Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana

Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana Subhankar Mohanty, Dinesh Pradhan, Prashanth Suravajhala, Giridara Kumar Surabhi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9184162/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Globally, bananas are a major staple food with significant nutritional and commercial potentials and are extremely perishable with a very short shelf life. Understanding the proteins expressed in different tissues during ripening can help in improving postharvest quality and shelf life. A gel-free comparative proteomic analysis was followed to investigate protein alterations in peel and pulp tissues during pre-climacteric and climacteric phases of banana. Total starch content was decreased when ripening progresses. In contrast, total sugar, sucrose, glucose and maltose content was increased several folds during the climacteric phase, in both peel and pulp tissues. The protein samples were subjected to orbitrap fusion mass spectrometry coupled with nano LC-MS/MS resulted in the identification of 950 and 1300 proteins in pulp and 1416 and 1279 proteins in peel tissues at pre-climacteric and climacteric stages, respectively. Mass-spectrometry identified proteins were categorised and they were involved in starch and sugar metabolism, cell wall modification, hormonal regulation and detoxification of reactive oxygen species (ROS), which were more prevalent in the pulp tissue during the climacteric period as compared to the pre-climacteric stage. Proteins such as α-1,4-glucan (α-gluc) phosphorylase, pectin esterase (PE), β-galactosidase (β-gal), α-mannosidase (α-man), pectate lyase (PEL), xyloglucan endotransglucosylase/hydrolase (XTH), pectin acetylesterase and β-hexoaminidase (β-hex) were found to be more in number in climacteric stage, and could be responsible for pulp softening, de-greening of the peel, alterations in texture and fruit quality. The study found that proteins related to sugar metabolism, such as fructose bisphosphate aldolase and sucrose synthase (SUS), were more abundant during the climacteric than pre-climacteric stage. This implies that during fruit ripening, these proteins contribute to the synthesis of sugars and the disintegration of starch. Interestingly, some of the proteins that play a crucial role in hormonal regulation were identified in the form of cysteine synthase (CS) (#17), amino methyltransferase (#10), and a more significant number of CS proteins were identified in pulp tissue at the climacteric stage. Venn analysis of the proteins from different tissues and stages suggests the presence of unique set of proteins in tissue specific manner, and could have a specialized role. Further, the protein-protein interaction study confirms that the XTH could be an ideal candidate known to be associated with unique pathways in peel at the climacteric stage. RTqPCR analysis revealed greater transcript levels of XTH4 and SOD in pulp, and PEL in peel tissue at climacteric stage indicates cell wall disintegration and loosening at climacteric stage. The potential candidate proteins identified in this investigation could be of immense help to gain insights of the regulatory mechanism of banana ripening process. Identified proteins can be further validated using genome editing technology to assign individual functional roles. Post-harvest Proteomics Pulp softening Ripening Cell-wall modification Sugar metabolism RNAi Shelf-life Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1 Introduction Banana belonging to the family Musaceae is the fourth most important food crop after rice, wheat, and corn. Banana is a fruit crop cultivated across tropical and subtropical regions with overall consumption of 21 kgs per person per year. As a climacteric fruit, the physicochemical changes that occur during banana ripening are fast, resulting in a notably perishable fruit [ 1 – 3 ]. Although ripening is an irreversible, genetically regulated, and coordinated process, it produces the desired quality characteristics of edible fruit [ 4 , 5 ]. However, over-softening exacerbates handling damage, reducing fruit's shelf life and making it more vulnerable to post-harvest diseases [ 5 , 6 ]. About 35–40% of the produced fruit and vegetables in India are lost yearly due to excessive softening [ 7 ]. While a genome remains constant to a large extent, the proteins in any particular cell change substantially or drastically because of mRNA degradation, ineffective translation, or selective splicing as well as the post-translational modifications (PTMs) [ 2 , 8 – 11 ]. Thus, in fruit ripening and related post-harvest studies, the proteomic approach has emerged as a crucial technique [ 11 , 12 ]. As proteins are considered effectors of gene expression, they play a central role in regulating many metabolic pathways related to the ripening process [ 3 , 11 ]. In this context, by identifying several functional proteins impacted during the process, proteome studies can aid in elucidation of fruit ripening [ 3 ]. Additionally, expression levels and post-translational modifications may help to understand the complex cellular processes at the protein level in fruit biology research [ 6 , 9 ]. Indeed, there have been several gel-based and gel-free proteomic investigations on the ripening of diverse fruits, including climacteric fruit such as apricot, mango, peach, tomato, oil palm, melon, and non-climacteric fruit such as grapes, chinese bayberry, strawberry and olive [ 6 , 9 , 13 – 21 ] However, in the case of bananas, few studies are reported on the proteome of different organs or tissues, such as meristem of the plant, during drought in leaf tissue and chilling injury mechanism in peel tissues [ 22 – 24 ]. Further, Dominguez-Puigjaner et al. [ 25 ] studied the analysis of proteins from banana pulp tissue limited to separating proteins extracted from the fruit during four stages of maturation. These samples subjected to 2D gel electrophoresis resulted in only five proteins of the same molecular