateful to Siyang Cotton Seed Farm and Institute of Biotechnology, Chinese Academy of Agricultural Sciences (CAAS), China, for experimental materials support. Authors’ contributions Y.C., X.Z., D.C. and Z.L. designed the study. S.D., Y.D. and Z.L. performed the experiments. Y.C. and Z.L. analyzed the data and wrote the manuscript. Y.C., Y.L.C. and Z.L. checked and revised the manuscript. All authors reviewed the manuscript. Funding The work was supported by the National Natural Science Foundation of China (31901462), the China Scholarship Council (202308320440), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX22_3508). Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. Plant samples were collected from university research area. Study protocol must comply with relevant institutional, national, and international guidelines and legislation. 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Cite Share Download PDF Status: Published Journal Publication published 15 Aug, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 27 Jun, 2024 Reviews received at journal 24 Jun, 2024 Reviews received at journal 19 Jun, 2024 Reviews received at journal 19 Jun, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers agreed at journal 05 Jun, 2024 Reviews received at journal 30 May, 2024 Reviewers agreed at journal 24 May, 2024 Reviewers agreed at journal 22 May, 2024 Reviewers invited by journal 22 May, 2024 Editor assigned by journal 22 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4450426","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309460703,"identity":"8d66f2dc-aeac-4668-bdb6-e8757e366168","order_by":0,"name":"Yuan Chen","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Chen","suffix":""},{"id":309460704,"identity":"eb1964ca-206f-4cf3-b48e-0a3a3c905f7b","order_by":1,"name":"Shu Dong","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"Dong","suffix":""},{"id":309460705,"identity":"8ca1a0d0-8441-4bc9-9006-b7f1cde56a89","order_by":2,"name":"Yuyang Dai","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yuyang","middleName":"","lastName":"Dai","suffix":""},{"id":309460709,"identity":"d333ee98-f97f-48e9-a804-e773cc11f54c","order_by":3,"name":"Xiang Zhang","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Zhang","suffix":""},{"id":309460711,"identity":"cdc67dc7-77e7-4d0c-8cd9-70519c3e8848","order_by":4,"name":"Dehua Chen","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Dehua","middleName":"","lastName":"Chen","suffix":""},{"id":309460713,"identity":"f6933417-1cfa-433b-a9b6-4465c20691e4","order_by":5,"name":"Yinglong Chen","email":"","orcid":"","institution":"The University of Western Australia","correspondingAuthor":false,"prefix":"","firstName":"Yinglong","middleName":"","lastName":"Chen","suffix":""},{"id":309460714,"identity":"7cae8f7e-0035-46de-8200-4c38ceefcd40","order_by":6,"name":"Zhenyu Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACPmYGBmYwi5mx8YFEhYScPCEtbHAt7MzNBhZnLIwNGwhpYYBp4Wdvk6hsq0hkOEBICzuP8eeCijuJGw4zNkjcnCeRwNjA/PDRDbwO4zEwnnHmGViL4cxtEnnsDGzGxjkEtCTzth0Ga0mW3CZRzNjAwyZNSMth3n8QLYf/zpFIbDhAWIthM28DWEtjg2QDUVrYipl5jh02nnmYsZlB4piEsWEzAb/w8x/e/Jmn5rBs3/njz39I1NTJybM3P3yMTwsMODbAmcxEKAcBeyLVjYJRMApGwUgEAKxQR9QMJ9WWAAAAAElFTkSuQmCC","orcid":"","institution":"Yangzhou University","correspondingAuthor":true,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-05-20 17:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4450426/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4450426/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05500-y","type":"published","date":"2024-08-15T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57596121,"identity":"00f50621-a854-40e9-ae60-a7aa1fcfa40e","added_by":"auto","created_at":"2024-06-03 06:52:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":499790,"visible":true,"origin":"","legend":"\u003cp\u003eStatistics on the number of days with daily mean temperature less than or equal to 20℃ in the main cotton areas of Xinjiang, China from 2014 to 2023\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/f305c4a777eb9fe22cb5ddb4.jpg"},{"id":57596126,"identity":"f034d9c4-67c6-4d19-9138-8c181745494f","added_by":"auto","created_at":"2024-06-03 06:52:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20457817,"visible":true,"origin":"","legend":"\u003cp\u003eThe decreasing range in Cry1Ac endotoxin protein content in boll shell, fiber, and seed under varying temperatures and stress durations treatment compared with CK (27 ℃). SK-1 and SK-3 represent cultivars Sikang 1 and Sikang 3, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/e7cf3a4112ad1b522f5fb7f3.jpg"},{"id":57596123,"identity":"a19d4ccd-7910-4606-8290-45a97a7b05b5","added_by":"auto","created_at":"2024-06-03 06:52:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5759533,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of low temperature stress on protein turnover in cotton seed in 2020 and 2021. FW represents fresh weight. A: soluble protein contents. B: free amino acid contents. C: GOT activities. D: GPT activities. E: protease activities. F: peptidase activities. Different lowercase letters indicate significant differences between different temperature treatments under the same stress time and variety (P\u0026lt;0.05). SK-1 and SK-3 represent cultivars Sikang 1 and Sikang 3, respectively.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/bd1fc58c6f02c1064069b47f.jpg"},{"id":57596127,"identity":"3e86f80c-7126-4831-be8a-bc4825bead84","added_by":"auto","created_at":"2024-06-03 06:52:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20457817,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between Cry1Ac endotoxin content and nitrogen metabolism related parameters in 2020 and 2021. Cry1Ac: Cry1Ac endotoxin content. SP: soluble protein. AA: free amino acid. * and ** represent the significance levels of 5% and 1%, respectively.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/d0f98f662de045e0de3de452.jpg"},{"id":57596122,"identity":"bdbbfc4a-7b86-486d-a16c-aacc78f82ed5","added_by":"auto","created_at":"2024-06-03 06:52:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":373658,"visible":true,"origin":"","legend":"\u003cp\u003ePath coefficients of key physiological index\u003c/p\u003e\n\u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e represents AA content. X\u003csub\u003e6\u003c/sub\u003e represents peptidase activity. Y represents seed Cry1Ac endotoxin content. Black numbers indicate correlation (combined effect). Red numbers indicate direct effect. The indirect effect of X\u003csub\u003e2\u003c/sub\u003e on Y through X\u003csub\u003e6\u003c/sub\u003e is 0.491, and the indirect effect of X\u003csub\u003e6\u003c/sub\u003e on Y through X\u003csub\u003e2\u003c/sub\u003e is -1.049.