Date of Defense
29-4-2025 5:00 PM
Location
F3-110
Document Type
Dissertation Defense
Degree Name
Doctor of Philosophy in Horticulture Science
College
CVAM
Department
Integrative Agriculture
First Advisor
Prof. Khaled Masmoudi
Keywords
DHNs, heterologous expression, heat, salinity, dehydration, abiotic stress tolerance, enzymatic activity, toxin proteins
Abstract
Dehydrins (DHNs) are hydrophilic, glycine-rich proteins that accumulate in vegetative tissues of plants in response to abiotic stress and during the late stages of seed maturation. In desert plants, these proteins likely contribute to their specialized adaptation under extreme environmental conditions. To examine the genetic mechanisms underlying DHN associated abiotic stress tolerance in desert plants, this study aimed to isolate them and investigate the architectural features and functional mechanisms related to them. It involved cloning of full-length cDNA sequences of DHN encoding genes, PcDHN1, CcDHN1, PdDHN1, and PdDHN2, from three desert plants Prosopis cineraria, Citrulus colocynthis, and Phoenix datcylifera, followed by bioinformatic analysis and structural modeling. Functional characterization included heterologous expression of these DHNs into yeast mutants and Arabidopsis plants to study their role in heat, salinity, and dehydration tolerance. Recombinant DHN proteins were further produced to examine their protective effects on enzymes, lactate dehydrogenase (LDH) and β-glucosidase (bglG), and Bt biopesticide formulations under adverse conditions. Through sequence analysis, it was found that PcDHN1 and CcDHN1 belonged to Y2SK2 and Y3SK-type DHNs, respectively, whereas PdDHN1 and PdDHN2 were FSK3-type. The four DHN proteins exhibited high hydrophilicity, as indicated by GRAVY scores of less than 0. Structural modeling predicted the presence of wide disordered regions in the DHNs, with random coils and short segments of α-helix constituting a major portion of their structure. In yeast knockout mutants (Cdc25 and AXT3K), the DHNs expression significantly enhanced their tolerance to heat and salinity stress. DHN transformed AXT3K yeast cells showed a selective accumulation of K+ with an increase of 54.28% to 66.64% and decline in Na+ content by 44.96% to 50.67% under saline conditions, providing a 4-fold increase in K+/Na+ ratio. The generated transgenic Arabidopsis lines for dehydration stress tolerance exhibited significantly high proline content and reduced accumulation of reactive oxygen species (ROS), specifically hydrogen peroxide (H₂O₂) and the superoxide radical (O₂⁻), which was associated with an increase of 2-fold in the activity of the antioxidant enzyme system. The overexpression of DHNs in Arabidopsis also enhanced the expression of stress-responsive genes such as RD29A, RD29B, NCED3, and HVA22D that improved their adaptation. Furthermore, these DHN proteins effectively preserved LDH function by a 3- to 4-folds under heat stress, dehydration-rehydration cycles, and freeze thaw treatments, preventing about 20% to 70% of LDH aggregation. While for bglG, it enhanced and protected around 2- to 7-folds of its activity under the heat stress condition. Moreover, the δ- endotoxin Bt formulation complemented with purified DHN proteins showed enhanced efficacy in controlling red palm weevils (RPWs), decreasing their egg hatching by 43.54% to 61.38%, and increasing the larval and adult mortality rates by 2-fold under high temperature stress. Thus, the present study interpreted the potential ability of DHNs to alleviate abiotic stress and protect biomolecules under adverse conditions.
Included in
RESPONSIVENESS OF DESERT PLANT DEHYDRIN GENES IN THE ALLEVIATION OF ABIOTIC STRESSES
F3-110
Dehydrins (DHNs) are hydrophilic, glycine-rich proteins that accumulate in vegetative tissues of plants in response to abiotic stress and during the late stages of seed maturation. In desert plants, these proteins likely contribute to their specialized adaptation under extreme environmental conditions. To examine the genetic mechanisms underlying DHN associated abiotic stress tolerance in desert plants, this study aimed to isolate them and investigate the architectural features and functional mechanisms related to them. It involved cloning of full-length cDNA sequences of DHN encoding genes, PcDHN1, CcDHN1, PdDHN1, and PdDHN2, from three desert plants Prosopis cineraria, Citrulus colocynthis, and Phoenix datcylifera, followed by bioinformatic analysis and structural modeling. Functional characterization included heterologous expression of these DHNs into yeast mutants and Arabidopsis plants to study their role in heat, salinity, and dehydration tolerance. Recombinant DHN proteins were further produced to examine their protective effects on enzymes, lactate dehydrogenase (LDH) and β-glucosidase (bglG), and Bt biopesticide formulations under adverse conditions. Through sequence analysis, it was found that PcDHN1 and CcDHN1 belonged to Y2SK2 and Y3SK-type DHNs, respectively, whereas PdDHN1 and PdDHN2 were FSK3-type. The four DHN proteins exhibited high hydrophilicity, as indicated by GRAVY scores of less than 0. Structural modeling predicted the presence of wide disordered regions in the DHNs, with random coils and short segments of α-helix constituting a major portion of their structure. In yeast knockout mutants (Cdc25 and AXT3K), the DHNs expression significantly enhanced their tolerance to heat and salinity stress. DHN transformed AXT3K yeast cells showed a selective accumulation of K+ with an increase of 54.28% to 66.64% and decline in Na+ content by 44.96% to 50.67% under saline conditions, providing a 4-fold increase in K+/Na+ ratio. The generated transgenic Arabidopsis lines for dehydration stress tolerance exhibited significantly high proline content and reduced accumulation of reactive oxygen species (ROS), specifically hydrogen peroxide (H₂O₂) and the superoxide radical (O₂⁻), which was associated with an increase of 2-fold in the activity of the antioxidant enzyme system. The overexpression of DHNs in Arabidopsis also enhanced the expression of stress-responsive genes such as RD29A, RD29B, NCED3, and HVA22D that improved their adaptation. Furthermore, these DHN proteins effectively preserved LDH function by a 3- to 4-folds under heat stress, dehydration-rehydration cycles, and freeze thaw treatments, preventing about 20% to 70% of LDH aggregation. While for bglG, it enhanced and protected around 2- to 7-folds of its activity under the heat stress condition. Moreover, the δ- endotoxin Bt formulation complemented with purified DHN proteins showed enhanced efficacy in controlling red palm weevils (RPWs), decreasing their egg hatching by 43.54% to 61.38%, and increasing the larval and adult mortality rates by 2-fold under high temperature stress. Thus, the present study interpreted the potential ability of DHNs to alleviate abiotic stress and protect biomolecules under adverse conditions.