weight identified by immunoblotting as polygalacturonase-related proteins [ 25 ]. Despite the current application of proteomic technique, investigating banana fruit ripening through proteomics is rather scanty. The accurate quantification of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics [ 26 ]. The mass spectrometry-based platforms have provided significant advances in accuracy, sensitivity, and the ability to multiplex vastly complex samples through bioinformatic tools [ 27 ]. Toledo et al. [ 28 ] have identified 26 proteins in the pulp tissue of both pre-climacteric and climacteric stages using nano-LC-MS/MS analysis. Another study by Yun et al. [ 5 ] identified 94 proteins in the peel tissue of bananas at 1, 8, 15, 17, 19, and 21days after harvest by a combined 2DE and MALDI-TOF/TOF-MS based proteome study. The orbitrap fusion mass spectrometry is a high-throughput technique that allows a greater number of peptide coverage with a high resolution at a faster rate with more accuracy [ 29 ]. The sensitivity of the mass spectrometry technology used in this investigation have demonstrated by the identification of 950–1416 proteins in various banana tissue types and stages using orbitrap-fusion mass spectrometry coupled with nano-LC-MS/MS. Previous studies by Inaba et al. [ 30 ] have clearly demonstrated that ethylene signalling was negatively controlled in banana pulp tissue, whereas positively controlled in banana peel [ 30 ]. There are currently few tissue-specific proteome studies, and more research is needed to understand the many plant parts and metabolic processes, such as the starch synthesis and its breakdown into sugars, which are promoter-specific in various tissues [ 31 , 32 ]. The current proteomic study provides information on glucose, maltose and sucrose, protein expression, functional category, protein-protein interactions, and gene expression for selective proteins of diverse metabolic pathways during pre-climacteric and climacteric stages and tissues in banana. The main objectives of this study were (i) to investigate the stage and tissue-specific proteome changes, and (ii) to investigate the proteins involved in various metabolic processes, and protein-protein interactions of the identified proteins that were operational during the ripening process. Our study provides an overview of critical biological processes that was operational during banana fruit ripening. Nevertheless, this is the first comprehensive gel-free proteome analysis of banana fruit tissues that has led to the identification of proteins implicated in various metabolic processes affecting the cell wall re-modelling, sugar and starch metabolism, hormone control, signalling and stress, and defence processes that cause the changes in texture that take place when banana ripens. 2 Materials and methods 2.1 Plant materials At 90- days after flowering (DAF), the Cavendish banana fruit ( Musa acuminata cv. Grand Naine) was collected from OUAT banana germplasm unit, Bhubaneswar, Odisha. Fully developed mature banana fruit at 90-DAF were allowed to ripen at ambient temperature for different time points, i.e., 2, 4, 6, 8, 10, and 12 days after ripening (DAR). Fruit samples were selected for uniformity in size, shape, colour, firmness, and free from visual defects. After separating the peel and pulp tissues from the pre-climacteric and climacteric stages, samples were flash frozen in liquid N 2 and kept at -80°C until further experiment. For the biometric, biochemical, and proteomic analyses, banana fruit tissues (pulp and peel) during pre-climacteric and climacteric phases were taken into consideration. Banana fruit samples from 90-DAF and 12-DAR were considered for the proteome analysis. At the same time, banana fruit samples from different developmental stages, i.e., 20, 40, 60, 80, and 90-DAF and ripening stages, i.e., 2, 4, 6, 8, 10, and 12-DAR, were considered for the biometric and biochemical analysis. 2.2 Biometric assays 2.2.1 Measurement of fruit fresh weight and firmness Fresh weights were recorded in triplicate for each developmental and ripening stage (g kg-1) FW and plotted in the graph after the fresh weights of the banana fruit (whole fruit) and tissues (peel and pulp) from various stages were separated. Using a penetrometer (model no. FR-5120, Lutron, USA) with a 6 mm plunger tip, the fruit was punctured to determine its firmness. After tearing off a tiny piece of banana fruit peel, three identical fruits were examined for hardness, each with five distinct points. Newtons (N) were used to represent the maximal force needed to pierce the banana fruit with the plunger tip. 2.3 Biochemical assays 2.3.1 Starch estimation The starch content in banana peel and pulp tissues during the developmental and ripening stages was estimated according to Shafiee et al. [ 33 ]. 2.3.2 Total sugar content The total sugar content was analysed by following Franscistt et al. [ 34 ]. 2.3.3 Sample preparation and estimation of glucose, maltose and sucrose Fruit tissue samples were prepared by following the modified method of Nath et al. [ 35 ], and then 5 mL of Carrez I solution, 5 mL of Carrez II solution and 10 mL 100 mM of NaOH solution was added and mixed properly after each addition. Then the volumetric flask was filled to 50ml with distilled water, mixed properly and filtered with Whatman® paper no1. From the filtrate 0.1mL (100µl) of liquid sample was taken for sugar estimation. Free D-glucose, maltose and sucrose contents of the samples were estimated by the method described by megazyme K-MASUG assay kit (Megazyme International Ireland Ltd, Wicklow, Ireland). 