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/818b83ebfe6d39f5459e294d.jpg"},{"id":57596124,"identity":"580c3b02-b329-4bce-a5e8-9139a0bb2e7d","added_by":"auto","created_at":"2024-06-03 06:52:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":654924,"visible":true,"origin":"","legend":"\u003cp\u003eLocation and information map of meteorological stations in the main cotton areas of Xinjiang for this study\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/67367a11e61a854fac89361e.jpg"},{"id":63071499,"identity":"3a9b39c4-4a2d-48c3-bc99-34a2c7c4b98c","added_by":"auto","created_at":"2024-08-22 20:07:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":49064863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4450426/v1/2e723c2a-95fd-48c2-be47-4d56e2e26c0c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low temperature exposure decreases Cry1Ac insecticidal endotoxin content in cotton seeds","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) transgenic cotton, featuring the expression of the Cry1Ac endotoxin (Bt protein), represents a revolutionary cotton variety achieved by introducing the Bt gene into cotton tissue cells. It stands as one of the most extensively cultivated and widely distributed genetically modified crops globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Bacillus thuringiensis produces a parasporal crystal named δ-endotoxin during its metabolic process, exhibiting toxic effects on various pests, particularly lepidopteran pests like cotton bollworm [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The introduction of the first genetically modified cotton variety with Bt traits in Xinjiang at the end of the last century brought about significant economic and ecological benefits through large-scale cultivation. The expression of insect-resistant proteins notably inhibits the growth of lepidopteran pests, such as cotton bollworm, reducing their impact on cotton and consequently increasing yield. Simultaneously, it curtails the use of chemical pesticides, alleviating environmental pressures [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the widespread cultivation of Bt cotton in China, the instability in the expression of Bt cotton's insecticidal protein has emerged as a pressing concern in the current landscape of transgenic cotton in China [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cry1Ac endotoxin content gradually decreases throughout the growing season, reaching its lowest concentration during the boll-setting stage [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, Cry1Ac endotoxin content varies across different cotton organs, with higher concentrations in the leaves and lower levels in reproductive organs like squares, flowers, and bolls [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Adverse environmental conditions, including extreme temperatures, especially low temperatures, can significantly decrease the expression of insecticidal proteins [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among these adverse environments, prolonged exposure to low temperatures has been shown to decrease the Cry1Ac endotoxin content in leaves throughout the growth period, with a more pronounced decrease during the boll-setting period [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Field experiments have demonstrated that continuous low temperatures lead to a decrease in Cry1Ac endotoxin content, exacerbating cotton bollworm damage [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, under low-temperature conditions, the insecticidal protein content in leaves changes early and significantly decreases throughout the entire stress period [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the shift of China's cotton production regions to Xinjiang, over 80% of cotton production in China now originates from Xinjiang, constituting nearly one-fifth of global cotton production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Specifically, in 2020, Xinjiang's cotton planting area and output accounted for 79.0% and 87.3% of China's respective values [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, low-temperature stress occurs more frequently in Chinese cotton production due to the high latitude of Xinjiang, with an increasing trend in the frequency and intensity of extreme low temperatures [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Temperatures below 15\u0026deg;C can impede the growth and development of cotton, and temperatures below 20\u0026deg;C in later stages are detrimental to cotton maturation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In July and August, the critical boll development stage, Xinjiang often experiences nightly temperatures around 15\u0026deg;C, even consecutive days with average temperatures below 20\u0026deg;C [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. On July 9, 2022, Kashgar experienced temperatures as low as 9\u0026deg;C, while in mid-August 2022, Altay encountered a week-long period with average temperatures below 15\u0026deg;C.\u003c/p\u003e \u003cp\u003eDue to the occurrence of low temperatures in the reproductive growth stage and the relatively lower expression of insecticidal protein in reproductive organs, it is crucial to explore the changes in insect resistance of Bt cotton in reproductive organs under low-temperature stress. However, prior studies have predominantly focused on leaves under low-temperature stress, and the impact of low temperatures on different components of cotton bolls, such as boll shells, fibers, and seeds, has been scarcely explored. Therefore, this study aimed to simulate low-temperature conditions that may occur in Xinjiang, compare the changes in the expression of Cry1Ac endotoxin in Bt cotton boll components under different degrees and durations of low temperatures, and investigate the associated physiological mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLow temperature occurrence during boll development stage in Xinjiang\u003c/h2\u003e \u003cp\u003eSummarizing the daily average temperatures from July 1 to August 31 in the past decade (2014\u0026ndash;2023), the East Xinjiang Cotton Region generally experienced higher temperatures with a lower threat of low temperatures. In recent years, both the North Xinjiang Cotton Region and the South Xinjiang Cotton Region have faced greater challenges from low temperatures during the boll-setting period, with particular attention needed in the North Xinjiang Cotton Region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to the previous five years (2014\u0026ndash;2018), in the last five years (2019\u0026ndash;2023), the total number of days with a daily average temperature lower than 20\u0026deg;C increased by 72.73% in Changji Hui Autonomous Prefecture, by 35.56% in Tacheng Prefecture, and by 54.84% in Bortala Mongol Autonomous Prefecture. It is evident that, in recent years, with climate change, the cotton-growing areas of Xinjiang, especially in Northern Xinjiang, have experienced more frequent occurrences of cold weather during the peak boll-setting period of Bt cotton. Consequently, investigating the impact of low temperatures on the Cry1Ac endotoxin content in cotton bolls holds significant importance for cotton security in China and globally.