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

The effects of mepiquat chloride (DPC) on the Cry1Ac protein content in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and -efficiency cotton cultivation.  This study was conducted using Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, Jiangsu Province, China.  Potted cotton plants were exposed to high temperature and drought stress, and sprayed with either 20 mg L⁻¹ DPC or water (CK).  Seven days after treatment, the Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed.  DESeq was used for differential gene analysis.  Under the DPC treatment, the Cry1Ac protein content increased by 4.7–11.9% compared to CK.  The α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased.  Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs. CK.  Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses showed that the differentially expressed genes were mainly involved in biological processes, such as carbon and amino acid metabolism.  For example, genes encoding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated.  The DPC treatment increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.  It also significantly upregulated the genes encoding glutamate synthase, pyrrolidine-5-carboxylic acid dehydrogenase, glutamate oxaloacetate transaminase, and N-acetylglutamate synthetase, while it downregulated the genes encoding glutamine synthetase.  Therefore, the synthesis of aspartic acid, glutamic acid, pyruvate, and arginine increased after treatment with DPC, and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.