2.4 Extraction of proteins Only molecular biology-grade chemicals were used in the current proteome study. Protein extraction was done by following Carpentier et al. [ 36 ] with slight modification, the proteins from the pulp and peel tissue of banana fruits at 90-DAF and 12-DAR were phenol extracted. One gram of pulp and peel (separately) tissue samples were ground in liquid N2 and added 5.0 mL of ice-cold extraction buffer (50 mM Tris-HCl (pH 8.5), 5 mM EDTA, 100 mM KCl, 1% w/v dithiothreitol [DTT], 30% w/v sucrose), containing a protease inhibitor cocktail (Sigma-Aldrich, USA, P9599). After adding an equivalent amount of cold Tris-HCl buffered phenol (pH 8.0), the mixture was stirred for 15 min at 4°C. Centrifugation at 6000×g for 30 min at 4°C was used to recover the phenol phase, which was then extracted again using the aqueous buffer. The samples were kept overnight at -20°C to precipitate the proteins using five volumes of 100 mM ammonium acetate in methanol. The proteins were then recovered by centrifugation (16,000 ×g, 45 min, 4°C). The pellets were washed with 0.2% w/v DTT in acetone and air dried at RT. 2.5 Sample preparation 300 µl of 1×PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) was used to dissolve 30 mg of dry protein pellet samples. The mixtures were then vortexed briefly for five minutes. The pellet sample of dissolved proteins was centrifuged for five minutes at 4°C at 12,000×g. For later usage, the clear supernatant was transferred to 1.5 mL eppendorf tube and kept at -80°C. 2.6 Quantification of protein samples Protein concentration was determined by using Quick start 1X Bradford assay kit (BioRad, USA, 5000205) and BSA as a standard reference. At 595nm, the absorbance value of unidentified protein samples was determined. A standard curve was plotted based on the absorbance and BSA content. 2.7 SDS-PAGE separation of proteins 100 µg of protein samples were dissolved in loading buffer (0.5 M tris-HCl [pH 6.8], 10% SDS, glycerol, and β-mercaptoethanol) before being incubated for four minutes at 95˚C in a water bath for SDS-PAGE analysis. Protein samples were added to the first lane, followed by the marker/protein ladder (Bio-Rad, USA, 1610317) with a molecular mass (Mw) range of 6.5–200 kDa. The protein samples were then resolved on a 12% polyacrylamide gel and stained with the GelCode blue dye reagent (Pierce, USA, 24590). 2.8 LC-MS/MS analysis Proteins were reduced, alkylated, and digested at 37°C in ammonium bicarbonate and with trypsin, as described in Ray et al. [ 37 ]. Orbitrap fusion mass spectrometry was used to analyse the peptides after they had been desalted using spin columns. One microgram of digested peptides was placed onto a pre-analytical column (100 µm × 2 cm, nanoViper C18, 5 µm, 100 A; Thermo Fisher Scientific) after each fraction was reconstituted in 15 µl of solvent A. The peptides were eluted with an isocratic gradient from 5% to 30% B for 110 min, 30–95% B for 2 min, and then held at 95% B for 8 min at 300 nl/min flow rate on an analytical column (75 µm × 50 cm, 3 µm particle, and 100 Å pore size; Thermo Fisher Scientific) coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA). The MS parameters were as follows: for full MS spectra, the scan range was m/z 375–1700 with a resolution of 60000 at m/z 100. MS/MS acquisition was carried out in full speed mode with a cycle time of three seconds. At m/z100, the resolution was 15000. The maximum injection time was 50 ms, and the intensity threshold was 5000. The isolation window was 1.2 m/z, and the AGC target was set to (4.0e5) 400000. Higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 30% successively broke apart ions having charges of 2+, 3+, and 4+. 40 seconds was chosen as the dynamic exclusion duration. 2.8.1 Data analysis Proteome Discoverer (PD) version 2.2 (Thermo Fisher Scientific) was used to process raw instrument files. The Sequest HT and Mascot (v2.6.0) search engines were used to search MS2 spectra against Musa acuminata fasta from the Uniprot database. Every search was set up with static modifications such as carbamidomethyl (+ 57.021Da) on cysteine, monoisotopic masses, and trypsin cleavage (maximum two missed cleavages) and dynamic modifications on methionine residues (+ 15.9949Da). The MS/MS tolerance was 0.05Da and the peptide precursor mass tolerance was 10 ppm. For proteins, peptides, and peptide spectral matching (PSMs), the false discovery rate (FDR) was maintained at 1%. Proteome Discoverer 2.2 was used to export the protein quantification values. 2.8.2 Protein-protein interactions To determine the functions and interactions of the identified proteins, a protein-protein interaction network (PPI) was analysed using Gene Mania.org [ 38 ]. The orthologous interaction pairings (interologs) were obtained by mapping the pulp/peel DAF and DAR differentially expressed proteins to GeneMania Arabidopsis thaliana. Venny ( https://bioinfogp.cnb.csic.es/tools/venny/ ) was used to analyze the common and unique proteins, and additional pathway maps between them were examined. Although the Musa acuminata genes were mapped to GeneMania, String-db.org ( www.string.embl.de ) was used to directly verify these interologs in the Musa database, which was mapped a relatively few. We use cytoHubba [ 39 ] to find the top-ranking genes based on the clustering coefficient and the coherent pathways and clusters associated with these distinct protein-coding genes. While betweenness, closeness, and centralities could form the other ranking coefficients, we felt that the clustering coefficient would yield more distinct interaction partners. 2.8.3 Functional categorization Proteins identified through mass spectrometry were functional categorized based on the literature available on different climacteric fruit in connection with the ripening process. 2.9 RNA isolation and cDNA synthesis The RNA isolation from both the tissue of banana was carried out by following the method of Meisel et al. [ 40 ]. First-strand cDNA was synthesized using total RNA by using Superscript-II reverse transcriptase first strand cDNA synthesis Kit (Invitrogen, USA) and Oligo dT primer (Sigma) for random amplification of unknown RNA according to the manufacturer’s recommendation. 2.10 Quantitative real-time PCR (RTqPCR) validation The RTq-PCR study was implemented with Invitrogen Quantstudio-5 (Applied Biosystems, USA) under the following condition: step (1) 50˚C 2 min, step (2) 95˚C 2 min, step (3) (95˚C 0.15 min, 50–60˚C 0.15 min, 70 ˚C 1min) × 40 cycles, followed by the thermal dissociation curve [ 41 ]. The relative expression level was analyzed using the 2-ΔΔCt method and normalized with reference housekeeping gene cyclophilin (CYP) [ 42 ]. 