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCry1Ac endotoxin content\u003c/h2\u003e \u003cp\u003eAccording to the low-temperature levels and frequency in Xinjiang, we set the low-temperature range from 16 to 20\u0026deg;C and tested the effect of different levels and durations of low temperature on Cry1Ac endotoxin contents in cotton bolls. Under low temperatures, the Cry1Ac endotoxin content in boll shell, fiber, and seed decreased, and with the decrease in temperature, their content showed a decreasing trend (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In 2020, compared with CK (27\u0026deg;C), after 12 hours of stress, the Cry1Ac endotoxin content in SK1 and SK3 boll shell significantly decreased at 16\u0026deg;C-17\u0026deg;C, fiber at 16\u0026deg;C, while the seeds showed no significant difference from CK. After 24 hours of stress, the Cry1Ac endotoxin content in SK1 boll shell, fiber, and seeds significantly decreased at 16\u0026deg;C-18\u0026deg;C, and the Cry1Ac endotoxin content in SK3 boll shell significantly decreased at 16\u0026deg;C-18\u0026deg;C, fiber at 16\u0026deg;C-17\u0026deg;C, and seeds at 16\u0026deg;C-18\u0026deg;C. After 48 hours of stress, the Cry1Ac endotoxin content in SK1 boll shell significantly decreased at 16\u0026deg;C-20\u0026deg;C, fiber at 16\u0026deg;C-18\u0026deg;C, seeds at 16\u0026deg;C-19\u0026deg;C, the Cry1Ac endotoxin content in SK3 boll shell significantly decreased at 16\u0026deg;C-20\u0026deg;C, fiber at 16\u0026deg;C-17\u0026deg;C, and seeds at 16\u0026deg;C-18\u0026deg;C. From the above results, it can be concluded that the temperatures causing a significant decrease in Cry1Ac endotoxin content in boll shell, fiber, and seed increased with the prolonged duration of low-temperature stress. Additionally, with the prolonged duration of stress, the Cry1Ac endotoxin content in boll shell, fiber, and seeds under each low-temperature treatment showed a gradual decrease.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of varying temperatures and stress durations on the Cry1Ac endotoxin content in cotton boll shell, fiber, and seed (ng g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" 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align=\"left\" colname=\"c12\"\u003e \u003cp\u003e250.43b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106.90b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95.74c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.65a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.08b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e52.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e 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align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50.49b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e276.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e216.74b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e200.99d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111.35b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.72c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e83.55d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.99b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e47.23d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e46.26c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e273.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e202.09b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e180.42e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e133.10a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e132.82a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e132.12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65.12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e65.95a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64.76a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e413.65a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e404.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e386.83a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e132.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e128.69a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e121.17a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e63.88a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e62.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e405.66a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e385.21ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e370.04a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e130.73ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e119.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e107.83b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.61a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.99a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e56.40bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e406.19a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e359.75b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e330.47b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.68b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e113.90b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95.31c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.34ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e58.85b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e55.44bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e403.82a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e320.57c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e294.26c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16℃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.44b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.08c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.73c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e59.50b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55.07c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50.57c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e400.2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e310.14c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e269.08d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eNote: FW represents fresh weight. Different lowercase letters indicate significant differences between treatments of each variety at P\u0026lt;0.05 in the same year. SK-1 and SK-3 represent cultivars Sikang 1 and Sikang 3, respectively.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn 2020, after 12\u0026ndash;48 hours of stress at 20\u0026deg;C, Cry1Ac endotoxin content decreased by 0.89%-7.72% in the SK1 boll shell, 1.57%-3.23% in fiber, and 1.11%-8.26% in seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, after 12\u0026ndash;48 hours of stress at 19\u0026deg;C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 0.71%-8.56%, 1.69%-6.46%, and 1.87%-23.88%, respectively. This trend continued with 12\u0026ndash;48 hours of stress at 18\u0026deg;C, where Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 1.10%-17.58%, 1.22%-18.16%, and 1.87%-30.76%, respectively. The pattern persisted with further decreases in temperature; after 12\u0026ndash;48 hours of stress at 17\u0026deg;C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 11.18%-26.70%, 3.42%-19.08%, 2.28%-38.13%, respectively. Similarly, after 12\u0026ndash;48 hours of stress at 16\u0026deg;C, Cry1Ac endotoxin content in the SK1 boll shell, fiber and seeds decreased by 10.49%-27.66%, 13.63%-27.77%, and 3.37%-46.44%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe comparison of the decrease in Cry1Ac endotoxin content in the cotton boll, fiber, and seeds after the same duration of stress revealed that in 2020, Cry1Ac endotoxin content in SK1 boll shell, fiber, seeds decreased by 0.89% \u0026minus;\u0026thinsp;10.49%, 1.57% \u0026minus;\u0026thinsp;13.63%, and 