Computational Molecular Biology 2024, Vol.14, No.5, 191-201 http://bioscipublisher.com/index.php/cmb 193 3 Proteomics Approaches in Studying Drought Stress 3.1 Overview of proteomics techniques Proteomics techniques have become indispensable in studying plant responses to drought stress (Liu et al., 2019). High-performance liquid chromatography attached with tandem mass spectrometry (HPLC-MS/MS) and 2D-PAGE are the most commonly used methods. HPLC-MS/MS allows for the high-throughput identification and quantification of proteins, providing detailed insights into the proteome's composition and dynamics under stress conditions. similarly, 2D-PAGE method can separate proteins based on their isoelectric point and molecular weight, enable the detection of post-translational modifications and change in protein abundance. These techniques, combined with bioinformatics tools, facilitate the comprehensive analysis of protein expression and function in response to drought stress. 3.2 Quantitative proteomics in drought stress research Quantitative proteomics has been instrumental in understanding the molecular mechanisms underlying drought tolerance in rice. Techniques such as label-free quantification and tandem mass tag (TMT) multiplexing have been used to accurately measure changes in protein abundance. A study using label-free quantitative proteomics identified significant alterations in the rice roots proteome under drought conditions, highlighting the upregulation of stress-related proteins and the downregulation of photosynthetic machinery (Wu et al., 2016; Shi et al., 2018). Similarly, TMT-based approaches have revealed the differential expression of proteins (DEPs) involved in various metabolic pathways, providing insights into the adaptive responses of rice to water deficit (Mirzaei et al., 2012). 3.3 Identification of drought-responsive proteins in rice Several studies have identified proteins that respond to drought stress in rice. Paul et al. (2015) and Agrawal et al. (2016) performed proteomic analysis of rice leaves under drought conditions identified proteins involved in energy metabolism, cell defense, and signal transduction as being differentially expressed. Notably, proteins namely actin depolymerizing factor, chloroplastic glutathione-dependent dehydroascorbate reductase, and caffeoyl-CoAO-methyltransferase have been reported to increase in abundance during drought stress (Salekdeh et al., 2002; Ali and Komatsu, 2006). Additionally, dehydrogenase and pyruvate dehydrogenase proteins related to carbohydrate and energy metabolism, have been implicated in enhancing drought tolerance. These findings underscore the complex network of protein interactions and regulatory mechanisms that enable rice to adapt to drought stress. 4 PPI Networks 4.1 Definition and significance of PPI networks PPI networks are conceptualized as maps of the physical and functional interactions between proteins within a cell. These networks are of great importance for the comprehension of the intricate biological processes and pathways that regulate cellular functions. In the context of rice subjected to drought stress, PPI networks can elucidate the mechanisms through which proteins interact to mediate stress responses, thereby potentially identifying key proteins that contribute to drought tolerance. One of the study on Brachy podium distachyon roots and leaves under drought stress demonstrated the significance of PPI networks in generating synergistic responses to stress (Bian et al., 2017). Similarly, the hybrid protein interactome in rice has been showed to contribute to heterosis, with specific PPIs potentially enhancing stress resilience (Li et al., 2019). 4.2 Techniques for constructing PPI networks A variety of experimental techniques are employed in the construction of PPI networks. The yeast two-hybrid (Y2H) assay is a method used to detects physical interactions between two proteins. This is achieved by reconstituting a functional TF in yeast. It has been employed extensively for the mapping of large-scale PPI networks. Co-Immunoprecipitation (Co-IP) employs antibodies to precipitate a protein of interest along with its interacting partners from a cell lysate, thereby facilitating the identification of protein complexes. The label-free shotgun proteomics can be employed to identify and quantify proteins and their interactions under different conditions. For example, this approach has been used to examine the effects of drought stress in rice (Hamzelou et al., 2020; Bai et al., 2021).
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