3 Results The present study considered banana fruit from pre-climacteric and climacteric stage for the proteome study. The fruit was selected based on its size, shape, texture, colour, and visual appearance. The fruit size was larger and has slight ridges on the pedicel parts in the 90-DAF (pre-climacteric stage) stage and appears to be green with faint yellow traces. In contrast, fruit in the 12-DAR (climacteric stage) had no discernible ridges, the pedicle portion turned black and dried, the fruit was brown with spots, and it had a softer texture than fruit in the 90-DAF stage (Fig. 1 ). For the biometric and biochemical analysis, banana fruit tissues (pulp and peel) during pre-climacteric and climacteric phases were taken into consideration. A graph was created by measuring the fresh weights of the banana fruit (whole fruit) and tissues (peel and pulp) from various developmental and ripening stages and fresh weights were recorded in triplicate for each stage (Fig. 2 A). Weight loss of banana fruit increased with the progress of ripening and the maximum weight loss recorded after 6-DAR. The maximum increase in the weight of pulp tissue was recorded at 90-DAF. The fruit firmness results are depicted in Fig. 2 B. In the current study, the starch content of banana fruit tissue increased during developmental stages (40, 60, and 90-DAF) and reduced during various ripening stages (2, 4, 6, 8, 10, and 12-DAR) (Fig. 3 A), describing the process of starch hydrolysis and sugar synthesis. Notably, the current study revealed an ascending trend in total sugar content from the developmental to the ripening stages of banana, signifying that sugar accumulation peaks at the commencement of ripening (Fig. 3 B). Further, the total sugar content pattern mirrored the expression patterns of sugar metabolism proteins, indicating that starch degradation into simple sugars caused textural alterations during banana fruit ripening. In consistence with the total sugar content, monosaccharides (D-glucose), disaccharides (maltose), and polysaccharides (sucrose) in banana peel and pulp tissues dramatically increased at the climacteric stage compared to the pre-climacteric stage (Fig. 4 ). Phenol-based extraction method was followed to extract proteins from banana pulp and peel tissues in both the pre-climacteric and climacteric stages. Samples were considered for the experiment from three independent biological replicates. Further, extracted proteins were quantified by Bradford assay using BSA as a standard. All protein samples extracted with phenol from the pulp and peel tissues from different stages yielded high concentrations. For quality check, the known amount of protein (100µg) was separated on a 12% SDS-PAGE and visualized using gel code blue stain reagent staining. Proteins were well separated with distinct bands, reflecting protein’s excellent quality without any degradation. Most of the protein bands fell between 14–200 kDa (Fig. 5 ). A gel-free proteome analysis of banana peel and pulp tissues using Orbitrap Fusion mass spectrometry resulted 950 to 1416 proteins in the pre-climacteric and climacteric stages, respectively. Proteins participated in metabolic processes including signal transduction, cell wall modification, carbohydrate metabolism, stress response and defence, hormone regulation, and redox homeostasis. The identified proteins were catalogued together with their corresponding accession numbers, total peptide scores, peptide coverage percentages, amino acid, molecular weights in kilo-daltons (kDa), unique peptide counts, and peptide sequences. (Supplementary Table 2–5). The functional classification was done for the mass spectrometry-identified proteins, which showed the abundance of the peel and pulp tissues of pre-climacteric and climacteric stages (Fig. 6 ). Proteome analysis revealed that the proteins involved in transport and stress/defense mechanisms were found to be abundant in the pulp tissue of pre-climacteric and peel tissue of climacteric stage, respectively. At the same time, the same proteins were low in abundance in peel tissue of pre-climacteric and pulp tissue of climacteric stage. Cell wall degrading proteins displayed a high abundant in peel and pulp tissues of climacteric stage, whereas low in peel tissue of pre-climacteric stage. However, compared to the pre-climacteric stage, it was revealed that the pulp and peel tissues of the climacteric stage had higher protein abundant linked to sugar metabolism (Fig. 6 A-D). Table 1 represents the proteins with different accession numbers (ID) identified through mass spectrometry which was commonly found in banana tissues at pre-climacteric and climacteric stages with varied peptide coverage (%). Changes in protein quantity or modulation were noted in the pre-climacteric and climacteric banana peel and pulp tissues based on the peptide coverage (%). Cell wall modification proteins such as β-hex and β-gal were higher in the pulp tissues of pre-climacteric and climacteric stages than in peel tissue. This study identified one number of β-hex (#M0T2F6) in the peel tissue of climacteric stage. One β-hex protein in pre-climacteric (# M0T2F6) and 3 β-hex proteins in climacteric stage (# M0T2F6, M0RQ03, M0S3Q7) were identified in the banana pulp. Further, 4 (#M0S9W4, M0RVL3, M0SQP6, M0SX47) and 1 β-gal enzyme (M0SQP6) was identified in peel tissues of banana fruit at the pre-climacteric and climacteric stage, respectively. Likewise, other cell wall degrading proteins such as PE and POD were found to be high in peel tissue in the climacteric stage compared with pulp tissue of both stages. Interestingly, in our study 1 number of PE (#M0RPM3) in pre-climacteric and 4 numbers of PE (#M0SC42, M0RPM3, M0TGN6, M0SC43) in the climacteric stage were identified in the peel. One PE (# M0RPM3) protein was commonly identified in the banana