Proteomics plays a crucial role in studying cellular differentiation, signal transduction, cancer development, and treatment. In recent years, it has also been increasingly important in plant protein research. The vast functional, spatial, and temporal diversity of plant proteomes is regulated by multiple factors that continuously modulate protein abundance, modifications, interactions, localization, and activity to meet the dynamic demands of plants. The analysis of proteome complexity and its potential genetic variations has garnered growing research attention. Mass spectrometry (MS)-based proteomics has emerged as a powerful method for studying protein function and its systemic relationships on a global scale.
What Can Proteomics Based on MS Provide for Plant Research?
Currently, many bottom-up proteomics studies have been conducted for large-scale, quantitative, and qualitative analysis of plant proteins, resulting in extensive peptide lists from specific samples. Quantitative proteomics methods enhance the characterization of protein abundance and post-translational modifications (PTMs).
Accurate, systematic, and quantitative proteomics methods are critical prerequisites for the development of all mass spectrometry techniques. Data-dependent acquisition (DDA) is the most widely used proteomics analysis method. Data-independent acquisition (DIA) is an emerging MS-based proteomics technique that greatly improves proteome coverage and detection accuracy. The complexity of high-abundance proteins and protein sample complexity can affect the detection of mass spectrometry signals. Pre-fractionation can be used to reduce sample complexity and increase proteome coverage. Additionally, increasing chromatographic separation time and using optimized columns such as C4 columns can more efficiently separate peptide fragments.
Proteomics can characterize PTMs and cellular signaling. Enrichment of PTMs, such as metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) for phosphorylation, enhances the depth of phosphoproteome coverage. This overcomes challenges related to variations in the quality of modified peptides and low abundance. Phosphoproteomics analysis is widely used to monitor the dynamic phosphorylation of plants under developmental changes and biological or abiotic stress, elucidating the regulatory mechanisms of various environmental signals. Phosphoproteomics analysis can also identify typical phosphorylation events on specific amino acid residues, thus unraveling specific cellular processes.
Cellular organelles that make up plant cells are crucial for all biological processes. Therefore, capturing the proteome of organelles and comprehensively understanding the subcellular localization and dynamics of proteins is essential. In plants, organelle proteomics analysis has become a primary method for studying protein localization, organelle composition, dynamics, and function.
Organelle analysis involves the purification of target organelles and the identification and quantification of proteins in different enriched subfractions. Through clustering analysis, proteins associated with the target organelle exhibit similar distribution characteristics, allowing differentiation from contaminants. This facilitates the study of organelle, sub-organelle, protein distribution, and their relationship with metabolites.
Proteomics in Studying Plant Stress Tolerance
Plants experience various abiotic stressors such as high temperature, low temperature, drought, and high salinity during their growth and development. Upon sensing these stress signals, plants regulate the expression of stress-responsive proteins through signal transduction pathways, thereby adjusting their physiological or morphological traits to enhance their tolerance to abiotic stresses.
The changes in plant protein metabolism under low-temperature stress and their relationship with cold tolerance have drawn significant interest. Researchers have observed variations in protein content in the leaves of Toona sinensis seedlings under low-temperature stress. Different sources of plants exhibited inconsistent responses at different temperatures, and dynamic changes were also observed within the same source under different stress durations. The impact of low-temperature stress on protein in Toona sinensis was not only reflected in protein content but also through the increase in protein components to maintain tissue stability. New protein components appeared and accumulated in Toona sinensis leaves with increasing severity of low-temperature stress, suggesting their association with cold tolerance.
Plant responses to abiotic stresses (Khan et al., 2014).
In a study by Makoto Hashimoto, Setsuko Komatsu, and others, the protein content of rice seedlings was found to change under low-temperature stress, with 19 proteins showing increased content and 20 proteins showing decreased content. Minor changes in stress-responsive proteins were evident, and four proteins were newly synthesized under low temperature. In the leaves, proteins related to energy metabolism were upregulated, while defense-related proteins were downregulated. These findings indicate that energy products are activated under low temperature, leading to the rapid upregulation of stress-related proteins, while defense proteins disappear under prolonged low-temperature exposure. Cui and colleagues investigated the proteomic changes in rice seedlings under progressively decreasing temperatures from room temperature to 15, 10, and 5°C and identified 60 proteins that were induced by the progressive low-temperature treatment.