1.11% \u0026minus;\u0026thinsp;3.67% after 12h of stress at 16\u0026ndash;20℃. After 24h of stress, Cry1Ac endotoxin content decreased by 4.15% \u0026minus;\u0026thinsp;23.83%, 5.11% \u0026minus;\u0026thinsp;25.10%, and 5.25% \u0026minus;\u0026thinsp;33.69% in the cotton boll shell, fiber and seeds, respectively. Similar trend was observed with longer stress duration, after 48h of stress, Cry1Ac endotoxin content in the boll shell, fiber and seeds decreased by 7.22% \u0026minus;\u0026thinsp;27.66%, 3.23% \u0026minus;\u0026thinsp;27.77%, and 8.26% \u0026minus;\u0026thinsp;46.44%, respectively. The trend of change in SK3 was similar to SK1. The experimental results in 2021 were similar to those in 2020. Consequently, with decreasing temperature and prolonged stress duration, the Cry1Ac endotoxin content in seeds within cotton bolls exhibited the greatest decline, rendering it the most unstable compared to boll shell and fiber, particularly susceptible to low temperatures. Therefore, this study focused on the seed insecticidal protein level under low temperatures and the nitrogen metabolism to uncover the underlying mechanism.\u003c/p\u003e \u003cp\u003eIn summary, compared to bolls and fibers, low temperature exhibited a greater impact on the Cry1Ac endotoxin content in cotton seed. After 24h and 48h of stress, the temperatures causing significant changes in Cry1Ac endotoxin content in SK1 seed were 18℃ and 19℃, and for SK3 were 18℃ and 18℃ in 2020; in 2021, for SK1, they were 17℃ and 19℃ respectively, and for SK3 were 18℃ and 18℃ respectively. Therefore, extending the duration of low-temperature stress not only increased the temperature at which Cry1Ac endotoxin content significantly decreased but also further reduced the Cry1Ac endotoxin content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen metabolism\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eSoluble protein and amino acid content\u003c/h2\u003e \u003cp\u003eUnder low-temperature treatment, the soluble protein content in seeds decreased, and with the decrease in temperature and extension of stress duration, its content showed a declining trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In 2020, after 12 hours of stress, the soluble protein content in seeds under low-temperature showed no significant difference compared to CK; after 24 hours of stress, low-temperature 3 significantly decreased the soluble protein content in seeds at 16℃-17℃, with reductions of 23.10%-30.69% and 26.48%-29.60%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly decreased the SK1 soluble protein content in seeds at 16℃-18℃, and the SK3 soluble protein content in seeds at 16℃-19℃, with reductions of 16.32%-33.68% and 14.78%-37.11%, respectively, compared to CK. The experimental results in 2021 were similar to those in 2020. In conclusion, the temperature leading to a significant decrease in soluble protein content in seeds increased with the prolongation of low-temperature stress duration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder low-temperature treatment, the content of free amino acids in seeds increased, and with the decrease in temperature and prolongation of stress duration, its content showed an upward trend. In 2020, after 12 hours of stress, the free amino acid content in seeds for SK1 and SK3 under 16℃-20℃ treatment showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly increased the free amino acid content in SK1 seeds at 16℃-18℃, and significantly increased the free amino acid content in SK3 seeds at 16℃-17℃, with increases of 9.25%-22.45% and 21.54%-23.70%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly increased the free amino acid content in SK1 seeds at 16℃-19℃, and significantly increased the free amino acid content in SK3 seeds at 16℃-18℃, with increases of 8.08%-29.66% and 11.73%-31.92%, respectively, compared to CK. The experimental results in 2021 were similar to those in 2020. In conclusion, the temperature leading to a significant increase in free amino acid content in seeds increased with the prolongation of low-temperature stress duration.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eGOT and GPT activity\u003c/h3\u003e\n\u003cp\u003eUnder low-temperature treatment, the activities of seed GOT and GPT decreased, and with decreasing temperature and prolongation of stress duration, their activities showed a declining trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In 2020, after 12 hours of stress, the seed GOT activity of SK1 and SK3 under treatment at 16\u0026deg;C-20\u0026deg;C showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly decreased seed GOT activity at 16\u0026deg;C-17\u0026deg;C, with decreases of 25.50%-26.85% and 25.83%-28.13% compared to CK; after 48 hours of stress, low-temperature significantly decreased the SK1 seed GOT activity at 16\u0026deg;C-19\u0026deg;C, and significantly decreased the SK3 seed GOT activity at 16\u0026deg;C-18\u0026deg;C, with decreases of 7.45%-31.45% and 14.75%-32.65% compared to CK. In 2020, after 12 hours of stress, under treatment at 16\u0026deg;C-20\u0026deg;C, the seed GPT activity of SK1 showed no significant difference compared to CK, while low-temperature significantly decreased the SK3 seed GPT activity by 8.96% at 16\u0026deg;C; after 24 hours of stress, low-temperature significantly decreased seed GPT activity at 16\u0026deg;C-18\u0026deg;C, with decreases of 8.31%-23.71% and 11.17%-24.69% in SK1 and SK3 respectively compared to CK; after 48 hours of stress, low-temperature significantly decreased seed GPT activity at 16\u0026deg;C-18\u0026deg;C, with decreases of 11.56%-28.44% and 11.28%-36.34% in SK1 and SK3 compared to CK. The results of the 2021 experiment were similar to those of 2020. In summary, the temperatures leading to a significant decrease in seed GOT and GPT activities increased with the prolonged duration of low-temperature stress.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtease and peptidase activity\u003c/h2\u003e \u003cp\u003eUnder low-temperature treatment, the activities of seed protease and peptidase increased, and with decreasing temperature and prolonged stress duration, their activities showed an upward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In 2020, after 12 hours of stress, the seed protease activity of SK1 and SK3 at 16\u0026deg;C-20\u0026deg;C showed no significant difference compared to CK; after 24 hours of stress, low-temperature significantly increased seed protease activity at 16\u0026deg;C-17\u0026deg;C, with increases of 24.43%-27.79% and 19.92%-21.85% respectively in SK1 and SK3, compared to CK; after 48 hours of stress, low-temperature significantly increased seed protease activity at 16\u0026deg;C-19\u0026deg;C, with increases of 15.54%-40.00% and 14.40%-32.78% compared to CK.