pulp during pre-climacteric and climacteric stages. In the current study, one (#M0TE13) and four numbers of α-man (#M0TWG0, M0TLF4, M0U935, M0TWG0) were identified in the pulp tissue of pre-climacteric and climacteric stages, respectively. Three α-man proteins in the pre-climacteric stage (# M0TWG0, M0U935, M0T6Z4) and four α-man proteins in the climacteric stage (# M0TWG0, M0T6Z4, M0TLF4, M0U935) were identified in the banana peel. Further, one number of XTH proteins (accession no-M0U6S4) was placed in pulp tissue at the climacteric stages of banana fruit. Two numbers of XTH proteins in the pre-climacteric stage (# M0TTV0, M0U6S4, M0TZC7, M0RMW1) and four XTH in the climacteric stage (# M0RMW1, M0TQQ9, M0TGI2, M0TTV0) were identified in the banana peel. Overall, proteins such as XTH and α-man were present more in peel tissue of the climacteric stage than in pulp tissues of both stages. PL proteins were found in peel tissue of the pre-climacteric stage and pulp tissue of the climacteric stage. It is modulating more in peel tissue of the pre-climacteric stage than the climacteric stage. Overall, PL may play an active part in the ripening process because there is a higher peptide coverage in the climacteric stage than in the pre-climacteric stage. Two PL (# M0U687, M0TAX1) were identified in peel tissues of the climacteric and pre-climacteric stages and one (M0TAX1) in pulp tissues of both the stages, respectively. In the present study, one S-adenosylmethionine synthase (SAM-synthase) protein in peel and pulp tissue of pre-climacteric (#M0T0C8) and climacteric stage (#M0SCW4) was identified. A total of five (#M0S638, M0TN23, M0SE45, M0T0C8, M0S0A4) SAM-synthase proteins were identified in peel tissue of the climacteric stage. Sugar metabolism-related protein such as SUS was exclusively present in the peel tissues of the pre-climacteric and climacteric stage. A high peptide coverage in case of SUS during the ripening stage is clear evidence of its role in starch hydrolysis to form simple sugars during ripening. SUS proteins were found in banana pulp at the pre-climacteric (M0RJE1) and climacteric (M0RJE1, M0TSQ0) stages, respectively. Further, a total of seven numbers of fructose-bisphosphate aldolase in pre-climacteric and five number in climacteric stage were identified in banana peel tissue. Likewise, four fructose-bisphosphate aldolase proteins in pre-climacteric and seven in climacteric stage were identified in banana pulp tissue. Identified proteins of cell wall modification, ROS mechanism, and sugar metabolism (represented in bold) were interpreted in metabolic pathways to understand their possible role in fruit ripening (Fig. 7 ). Tissue types were shown in the legend, where proteins were abundant and present at particular stages. For pulp and peel tissues of the pre-climacteric and climacteric stages, distinct numerals or codes were assigned: 1-pre-climacteric peel; 2-climacteric peel; 3-pre-climacteric pulp; and 4-climacteric pulp (Fig. 7 ). Proteins such as XTH, PL, and PE were involved in cell wall modification. XTH acts as an intermediate to convert xyloglucan to xylose. At the same time, PE serves as an intermediate for converting pectin into PL. SUS is involved in sugar metabolism in pre-climacteric and climacteric stages of pulp tissues. SUS acts as intermediates for the conversion of sucrose into fructose. SOD and GPX were involved in the ROS mechanism and present in both the tissues (peel and pulp) and stages. Pyruvate dehydrogenase (PDH) is a convergence point in regulating the fine metabolic tuning between glucose and fatty acid oxidation. Pyruvate kinase (PK) catalyzes the final step in glycolysis, in which phosphoenolpyruvate is converted to pyruvate. The product pyruvate is then served to prime the TCA cycle. From the protein-protein interaction (PPI) networks, we observe that there are unique proteins specific to particular tissue type and stage, which we assume many of the sequences are novel. However, XTH protein 8-related (PTHR31062) is the ideal candidate that is known to be associated with unique pathways in peel at the climacteric stage. We argue that, while our results suggested that ROS-related proteins possibly cross-talk with cell wall metabolism by promoting the cell wall loosening (Fig. 8 ), one of the striking features associated with the pre-climacteric stage of peel and pulp is that the climacteric stage peel has a distinct set of genes mapped with XTH protein 8-related (PTHR31062) or XTH family members with XTH4 forming a central node of interacting partners. This could be because of its prominent ripening role in the peel instead of the pulp, which includes later. A host of other proteins, including CYTC-2 family members and APX3, are known to primarily shown to be interacting in this milieu. To check this, we sought to ask which among these genes form the top-ranking genes, and we used cytoHubba. Our results indicate that the Musa acuminata network has no well-annotated interactants. In contrast, the ones with Arabidopsis have distinct candidates that are well annotated, and the network is more robust as averse to the former (Fig. 8 A-D). From Fig. 9 , the higher the contrast, the greater the rank (maroon being the top and light yellow being the lowest among the top 10 ranking clusters). Among them, we observed that cytochrome-c oxidase sub unit Vb. Cytochrome-c oxidase (CcO), the terminal oxidase, is among the top as it is known to be a multi-chain transmembrane protein in mitochondria that aids oxidation. In addition, ascorbate peroxidases and rubredoxin are the other top candidates (AT1G80230 and AT3G15640; Fig. 9 ). This could be because PEs aid the xylem translocation during ripening in pedicels, void of water content. The Venn diagram (Fig. 10 ) depicts the detected number of common and distinct differentially expressed proteins identified from different banana tissue of 90-DAF and 12-DAR. In the case of 90-DAF peel and pulp, a total of 175 (14.6%) and 36 (3%) unique proteins, sharing 5 (0.4%) common for both tissues. This suggests that the presence of a specific set of proteins may have distinct functions in two tissues. Twenty-six (2.2%) proteins were common between pulp tissues of 90-DAF and 12-DAR. Thirty-six (3%) and 203 (16.9%) proteins were unique for 90-DAF pulp and 12-DAR pulp, respectively. 