Cotton (Gossypium hirsutum L.), a crucial global fiber and oil seed crop, faces a diverse biotic and abiotic stresses. Among these, temperature stress strongly influences its growth, prompting adaptive physiological, biochemical and molecular changes. In this study, we explored the proteomic changes underscoring the heat stress tolerance in the leaves of two locally developed cotton genotypes, i.e., heat tolerant (GH-Hamaliya H_tol) and heat susceptible (CIM-789 H_sus), guided by morpho-physiological and biochemical analysis. These genotypes were sown at two different temperatures, control (35ºC) and stress (45ºC) in glass house, in randomized complete block design (RCBD) in three replications. At the flowering stage, a label-free quantitative shotgun proteomics of cotton leaves revealed the differential expression of 701 and 1270 proteins in the tolerant and susceptible genotype compared to the control, respectively. Physiological and biochemical analysis showed that the heat-tolerant genotype responded uniquely to stress by maintaining the net photosynthetic rate (Pn)( 25.2-17.5 µmolCO₂m^-2S^-1), chlorophyll (8.5-7.8 mg/g FW), and proline contents (4.9-7.4 µmole/g) compared to control, supported by the upregulation of many proteins involved in several pathways including photosynthesis, oxidoreductase activity, response to stresses, translation, transporter activities as well as protein and carbohydrate metabolic processes. In contrast, the distinctive pattern of protein downregulation involved in stress response, oxidoreductase activity, and carbohydrate metabolism was observed in susceptible plants. To the best of our knowledge, this is the first proteomic study on cotton leaves that has identified more than 8000 proteins with an array of differentially expressed proteins responsive to the heat treatment that could serve as potential markers in the breeding programs after further experimentation.

Protein digestion and samples preparation for nano-LC-MS/MS

For in-solution digestion of extracted proteins, 100 µg of protein sample was utilized with some modifications of the method described earlier [29,30]. A 100-μg protein aliquot was taken, reduced with 10 mM DTT for 1 hour at 30 °C, alkylated with 40 mM iodoacetamide (IAA) for 30 minutes at room temperature, and then covered to protect from light. The urea concentration was diluted by adding 50 mM ammonium bicarbonate in a 1:9 (sample: ammonium bicarbonate). Trypsin (Promega) was then used to digest the proteins for 18 hours at 37 ºC. Trifluoroacetic acid (TFA) (10%) was added to the mixture to stop the digestion, and the peptides were passed through a C18 Poros R2 resin (Applied Biosystems) column for cleaning. The column was washed three times with 100% acetonitrile, followed by the application of the peptides. After passing, the peptides were cleaned of any salt and detergent residue using 200 µl of 0.1% TFA. The peptides were eluted in acetonitrile with 0.1% TFA, 50% ACN, and 50 µl of 0.1% TFA and 70% ACN. A Thermo Fisher SRF 110-speed vacuum concentrator was used to concentrate the eluted peptides. The peptides were solubilized in 15 µl of 0.1% formic acid to ensure peptide recovery.

Nano-LC-MS/MS analysis

Aliquots containing 6 µg of peptides from every sample were diluted with 0.1% FA to make a final concentration of 0.5 µg/µL. The samples were then analyzed in a Thermo Fisher Scientific nanoLC-EASY 1000 system connected online to an ESI-Q Exactive Plus Orbitrap mass spectrometer. A 4 µL sample was eluted from a C18 ReproSil-Pur (C18-AQ) trap column (Maisch GmbH in Ammerbuch, Germany). It was separated in a C18 column (20 cm length× 75 μm diameter) with a 3 μm particle diameter. The chromatographic separation was taken for 120 minutes with a separation flow rate of 200 nL/min, followed by a gradient of 5% to 30% of phase A (H₂O 95%, ACN 5%, FA 0.1%) for 100 minutes and 30% to 45% of phase B (H₂O 5%, ACN 95%, FA 0.1%) for 9 minutes, ending on isocratic 95% of phase B for 5 minutes. The peptides were positively polarized and ionized at 2.30 KV in an ESI source. Data was collected in DDA mode while the ion transfer capillary was maintained at 200 °C. The MS1 spectra comprised a complete scan in the m/z range of 350.0–1800.0, including a minimum intensity of 2000 and a resolution of 60,000 FWHM (for m/z 400). The 20 most intense precursor ions were fragmented by HCD for MS2 acquisition with a resolution of 7,500 FWHM (for m/z 400), normalized CE (collision energy) of 30 V, an isolation window of 2.5 m/z, and dynamic exclusion of 45 s.