\u003c/p\u003e \u003cp\u003eIn 2020, after 12 hours of stress, there was no significant difference in cotton seed peptidase activity at 16\u0026deg;C-20\u0026deg;C compared to the control (CK); after 24 hours of stress, low-temperature significantly increased the SK1 seed peptidase activity at 16\u0026deg;C-18\u0026deg;C, and significantly increased the SK3 seed peptidase activity at 16\u0026deg;C-19\u0026deg;C, with increases of 8.63%-16.88% and 4.13%-28.00%, respectively, compared to CK; after 48 hours of stress, low-temperature significantly increased the SK1 seed peptidase activity at 16\u0026deg;C-19\u0026deg;C, and significantly increased the SK3 seed peptidase activity at 16\u0026deg;C-18\u0026deg;C, with increases of 7.61%-31.52% and 11.68%-39.21%, respectively. The results of the 2021 experiment were similar to those of 2020. In summary, the temperatures leading to a significant increase in cotton seed protease and peptidase activity increased with the prolonged duration of low-temperature stress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCorrelation analysis and pathway analysis\u003c/h3\u003e\n\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, it can be observed that after 12 hours of low-temperature treatment, Cry1Ac endotoxin content in seeds was not correlated with the soluble protein content and peptidase activity. However, it showed a significant positive correlation with GOT, GPT, and protease activity, and a highly significant negative correlation with free amino acid content. After 24 hours of low-temperature treatment, the Cry1Ac endotoxin content in seeds showed a significant positive correlation with soluble protein content, GOT, and GPT activity, a highly significant negative correlation with free amino acid content, and a negative correlation with protease and peptidase activity, although not significant. After 48 hours of low-temperature treatment, the Cry1Ac endotoxin content in seeds exhibited a highly significant positive correlation with soluble protein content, GOT, and GPT activity, and a significant negative correlation with free amino acid content, as well as protease and peptidase activity. The above results indicated that low temperature primarily reduced protein synthesis capacity, and enhanced protein degradation capability, thereby affecting the Cry1Ac endotoxin content in seeds. Moreover, with the prolonged duration of stress, a more significant correlation was found between Cry1Ac endotoxin content in seeds and the content of key nitrogen metabolism substances and key enzyme activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing Cry1Ac endotoxin content (Y) as the dependent variable and SP (X1), AA (X2), GOT (X3), GPT (X4), protease (X5), and peptidase activity (X6) as independent variables, stepwise regression analysis was conducted, and the coefficient of determination of the regression equation was R2\u0026thinsp;=\u0026thinsp;0.682 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This indicated that the selected indicators reflected the main factors influencing the content of Cry1Ac endotoxin in seeds. To further clarify the effects of the indicators determined by stepwise regression on Cry1Ac endotoxin content, pathway analysis was performed on the selected indicators with Cry1Ac endotoxin content (Y) as the dependent variable. The results showed that AA (X2) and peptidase (X6) both had significant negative effects on Cry1Ac endotoxin content. Although peptidase had a direct positive effect on Cry1Ac endotoxin content, its indirect effect was \u0026minus;\u0026thinsp;1.049 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In summary, the increase in amino acid content and peptidase activity in cotton seeds under low-temperature stress mainly contributed to the decrease in Cry1Ac endotoxin content.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of stepwise regression analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDependent variable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSignificant level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThe order of stepwise\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRegression equation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCry1Ac endotoxin content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e, X\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eY\u0026thinsp;=\u0026thinsp;655.956-75.095X\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;21.308X\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: X2 represents AA content. X6 represents peptidase activity. Y represents seed Cry1Ac endotoxin content.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eThe increasingly frequent low-temperature stress reduced the Cry1Ac endotoxin content in cotton bolls, with the greatest impact observed on cotton seeds.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDue to the distribution of soil and topographical influences in China, regions like Xinjiang and Inner Mongolia, characterized by vast land and sparse population, have formed fertile and expansive plains suitable for mechanized cultivation of crops. However, the higher latitudes in regions like Xinjiang result in lower temperatures throughout the year. Previous studies have shown that temperatures below 15℃ seriously affect the growth and development of cotton [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Maho et al. found that the survival rate of cotton bollworm larvae was higher under low temperatures (14\u0026thinsp;~\u0026thinsp;22℃) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Zhou et al. studied the impact of temperature on the insecticidal activity of expressed insecticidal proteins in cotton plants, finding that at 16℃, the insecticidal activity of Bt cotton was significantly reduced [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Zhang et al. demonstrated that the entire growth period of Bt cotton was affected by low-temperature and high-humidity conditions. The decline in insecticidal protein content in Bt cotton leaves varied with the degree and duration of stress, with a faster decline during the boll development period compared to the flowering period [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Chen et al. demonstrated that low-temperature stress reduced the insecticidal protein content in cotton bolls, and prolonging the duration of low temperatures increased the temperature at which significant changes occur [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, it can be concluded that low-temperature stress during the cotton growth stages, especially during the boll development period, leads to a decrease in the insect resistance of Bt cotton.\u003c/p\u003e \u003cp\u003eOur current study indicated that in recent years, both Southern Xinjiang and Northern Xinjiang cotton regions frequently experience low temperatures during the Bt cotton boll development period. Moreover, low temperatures reduced the Cry1Ac endotoxin content in boll shell, fiber, and seeds of cotton bolls. As the temperature decreased, the Cry1Ac endotoxin content in boll shell, fiber, and seeds all showed a declining trend. Additionally, the temperature at which the Cry1Ac endotoxin content underwent significant changes increased with the prolonged duration of low-temperature stress. Moreover, as the most affected part of bolls by low-temperature stress, after 24h and 48h of low-temperature stress, the Cry1Ac endotoxin content in cotton seeds experienced the greatest decline, reaching up to 33.69% and 46.44% in SK1 and SK3, respectively, with a significant decrease occurring at temperatures between 17℃ and 19℃. The decrease in Cry1Ac endotoxin content would endanger the safety of cotton production in Xinjiang, and thus special attention should be given to low-temperature hazards, and appropriate practices should be applied when the temperature drops below 19℃ during the boll development period. The hybrid variety SK-3 had a smaller decrease compared to the conventional variety SK-1, possibly due to the better stress resistance of the hybrid variety. However, this conclusion needs further verification.