108 (9%) common proteins were identified between pulp (#203) and peel tissues (#131) of 12-DAR. 91 (7.6%) proteins are commonly studied for tissues and ripening stages. They reveal a complex nature and active metabolic pathways that are operational during ripening in pulp tissues of 12-DAR. Peel and pulp tissues at the pre-climacteric stage had higher levels of XTH4 expression than at the climacteric stage (Fig. 11 A). An averse, another cell wall modification gene, PEL expression was increased in the peel during the climacteric stage (Fig. 11 C). Peroxidase (POD), superoxide dismutase (SOD) and SUS expression was decreased at climacteric stage, compared to pre-climacteric stage in the pulp tissue (Fig. 11 B, E, D). 4 Discussion The present study revealed that the phenol-based method effectively extracted a high yield of proteins over the TCA/acetone method and resulted in good protein separation on SDS-PAGE. Fruit softening is dependent on changes to the structural characteristics of the cell wall, including the enormous depolymerisation and solubilisation of proteins, lignin, and polysaccharides (pectins, cellulose, and hemicelluloses) [ 2 , 5 ]. The starch content decreased with an increase in sugar content during the climacteric stage, suggesting the ripening phase starch hydrolysis and sugar synthesis [ 43 ]. However, the banana pulp presented significantly higher sugar content than the peel during the climacteric stage. Our results are inconsistent with the previous reports, where the total amount of mono-(D-glucose), di- (maltose), and polysaccharides (sucrose) in banana peel and pulp tissues increased significantly at the climacteric stage compared to pre-climacteric stage [ 44 ]. 4.1 Proteins associated with cell wall metabolism Cell wall disassembly, which is aided by several cell wall-degrading proteins such as PE, PL, PG, PME, and others, breaks down many polysaccharide networks and promotes banana ripening. The PE functions primarily by altering the localized pH of the cell wall resulting in alterations in cell wall integrity. This enzyme is known to extensively decrease the rigidity of cell wall structure and solubilization of pectins during fruit softening [ 9 ]. PE proteins were previously found in pulp tissues at two phases of ripening, mesocarp tissues at ripe and unripe stages, and pulp tissues at four ripening stages in peach [ 45 – 47 ]. Further, the high number of PE proteins in the present study during the climacteric stage suggests that these proteins may have key role in fruit softening at this phase. Likewise, PL is involved in the maceration and soft-rotting of fruit tissue [ 2 ]. At the climacteric stage of banana fruit, two PL proteins were up-regulated in the peel [ 5 ] and pulp tissue [ 8 ], which was consistent with the breakdown of cell wall components [ 5 , 8 ]. In the current investigation, peel tissues at both phases showed a high abundance of PL proteins. In addition, the peel and pulp tissues exhibited increased expression of the PL gene, indicating PL could play essential role in breakdown of pectin in banana peel tissues during ripening, and our findings are in consistent with the earlier reports on different fruits [ 5 , 8 ]. Fruit softening is facilitated by the ripening-specific N-glycan processing enzyme called β-hex [ 7 ]. Earlier, β-hex was identified in pericarp tissues of capsicum at four developmental and three ripening stages [ 7 ]. This protein was also identified in the pericarp tissues of tomatoes at four ripening stages [ 48 ]. Further, the RNAi mediated suppression of β-hex genes, resulted in extended shelf-life for 30-days in tomato and 7-days in capsicum [ 7 , 48 ]. In this study, β-hex proteins were identified in high abundance during the climacteric stage, indicating that they may have an active role in banana ripening. Another class of cell wall-modifying proteins called XTH, helps in maintaining the integrity of the cell wall through endotransglucosylase and weaken it during fruit ripening through hydrolase activity. XTHs involved in the cross talk regulatory mechanism of auxin and ethylene signalling to promote fruit ripening [ 49 ]. In this regard, XTH proteins were identified in other fruit tissues during ripening stages, such as pericarp tissues of tomatoes, kiwi, and mesocarp tissue of apricot [ 50 – 52 ]. Similarly, Kok et al. [ 6 ] revealed that oil palm had five different XTH proteins, all of which were up-regulated during the ripening stages. In consistent with earlier reports, in this study four XTH proteins were identified in the peel during ripening. A cell wall modifying protein called β-gal aids in the debranching of pectin and enhances the depolymerisation process as fruit softens [ 53 ]. Previously, the β-gal during fruit softening was reported in diverse fruit such as mango [ 54 ], chocolate vine [ 55 ], tomato [ 56 ] and strawberries [ 57 ]. In this regard, β-gal proteins showed a high abundance in banana peel and pulp tissues during the climacteric stage, which suggests that β-gal could play a role in degrading the pectin in the cell wall and breakdown of polysaccharides in banana leading to a softening of fruit during ripening. The changes in the activity of the cell wall-bound PODs during the fruit ripening determine the firmness of the fruit. Also, it inhibits cell wall tightening by breaking the cell wall bonds [ 58 ]. Escalante-Minakata et al. [ 58 ] have identified the PODs in both pulp and peel tissues of banana fruit during the developmental and ripening stages. In the present study, a high number of POD proteins were identified in banana peel at the climacteric stage, suggesting that POD may modulate the banana ripening process by removal of ROS, there by tightening the cell wall. 4.2 Protein associated with sugar metabolism