The integration of different biological methods has become an important trend in research. Among them, the integration of proteomics and metabolomics approaches has played a crucial role in studying the biochemical processes and metabolic networks of plants. One plant that has received significant attention in this regard is guava (Psidium guajava).
Proteomics involves the large-scale study of proteins, including their structure, function, and interactions within a biological system. By applying proteomics techniques to guava research, scientists have been able to identify and characterize the abundance, localization, and post-translational modifications of proteins involved in various physiological processes of the plant. This provides valuable insights into the molecular mechanisms underlying guava's growth, development, and response to environmental stimuli.
On the other hand, metabolomics focuses on the comprehensive analysis of small molecules, such as metabolites and their intermediates, within a biological system. By employing metabolomics approaches, researchers have been able to investigate the metabolic pathways and profiles in guava, elucidating the composition and changes in metabolites under different growth conditions, stress responses, and fruit ripening stages. This enables a better understanding of the metabolic regulation and bioactive compound synthesis in guava.
The integration of proteomics and metabolomics methodologies in guava research allows for a more holistic and comprehensive analysis of the plant's biological processes. By correlating the changes in protein expression with alterations in metabolite levels, scientists can establish a more complete picture of the complex interactions and regulatory networks underlying guava's physiology and metabolism. This integrated approach not only enhances our understanding of guava's biology but also provides valuable information for improving its cultivation, nutritional value, and potential applications in industries such as food and pharmaceuticals.
Case. Integration of proteomics and metabolomics approaches to elucidate the ripening process of white guava (1)
Proteomic analysis
In this study, white guava fruits (Psidium guajava L. cv "criolla") were collected at Piedra de Agua, Jilotepec, Veracruz. The fruits were categorized into two ripening stages: mature green (MG) and mature yellow (MY). The peel, pulp, and seeds (including mucilage for MY) were separated and frozen in liquid nitrogen. Proteomics analysis was performed on pooled samples from three independent trees.
For proteomics, the peel and pulp tissues (2 g) were ground in liquid nitrogen and suspended in an ice-cold extraction buffer. The samples were homogenized and mixed with phenol solution. After centrifugation, the proteins were precipitated with acetone. The resulting pellet was dissolved in phosphate-buffered saline (PBS) with SDS. Protein concentration was determined using the BCA Assay Kit.
Following reduction and alkylation, 100 µg of protein was digested with TCEP and IA. The remaining IA was quenched with DTT. After protein precipitation and drying, the protein pellet was resuspended in TEAM with SDS. Trypsin digestion was performed, and the peptides were labeled with TMT 6plex reagent. The labeled samples were pooled and fractionated using strong cation exchange and high pH reverse-phase C18 cartridges. Each fraction was desalted and dried.
For nano LC-MS/MS analysis, the dried samples were resuspended in formic acid and injected into a nano LC platform. The peptides were separated on a C-18 RSLC column using a gradient of solvent A and solvent B. The analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer with SPS-MS3. Detailed information on data analysis can be found in the Supplementary Materials and Methods of the study.
Researchers analyzed mature green (MG) and mature yellow (MY) guava peel and pulp. Specific protein profiles were observed using SDS-PAGE. Proteomics analysis identified 2,695 proteins in the peel and 3,166 proteins in the pulp, with a core proteome of 2,275 proteins shared between the two tissues. Comparative proteomics revealed significant down-accumulation of proteins during ripening in both peel and pulp, while the MY peel showed protein over-accumulation. Differential protein analysis identified 859 and 817 proteins in the peel and pulp, respectively, with a small overlap. Gene ontology analysis revealed positive regulation of processes such as glucose metabolism and lignin biosynthesis in MY guava samples, while negative regulation was observed in photosynthesis. The study validated the findings through comparative analysis with other fruits during ripening.
Untargeted metabolomics and target metabolomics analysis
In this study, the sample preparation involved collecting mature green (MG) and mature yellow (MY) guava fruits and separating the peel, pulp, seeds, and mucilage surrounding the seeds. The samples were frozen and stored at -80°C until analysis. For metabolomics analysis, the samples were lyophilized, ground, and mixed with diatomaceous earth before extraction with methanol using an accelerated solvent extraction system.
Untargeted metabolomics analysis was performed on methanolic extracts of MG and MY whole fruit samples using an ultra-high resolution chromatograph coupled to a high-resolution mass spectrometer. The chromatographic separation was done using a reversed-phase column, and the analysis was conducted in both positive and negative ionization modes. The acquired data were subjected to statistical analysis and fold change analysis to identify over-accumulated and down-accumulated features in MY compared to MG samples. Tentative identification of metabolites was performed using the Functional Analysis module, and pathway analysis was conducted using the Pathway Analysis module of the MetaboAnalyst bioinformatics platform.
In addition to untargeted metabolomics, targeted metabolomics analysis were carried out for pigment quantification and the quantification of ACC, ABA, and phenolics. For pigment quantification, dried MG and MY peel samples were resuspended in methanol:acetone and analyzed by UPLC-QTOF-HRMS. The quantification of ACC, ABA, and phenolics was performed in methanolic extracts of MG and MY whole fruit as well as separate tissue samples using a UPLC-QqQ-mass spectrometer.
Untargeted metabolomics analysis of MG and MY fruits revealed significant changes in metabolite profiles. MY fruits showed over-accumulation of metabolites related to porphyrin and chlorophyll metabolism, starch and sucrose metabolism, carotenoid biosynthesis, anthocyanin biosynthesis, and flavonoid biosynthesis. Down-accumulated metabolites were associated with flavonoid biosynthesis, secondary metabolism, glucosinolate biosynthesis, and other pathways. Targeted analysis confirmed lower pigment levels in MY peel and higher levels of ACC, ABA, and phenolics in MY fruits. Overall, MY fruits exhibited distinct metabolite profiles compared to MG fruits.
Combined analysis of proteomics and metabolomics
The integration of proteomics and metabolomics data provided insights into the metabolic pathways involved in white guava ripening. Ethylene, carotenoid-ABA, and phenolics biosynthesis pathways played key roles in the ripening process. Over-accumulation of ACC and ACC oxidase indicated increased ethylene production. Decreased carotenoid precursors and increased ABA levels suggested alterations in carotenoid-ABA biosynthesis. Phenolic compound biosynthesis was traced from chorismate, with positive accumulation of enzymes in the pathway. Flavonoid biosynthesis showed changes in naringenin and downstream metabolites. Anthocyanin biosynthesis involved the accumulation of specific enzymes and pigments. These findings highlight the complex metabolic changes occurring during guava ripening.
This paper investigated the regulation of undescribed metabolic pathways during ripening of white guava by using proteomics and metabolomics techniques on different fruit tissues from two ripening stages of white guava. The results revealed that ethylene and abscisic acid (ABA) signaling have positive effects on the regulation of biochemical changes during the ripening process of white guava. This study provides a preliminary understanding of the molecular mechanisms involved in external sensing and signaling during ripening of white guava, in which growth regulators can fine-tune the biochemical changes in fruit traits.
Reference
- Monribot-Villanueva, Juan L., et al. "Integrating proteomics and metabolomics approaches to elucidate the ripening process in white Psidium guajava." Food Chemistry 367 (2022): 130656.
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