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLow-temperature stress reduced the protein synthesis capacity and enhanced the protein degradation capability, leading to decreased Cry1Ac endotoxin content in cotton seeds.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtein metabolism in living organisms is in dynamic balance. While new proteins are synthesized, there is also degradation of old proteins, and the amino acids produced from degradation can be utilized for protein synthesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Multiple studies have shown that the concentration of Cry1Ac endotoxin protein content is closely related to protein turnover in plants [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Chen et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] proved the altered square nitrogen metabolic intensity was main physiological cause of changed square growth and Cry1Ac endotoxin content. The changes in seed Cry1Ac endotoxin content under nitrogen increasing and nitrogen deficient conditions have also been shown to be the result of protein turnover [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn terms of environment, the protein synthesis in the alternating high temperature cotton boll shell decreases and degradation increases, thereby reducing the Cry1Ac endotoxin content [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Under high-temperature conditions, the insecticidal protein content in Bt cotton leaves decreased, possibly due to the greater degradation of soluble proteins in leaves by high temperatures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. When the temperature was above 38℃, and diurnal temperature variations persisted for more than 7 days, the ability of protein degradation in squares and bolls exceeded the synthetic capacity, resulting in a significant decrease in insecticidal protein content [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, high-temperature stress accelerates protein degradation, slows down protein synthesis, and ultimately leads to a decrease in insecticidal protein content.\u003c/p\u003e \u003cp\u003eThis study yielded consistent findings in low-temperature conditions. Specifically, we observed peptidase and protease activities increased under low temperatures, leading to increased protein degradation and a subsequent rise in free amino acids. Concurrently, diminished activities of enzymes such as GOT and GPT directly contributed to reduced protein synthesis and a decline in soluble protein content, consequently leading to a decrease in Cry1Ac endotoxin content. Cry1Ac endotoxin content exhibited a significant positive correlation with soluble protein content, GOT activity, and GPT activity, while displaying a significant negative correlation with free amino acid content, protease activity, and peptidase activity.\u003c/p\u003e \u003cp\u003eIn summary, low temperatures impaired protein synthesis capacity while augmenting protein degradation capacity, ultimately resulting in a reduction in Cry1Ac endotoxin content.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe increase of free amino acid content and peptidase activity in cotton seeds mainly contributed to the deduction in Cry1Ac endotoxin content\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile previous studies have highlighted the relationship between Cry1Ac endotoxin content and protein metabolism, few have delved into a detailed analysis. Our study utilized stepwise regression and pathway analysis to elucidate this relationship further. We found that the rise in free amino acid content exerted a significant direct negative effect on Cry1Ac endotoxin content, while the indirect negative effect of peptidase activity was even more pronounced. In summary, the elevation of free amino acid content and the increase in of peptidase activity in seeds under low-temperature stress primarily contributed to the decline in Cry1Ac endotoxin content. These findings offer novel insights and a theoretical foundation for mitigating the detrimental effects of low-temperature stress on the insect resistance of Bt cotton.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn recent years, both the Northern and Southern Xinjiang cotton regions in China have experienced a growing incidence of low-temperature stress during the boll development period. These low temperatures have had a significant impact on the Cry1Ac endotoxin content in cotton bolls. Moreover, as the duration of stress persisted, the threshold temperature leading to a significant decrease in Cry1Ac endotoxin content had risen. Notably, cotton seeds were susceptible to low-temperatures, highlighting the need for practices to bolster the resistance of Bt cotton to temperatures\u0026thinsp;\u0026le;\u0026thinsp;19℃. Protein metabolism analysis revealed that under low-temperature conditions, the decrease in Cry1Ac endotoxin content was primarily related to enhanced protein degradation and diminished synthesis ability. Notably, the levels of free amino acids and peptidase activity had played a key role in this process, underscoring their significant contribution to decreased Cry1Ac endotoxin content.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and experimental design\u003c/h2\u003e \u003cp\u003eThe study was conducted using two Bt transgenic cotton cultivars, \u0026lsquo;Sikang1\u0026rsquo; (conventional, SK1) and \u0026lsquo;Sikang3\u0026rsquo; (hybrid, SK3), at Yangzhou University, Yangzhou, China (32\u0026deg;30\u0026rsquo;N, 119\u0026deg;25\u0026rsquo;E) during the 2020\u0026ndash;2021 cotton growing season. Seeds were sowed in the greenhouse on April 15th, 2020, and April 18th, 2021, and the seedlings were transplanted to pots (50 cm height, 40 cm diameter, 62.8 L volume) at 35 days after sowing. The pots were filled with 20 kg sandy loam soil (Typic fluvaquents, Entisols), containing 18.8 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e organic matter and available N-P-K at 135.2, 22.8, and 80.9 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Plants were watered thoroughly on a daily basis.\u003c/p\u003e \u003cp\u003eOn the day of transplanting (May 17th), 1.5g N as urea, 0.7g P as single superphosphate, and 2.6g as KCl were mixed into the soil of each pot, and one seedling was transplanted in each pot. At 46 days after transplanting, 1.6g N as urea, 0.7g P as single superphosphate, and 2.6g as KCl were top-dressed into each pot. At 68 days after transplanting, 2.0g N as urea was top-dressed into each pot.