The fruit goes through a number of physiological and biochemical changes as it ripens, which breaks down the starch and causes an accumulation of sugar or sucrose [ 53 ]. Once sucrose reaches the sink cells, it hydrolysed by SUS or invertases into glucose and fructose. SUS is mainly involved in synthesizing carbohydrate polymers, i.e., starch or cellulose, or in the generation of active compounds which help in fruit development [ 59 ]. In this regard, Tian et al. [ 59 ] have identified SUS proteins in kiwi fruit tissues (exocarp) at different developmental and ripening stages. In the present investigation, a high number of SUS proteins in the climacteric stage of banana pulp suggests that sugar synthesis is quite active during the ripening process. In contrast, reduced SUS transcript level was recorded at climacteric stage in both peel and pulp tissues, it could be due to the post translational modification, such as selective pre-mRNA splicing, methylation and de-methylation process that might occured at transcript level [ 60 – 64 ]. An essential component of sugar metabolism, fructose-bisphosphate aldolase (FBA) also controls the sink metabolism of fruit tissue during ripening [ 21 , 64 ]. A total of four up-regulated proteins of FBA were identified in watermelon fruit at three developmental stages. The FBA proteins in melon have significantly increased, which implies that this protein may play a critical role in sucrose metabolism during fruit ripening [ 64 ]. Further, using TMT labelling coupled with LC-MS/MS analysis, Li et al. [ 21 ] have revealed that FBA proteins actively participate in the sugar metabolism and are found to be increased in abundance during blueberry ripening [ 21 ]. In this investigation, a total of four and seven FBA proteins were identified in pulp tissues of pre-climacteric and climacteric stages, respectively. Whereas, in the case of peel tissues, three and five FBA proteins were identified in both the stages of banana, respectively. Overall, our study concluded that sugar metabolism-related proteins were highly modulated during the banana fruit ripening. 4.3 Proteins associated with hormonal regulation Various plant hormones regulate fruit development and ripening process in the cell. Several studies have attempted to identify receptor proteins and signaling components in connection with multiple plant hormones. The CS protein is involved in the formation of methionine which is generally involved in ethylene biosynthesis through the intermediary’s cystathionine and homocysteine [ 14 ]. CS enzyme was down-regulated during the ripening stages of tomato pericarp and also in mango pulp [ 14 , 65 ]. In the present study, one CS protein in the pre-climacteric and six CS proteins in the climacteric stage were identified in the banana pulp. Likewise, six CS proteins in the pre-climacteric and four CS proteins at the climacteric stage were identified in peel tissue. However, based on our result, it is speculated that by participating in the ethylene biosynthesis pathway, CS proteins may play a significant role in the ripening process. During fruit ripening, S-adenosylmethionine synthase (SAM-synthase) proteins participate in various polyamine processes as well as the synthesis of ethylene [ 66 ]. SAM serves as a precursor of polyamine and ethylene, which are known to regulate fruit ripening [ 66 ]. Proteomic investigations in the pericarp of cherry tomatoes also revealed an increase in the quantity of SAM- synthase proteins during fruit ripening [ 67 ] and peach mesocarp [ 68 ]. According to Choi et al. [ 66 ], there was a comparatively high expression of SAM-synthase in the early stages of tomato ripening as opposed to the later stages. In the present study, SAM-synthase proteins (#6) showed a high abundance in the climacteric stage peel tissue. Our findings are inconsistent with earlier reports on other fruits during ripening. In this scenario, SAM-synthase may be essential for ethylene production. Signalling by auxin and ABA is mediated by tetratricopeptide repeat (TPR) domains proteins. The ETO1 (ETHYLENE-OVERPRODUCER1) protein in Arabidopsis has been shown to directly interact with a 1-aminocyclopropane-1-carboxylate synthase isoform through its TPR domains, thereby negatively regulating ethylene production in seedlings [ 69 ]. TPR proteins precise function in fruit ripening is still unknown. Based on previous reports, it was observed that ABA regulates ethylene biosynthesis, and ABA-ethylene interaction triggers the fruit ripening process [ 70 ]. ABA governs the transformation of 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene during fruit ripening by ethylene-dependent or -independent mechanisms [ 71 ]. Nevertheless, no molecular mechanism examining the relationship between ABA and ethylene during fruit ripening not been published yet. In the present study, interestingly, TPR proteins showed high abundance in peel tissue of climacteric stage (#7), and we speculate that it may have some role in hormonal regulation during ripening process. 4.4 Proteins associated with stress, defense, ROS mechanism The climacteric fruit ripening process involves an increase in the respiration and, consequently, alteration of redox homeostasis in the cell, with reactive oxygen species (ROS) build-up, which in turn determines lipid peroxidation, protein denaturation, and metabolism deterioration to achieve a final degradation state functional to seed release [ 72 ]. SOD is known to be involved in many biological processes, such as oxidizing the lipids and denaturation of DNA fragments [ 73 ]. In higher plants, SODs act as antioxidants and protect cellular components from being oxidized by ROS [ 74 ]. The compartmentalization of different forms of SOD throughout the plant makes them counteract stress very effectively [ 75 ]. SOD enzyme was identified in the pulp tissues of peach during two developmental and two ripening stages, and their results showed high O 2 and H 2 O 2 contents in the middle stage of peach fruit development. In another study, SOD was identified