When exposed to high-temperature stress, plants can induce the expression of a large number of heat shock proteins (HSPs). These proteins are essential components for plants' short-term adaptation to stress and help mitigate the damage caused by high-temperature stress. Yves Meyer and Yvette Charties studied the proteome of tobacco leaf mesophyll protoplasts after heat treatment and found that protein synthesis was largely unaffected, but the synthesis of certain proteins was significantly reduced. Additionally, 17 specific heat shock proteins were newly induced.
Proteomics in Plant Development and Growth
Proteomics has significantly contributed to our understanding of the molecular mechanisms involved in plant development and growth processes. By employing proteomic analysis techniques, researchers have been able to investigate the changes in protein expression throughout various stages of plant development, enabling the identification of key proteins associated with important developmental events.
During plant development, differentiating cells undergo specialized processes such as cell differentiation, organ formation, flowering, and fruit development, which are regulated by specific proteins. Proteomic studies have allowed researchers to identify and characterize these proteins, providing insights into their functions and regulatory roles in plant development.
Workflow of Proteomics (Yadav et al., 2023)
For example, in a study on seed germination in Pinus tabuliformis, the changes in nucleic acids and proteins were examined. The results revealed that protein content exhibited varying degrees of increase or decrease during germination, with pronounced changes observed in embryo protein components. Acidic proteins in the lower isoelectric point range (pH 4.3-5.2) showed continuous accumulation, while alkaline proteins in the higher isoelectric point range (pH 6.5-8.0) and low molecular weight (below 4.3×10 kDa) gradually degraded and disappeared. These proteins were identified as a group of glycoproteins based on PAS staining. The dynamic presence and absence of these proteins during different stages of seed embryo development are indicative of their specific roles in regulating embryo growth and the establishment of healthy seedlings.
By studying the proteome during plant development, researchers can gain a comprehensive understanding of the molecular events that drive growth and morphogenesis. This knowledge is vital for unraveling the intricate regulatory networks that govern plant development and may have implications for crop improvement and agricultural practices.
Proteomics in Plant Metabolic Pathways
One application of proteomics in plant metabolic pathways is the identification of enzymes and proteins involved in primary metabolism, including carbohydrate metabolism, amino acid metabolism, and lipid metabolism. By mapping the proteomes of different tissues or developmental stages, researchers can identify key enzymes and regulatory proteins that control metabolic fluxes, substrate utilization and product accumulation. This knowledge contributes to the understanding of the regulation of key metabolic pathways and facilitates the development of strategies to improve crop traits such as improved yield and nutrient content.
In addition to primary metabolism, proteomics has elucidated the complex network of enzymes and proteins involved in secondary metabolism, which produces a variety of specific compounds, including phytochemicals, pigments, and defense compounds. Through proteomic analysis, researchers have successfully identified and characterized enzymes, transporter proteins, and regulatory proteins involved in the biosynthesis of secondary metabolites. This knowledge allows the manipulation of metabolic pathways to enhance the production of secondary metabolites with pharmaceutical, nutritional or industrial applications.
Proteomics in Crop Improvement
Through proteomic analysis, researchers can identify proteins involved in key physiological processes, such as photosynthesis, carbon assimilation and nutrient uptake. By understanding the proteomic basis of high-yielding varieties, breeders can select or genetically engineer crops to improve energy capture and utilization efficiency, ultimately increasing productivity.
Proteomics of model and crop plant species (Vanderschuren et al., 2013)
Proteomics has also proven valuable in improving crop resistance to pests, diseases, and abiotic stresses. By comparing the proteomes of resistant and susceptible varieties, researchers can identify proteins associated with defense responses, stress tolerance and signaling pathways. This knowledge can be used to develop crops with enhanced resistance to biotic and abiotic stresses through conventional breeding or genetic engineering approaches.
In addition, proteomics can help to improve the nutrient use efficiency of crops. By studying the proteome of plants under different nutrient availability, researchers can identify proteins involved in nutrient uptake, transport and utilization. This information can guide the development of crop varieties to improve nutrient uptake efficiency, minimize fertilizer requirements and reduce environmental impact.
References
- Khan, PS Sha Valli, et al. "Abiotic stress tolerance in plants: insights from proteomics." Emerging technologies and management of crop stress tolerance. Academic Press, 2014. 23-68.
- Yadav, Bal Govind, et al. "Understanding the Proteomes of Plant Development and Stress Responses in Brassica Crops." Journal of Proteome Research 22.3 (2023): 660-680.
- Vanderschuren, Hervé, et al. "Proteomics of model and crop plant species: status, current limitations and strategic advances for crop improvement." Journal of proteomics 93 (2013): 5-19.
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