\u003c/p\u003e \u003cp\u003eThe experiments were performed using a completely randomized design with six replications, consisting of six temperature regimes (optimum temperature: 27\u0026deg;C and low temperatures: 16\u0026deg;C, 17\u0026deg;C, 18\u0026deg;C, 19\u0026deg;C, 20\u0026deg;C) in 2020 and five temperature regimes (optimum temperature: 27\u0026deg;C and low temperatures: 16\u0026deg;C, 17\u0026deg;C, 18\u0026deg;C, 19\u0026deg;C) in 2021. The temperature was set based on low-temperature variations in Xinjiang and the previous study by Chen et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. At the peak flowering stage (July 18th), flowers on the seventh to eighth fruiting branches were tagged, and temperature treatments were initiated fifteen days after flower appearance by transporting pots to environmentally controlled rooms with different temperature settings (14h d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e photoperiod at a photon flux density of 200 mmol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; and 70% relative humidity). Five labeled bolls of each treatment were collected at 12h, 24h, and 48h after treatments and stored at -80\u0026deg;C for later measurements. Each cotton boll was separated into three parts: shell, fiber, and seed. The boll shell, fiber, and seed were thoroughly mixed respectively before subsampling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeteorological data sources\u003c/h2\u003e \u003cp\u003eXinjiang (73.66\u0026deg;-96.38\u0026deg;E, 34.42\u0026deg;-49.17\u0026deg;N) is located in the border area of Northwest China. Xinjiang is the largest arid and semiarid region in China, characterized by a typical continental climate with a dry climate, little rainfall, sufficient sunshine time, and a large diurnal temperature range (12.9\u0026ndash;15℃) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Xinjiang cotton holds a significant position in global cotton production. Currently, based on different climate characteristics and vegetation types, Xinjiang's cotton-growing areas are divided into three cotton regions: East Xinjiang Cotton Region (Hami City, Turpan City), North Xinjiang Cotton Region (Changji Hui Autonomous Prefecture, Tacheng Prefecture, Bortala Mongol Autonomous Prefecture), and South Xinjiang Region (Aksu Prefecture, Kizilsu Kyrgyz Autonomous Prefecture, Kashgar Prefecture, Bayingol Mongol Autonomous Prefecture) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This data is sourced from the Xinjiang Statistical Yearbook [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Due to the cotton boll formation period being concentrated from July to August each year, we selected the daily average temperature data (T) from July 1st to August 31st in the past decade. The meteorological data is sourced from the National Oceanic and Atmospheric Administration (NOAA) of the United States (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.noaa.gov\" target=\"_blank\"\u003ewww.noaa.gov\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.noaa.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the National Center for Environmental Information (NCEI) (ncei.noaa.gov). We used the basic GAODE map data compiled from the DISHU Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dycharts.com\u003c/span\u003e\u003cspan address=\"https://dycharts.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which includes the boundary of Xinjiang, provincial boundaries, and national boundaries.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological measurements\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of Cry1Ac endotoxin content\u003c/h2\u003e \u003cp\u003eThe concentrations of the Cry1Ac endotoxin in the boll shell, fiber, and seed extracts were determined by immunological analysis using enzyme-linked immunosorbent assay (ELISA) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Tissue extracts were harvested by homogenizing the frozen tissue (1.5 g) in 2 mL of extraction buffer (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e 1.33 g, DTT 0.192 g, NaCl 1.461 g, and Vc 0.5 g dissolved in 250 mL of distilled water). The extracts were then transferred to 10-mL centrifuge tubes. The tubes were shaken by hand and stored at 4\u0026deg;C for 4 h. After centrifugation at 10,000\u0026times;g, the extracts were collected, and the filtered supernatants were collected for determination. Microtitration plates were coated with the standard Cry1Ac insecticidal endotoxin and samples, then incubated at 37\u0026deg;C for 4 h. Antibodies against the Cry1Ac insecticidal endotoxin were added. After that, horseradish peroxidase-labelled goat anti-rabbit immunoglobulin was added, and the samples were incubated for 30 min at 37\u0026deg;C. Finally, the buffered enzyme substrate (1,2-phenylenediamine) was added. Fifteen minutes later, the reaction was terminated using 50 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (3 mol L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e). The results from absorbance measurements at 490 nm were recorded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAssay of soluble protein (SP) and free amino acid (AA) content\u003c/h2\u003e \u003cp\u003eFresh seed samples were stirred at 4\u0026deg;C in 3 mL of cold water (Milli-Q reagent grade) and centrifuged. The supernatant was stored on ice. The total free AA content was determined by the ninhydrin assay according to Yemm et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The total soluble protein content was determined by the Coomassie Blue dye-binding assay of Zou [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The absorbance readings were converted into protein concentrations using bovine serum albumin in the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eActivities of glutamic-pyruvic transaminase (GPT) and glutamic oxaloacetate transaminase (GOT) assay\u003c/h2\u003e \u003cp\u003eFresh seed samples were homogenized in 0.05 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Tris-HCl (pH 7.2), the homogenate was centrifuged, and the supernatant was analyzed for GOT activity. A mixture of 0.5 mL of 0.8 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e alanine in 0.1 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Tris-HCl (pH 7.5) together with 0.1 mL of 2 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e pyridoxal phosphate solution was used, and 0.2 mL of 0.1 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 2-oxoglutarate solution and 0.2 mL of the prepared enzyme were added to this mixture. The reaction mixture was incubated at 37\u0026deg;C for 10 min followed by termination of the reaction with 0.1 mL of 0.2 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e trichloroacetic acid solution, following which the pyruvate with chromogen was converted to pyruvate hydrazone. The color intensity of the hydrazone in saturated water toluene was measured at 520 nm. GOT activity was calculated simultaneously from authentic pyruvate standards. The procedure used for assaying the activity of GPT was identical to the GOT assay, except that in the GPT assay, 0.5 mL of 0.8 mol alanine in 0.1 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Tris-HCl (pH 7.5) was substituted for 0.5 mL of 0.1 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e buffered aspartate solution in the reaction mixture, and aniline citrate addition was omitted [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eActivities of protease and peptidase assay\u003c/h2\u003e \u003cp\u003eFresh seed samples were homogenized at 4\u0026deg;C in 1 mL of β-mercaptoethanol