Morpho-Physiological and Proteomic Response of Bt-Cotton and Non-Bt Cotton to Drought Stress

Abstract

Drought stress impacts cotton plant growth and productivity across countries. Plants can initiate morphological, cellular, and proteomic changes to adapt to unfavorable conditions. However, our knowledge of how cotton plants respond to drought stress at the proteome level is limited. Herein, we elucidated the molecular coordination underlining the drought tolerance of two inbred cotton varieties, Bacillus thuringiensis-cotton [Bt-cotton + Cry1 Ac gene and Cry 2 Ab gene; NCS BG II BT (BTCS/BTDS)] and Hybrid cotton variety [Non-Bt-cotton; (HCS/HDS)]. Our morphological observations and biochemical experiments showed a different tolerance level between two inbred lines to drought stress. Our proteomic analysis using 2D-DIGE revealed that the changes among them were not obviously in respect to their controls apart from under drought stress, illustrating the differential expression of 509 and 337 proteins in BTDS and HDS compared to their controls. Among these, we identified eight sets of differentially expressed proteins (DEPs) and characterized them using MALDI-TOF/TOF mass spectrometry. Furthermore, the quantitative real-time PCR analysis was carried out with the identified drought-related proteins and confirmed differential expressions. In silico analysis of DEPs using Cytoscape network finds ATPB, NAT9, ERD, LEA, and EMB2001 to be functionally correlative to various drought-responsive genes LEA, AP2/ERF, WRKY, and NAC. These proteins play a vital role in transcriptomic regulation under stress conditions. The higher drought response in Bt cotton (BTCS/BTDS) attributed to the overexpression of photosynthetic proteins enhanced lipid metabolism, increased cellular detoxification and activation chaperones, and reduced synthesis of unwanted proteins. Thus, the Bt variety had enhanced photosynthesis, elevated water retention potential, balanced leaf stomata ultrastructure, and substantially increased antioxidant activity than the Non-Bt cotton. Our results may aid breeders and provide further insights into developing new drought-tolerant and high-yielding cotton hybrid varieties.

Keywords: 2D difference gel electrophoresis; Cytoscape; MALDI-TOF/TOF MS; abiotic stress; qRT-PCR.

FIGURE 1

Performance of BT and Non-BT cotton plants against drought stress. (A) BT and Non-BT seed germination Control without mannitol, with 100 mM mannitol and with 200 mM mannitol. (B) BT and Non-BT cotton plants (60–65 days old) were subjected to drought (200 mM) for 15 days at 30°C. Later, the treated plants were allowed to grow under normal conditions for 20 days and later photographed.

FIGURE 2

Biochemical characterization of BT and Non-BT cotton lines against drought stress condition. For analyzing the stress tolerance ability of Bt and Non-Bt cotton plants at the 1-month stage, drought stress (200 mM mannitol) was applied for 10 days at 30°C. Later, the treated plants were allowed to grow under normal conditions. After 0, 4, 8, and 12 days of mannitol stress, the samples were used for analysis. Data on (A) chlorophyll content, (B) carotenoid, (C) APX, (D) catalase, (E) SOD, and (F) proline were recorded and photographed. In each treatment, 10 seedlings of BT and Non-BT varieties were used. Bar represents mean and I represents SE from three independent experiments. ^∗indicates significant differences in comparison with the C at P < 0.05, FW represents fresh weight.

FIGURE 3

Observations of leaf stomata under a scanning electron microscope of BT and Non-BT cotton.

FIGURE 4

(A) 2-DE analysis of protein. Proteome maps of Intra-hirsutum Hybrid (Gossypium hirsutum) cotton varieties (BT hybrid—BTCS and BTDS and Non-Bt varieties—HCS and HDS). The numbered protein spots were identified by MALDI-TOF from BTCS and HCS grown in well-watered conditions, and BTDS and HDS grown under drought stress with 200 mM mannitol. (B) Venn diagrams of BTCS/BTDS and HCS/HDS. Venn diagrams representing the overlap of identified DEP spots, which were underexpressed and overexpressed. Unique spots in between groups such as BTCS and BTDS, and HCS and HDS. (C) Venn diagrams of BTCS/HDS and BTCS/HCS. Venn diagrams representing the overlap of identified DEP spots, which were unique spots, different spots, and matched spots in between groups such as BTDS and HDS, and BTCS and HCS.

FIGURE 5

(A) The subcellular locations of the identified proteins. (B) GO classification of the identified DEPs. To reveal the functions of the identified eight DEPs between Bt and Non-Bt, GO functional analysis was carried out using Panther classification system software. The identified eight DEPs were classified into three main categories including cellular component, biological process, and molecular function with seven subgroups. The number of genes signifies that of proteins with GO annotations. (C) Interactions of these differentially accumulated proteins are extracted using the app GeneMANIA from Cytoscape.

FIGURE 6

Real-time PCR analysis of ATP Syn, NAT9, GTP-binding protein, DREB, WRKY, LEA, AP2/ERF, and NAC genes in BT and Non-BT cotton variety plants under drought stress conditions. Relative transcript levels of genes in BT and Non-BT cotton plants subjected to 2000 mM mannitol stressed condition were analyzed using real-time PCR. Actin gene was used as a reference. Bar represents mean, and I represents SE from three independent experiments. Asterisk indicates significant differences in comparison with Non-BT at P < 0.05.