Effect of exogenous substances on insecticidal protein contents of low temperature affected Bt cotton plants

Bt ( Bacillus thuringiensis ) cotton exhibits effective resistance against lepidopteran insects; however, substantial declines in both the yield and quality of cotton plants are induced by low-temperature stress, concurrently decreasing Bt protein expression and insecticidal efficiency. As a primary fiber crop of significant industrial interest, Bt cotton requires stable agronomic performance under abiotic stress. This study employed a three-factor completely randomized experiment within a controlled-environment chamber to evaluate the effects of seven composite treatment groups (a water control and six exogenous regulators administered at 16°C) across three recovery sampling times (48, 72, and 96 h) on Bt toxin levels and nitrogen metabolic status in boll shells of two Bt cultivars. Results demonstrated that foliar application of 20 mmol/L CaCl₂ was the most effective in maintaining Bt protein stability during stress, resulting in a measured content numerically comparable to the optimal temperature control; chitosan (CTS) and salicylic acid (SA) exhibited partial mitigation effects. During the recovery phase (48–96 h), measured Bt protein content under 6-BA treatment recovered significantly compared with the water-sprayed control based on pooled data across growing seasons and cultivars (Tukey's HSD, 𝑃 < 0 . 0 5 , 12 biological replicates).Partial least squares path modeling (PLS-PM) revealed that at the end of chilling stress (48 h), soluble protein content ( 0 . 3 6 0 * * ) and proteolytic enzyme activity ( − 0 . 3 7 0 * ) exerted significant direct positive and negative effects on Bt protein content, respectively. During the recovery phase (96 h), proteolytic enzyme activity was the primary direct inhibitory factor (-0.599**), whereas transamination (48 h: 0.918**; 96 h: 0.992**) and free amino acid content (48 h: 0.574**; 96 h: 0.942**) indirectly regulated Bt protein accumulation by altering soluble protein synthesis. This study reveals that exogenous regulators facilitate the shift of boll shell nitrogen metabolism from a ‘degradation-dominant’ to a ‘synthesis-dominant’ mode by enhancing protein synthesis and suppressing proteolysis.