extraction buffer (pH\u0026thinsp;=\u0026thinsp;6.8). Cell debris was removed by centrifugation, and the supernatant was placed on ice, and the protease activity determined spectrophotometrically at 400 nm using azocasein as a substrate. Peptidase activity was determined as described by Hu et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A total of 0.1 mL extract was added to 1-mL buffer containing 50 mM Tris-HCl (pH 8.0), 1 m Mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MnCl2, and 5 m Mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e peptide, and then 25 mL of the reaction mixture was incubated at 37\u0026deg;C for 30 min in 1 mL of a 1% ninhydrin solution containing 100 mg of cadmium acetate, 85 mL of ethanol, and 15 mL of acetic acid in a total volume of 100 mL. The optical density was measured at 505 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData processing and statistics\u003c/h2\u003e \u003cp\u003eExperimental data were analyzed using SPSS 25 (IBM Corp., Armonk, NY, USA). The significance of the differences between different treatments was tested with the least significant difference (P\u0026thinsp;\u0026le;\u0026thinsp;0.05), and the Pearson correlation coefficient was used to measure their correlations. The tables and figures were processed and plotted with Excel 2016 (Microsoft Corp., Redmond, USA) and Origin 2023 (OriginLab, Northampton, Massachusetts, USA).\u003c/p\u003e \u003cp\u003ePath analysis can decompose the direct correlation between the independent variables and the dependent variables, study their direct and indirect importance, and provide a theoretical basis for accurate statistical decisions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this paper, SPSS software, Pearson method, physiological indicators related to nitrogen metabolism (soluble protein, SP content, AA content, GOT activity, GPT activity, protease activity, and peptidase activity) as the independent variable (X), Bt protein content as the dependent variable (Y), and the direct and indirect effect of physiology related to nitrogen metabolism on Bt protein content.\u003c/p\u003e \u003cp\u003ePyi\u0026thinsp;=\u0026thinsp;bisi/sy; Pyij\u0026thinsp;=\u0026thinsp;rij\u0026times;Pyi\u003c/p\u003e \u003cp\u003eWhere Pyi and Pyij represent the direct diameter coefficient and the indirect diameter coefficient rij are the correlation coefficient between independent variable i and dependent variable j; bi is the partial regression coefficient of dependent variable y on independent variable i; si and sy represent the standard deviation of i and y respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Siyang Cotton Seed Farm and Institute of Biotechnology, Chinese Academy of Agricultural Sciences (CAAS), China, for experimental materials support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C., X.Z., D.C. and Z.L. designed the study. S.D., Y.D. and Z.L. performed the experiments. Y.C. and Z.L. analyzed the data and wrote the manuscript. Y.C., Y.L.C. and Z.L. checked and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Natural Science Foundation of China (31901462), the China Scholarship Council (202308320440), and the Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province, China (KYCX22_3508).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. Plant samples were collected from university research area. Study protocol must comply with relevant institutional, national, and international guidelines and legislation. Our experiment follows with the relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRout G, Swain R, Sahoo DP. Journey of genetically modified crops: Status and prospects. Magna Sci Adv Res Rev. 2023;7:103\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026eacute;ma O, Som\u0026eacute; HN, Traor\u0026eacute; O, Greenplate J, Abdennadher M. 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Agron Sinica. 2022;48(6):1537\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahmoud MW, Hussein E, Ashour K. Sequential path analysis for determining the interrelationships between yield and its components in peanut. Egypt J Agron. 2020;42:79\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"low-temperature, cotton seed, Cry1Ac endotoxin, protein metabolism","lastPublishedDoi":"10.21203/rs.3.rs-4450426/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4450426/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020\u0026ndash;2021, two Bt transgenic cotton cultivars, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20\u0026deg;C) with different durations (12h, 24h and 48h) were applied during the peak boll-setting period.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAs the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the temperature causing a significant change in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17\u0026deg;C to 19\u0026deg;C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn summary, when the daily average temperature was \u0026le;\u0026thinsp;19\u0026deg;C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production.\u003c/p\u003e","manuscriptTitle":"Low temperature exposure decreases Cry1Ac insecticidal endotoxin content in cotton seeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 06:52:29","doi":"10.21203/rs.3.rs-4450426/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-27T16:22:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-24T16:29:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T15:28:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T09:53:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208125660340368446377025781038159678045","date":"2024-06-14T16:36:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313229228175603546141592752273945367590","date":"2024-06-14T10:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247041707929969901204911640976073224288","date":"2024-06-13T14:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178264555714539092308861053083564313324","date":"2024-06-05T10:09:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-30T07:54:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117501779912645843579214575037053447129","date":"2024-05-24T17:59:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277514818067138333160547827752387934438","date":"2024-05-23T01:58:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-22T15:58:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T06:16:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T06:15:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-05-20T17:04:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8aa436f-f4ec-4c81-bbb1-68075dd0c6c9","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:41:53+00:00","versionOfRecord":{"articleIdentity":"rs-4450426","link":"https://doi.org/10.1186/s12870-024-05500-y","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-08-15 15:58:08","publishedOnDateReadable":"August 15th, 2024"},"versionCreatedAt":"2024-06-03 06:52:29","video":"","vorDoi":"10.1186/s12870-024-05500-y","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05500-y","workflowStages":[]},"version":"v1","identity":"rs-4450426","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4450426","identity":"rs-4450426","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}