in the pulp tissues of peach at 125-DAF. The results revealed that Mn-SOD plays a key role in mitochondria for regulating the ripening and senescence process in peach fruit [ 76 ]. Using multiple reaction monitoring (MRM), Song et al. [ 77 ] investigated the strawberry antioxidant defense system during ripening. They have identified three SODs through QTRAP-LC-MS/MS that were down-regulated during later stages of strawberry ripening. In the current investigation, banana peel tissues at the pre-climacteric stage had a significant number of SOD proteins (#7), inconsistent with the observation, gene expression studies further revealed the reduced gene expression in the pulp tissue at climacteric stage (Fig. 10 B). In this regard, our results agree with previous reports on different fruit, where SOD proteins may play a role in scavenging reactive oxygen species and H 2 O 2 content that are arising due to high respiration rates during banana developmental and ripening stages. It may also be a signaling molecule driving different metabolic processes, promoting fruit rapid development. During three ripening stages, three classes of GPX enzymes were identified in peach skin. The study revealed that GPX enzymes were up-regulated during later stages of peach ripening [ 78 ]. The proteome study has shown that GPX proteins were involved in ROS mechanism and, exhibited higher defense anti-oxidative capacities and protected the fruit from deterioration in chilli and strawberry fruit during ripening [ 77 , 79 ]. In this regard, we speculate that the presence of more GPX proteins in this study may play a protecting role in banana peel tissue during the climacteric stage. Glutathione reductase (GR) plays a key role in maintaining the cellular control of ROS. It acts as an antioxidative mechanism in the fruit ripening process, which requires a turnover of active oxygen species (AOS) such as hydrogen peroxide and superoxide anion [ 80 , 81 ]. GR protein levels were elevated in the tomato's pericarp during ripening [ 80 ]. In our study, two GR proteins were identified during the pre-climacteric stage, suggesting that GR protein may mediate the biochemical and physicochemical changes occurring during ripening. 4.5 Protein-protein interaction studies Additionally, a study on the protein-protein interactions has shown that the peel tissue of the climacteric stage has a distinct set of proteins mapped with XTH ​protein 8-related (PTHR31062) or XTH family members with XTH4 forming a central node of interacting partners. In addition, our results speculate that the banana peel ripening may be significantly influenced by XTH proteins by disassembling the cell wall and degradation of hemicelluloses, leading to a softening of the peel tissue, thereby reducing the shelf-life of the fruit. In contrast, XTH4 gene expression was reduced in the peel tissues at later stage of ripening, compared to pre-climacteric stage (Fig. 8 ). It could be due to the fact that mRNA expression is not always correlated to the quantity of expressed proteins due to varied regions [ 82 ]. 5 Conclusion The functional classification of identified proteins showed differential abundance between the pre-climacteric and climacteric stages of the banana. Functional categorization of proteins from banana peel and pulp tissues at pre-climacteric and climacteric stages revealed that proteins were involved in various metabolic pathways and biological processes such as amino acid metabolism, sugar metabolism, starch biosynthesis, hormonal regulation, stress, and defense mechanism, cell wall modification, signaling, transport, protein folding, energy, and carbohydrate metabolism, etc. Moreover, the presence of numerous cell wall modification proteins in the climacteric stage compared to the pre-climacteric stage of banana fruit indicates that cell wall modification proteins actively participate in the cell wall softening process. Identification of high numbers of sugar metabolism-related proteins in the climacteric stage exhibited the phenomena of starch breakdown to form simple sugars during fruit ripening. A large number of unique tissue specific protein sets (based on Venn) indicates the necessity of conducting tissue-specific proteome investigations. Interestingly, some of the proteins were identified in the form of β-adaptin, ferritin, 2-Hacid_dh_domain, PAP-fibrillin, ADK-lid domain, why domain, GLP, and clathrin protein, whose specific role in fruit ripening is still unexplored. Further, some uncharacterized proteins were also identified in our study, which could be considered as novel proteins (whose functional validation is not carried out) as sequence information is not available in the protein database. Some of the critical cell wall modification proteins identified through this investigation, such as α-man and β-hex, were identified in other fruit crops, and conformed enhancement in the fruit shelf-life, when corresponding genes were supressed using RNAi. However, further studies seek to validate and assign a functional role for these proteins in bananas. The protein-protein interaction study revealed that the XTH family members with XTH4 forming a central node of interacting partners and XTH ​protein 8-related (PTHR31062) are the ideal candidate associated with unique pathways in peel at the climacteric stage. XTH proteins were revealed to play a significant role in the post-harvest softening of fruit, auxin and ethylene-mediated signalling. It necessitates further investigation to unlock the functional role of XTH protein in banana fruit ripening. This study will shed light on the regulatory process of the ripening mechanism in bananas. Some of the key candidates identified can be further validated through the genome editing tools to assign functional roles in controlling ripening and to enhance fruit shelf-life in banana. Declarations Ethics approval and consent to participate The fruit samples were collected from OUAT banana germplasm unit, Bhubaneswar, Odisha with the consent of the competent authority of the institute. All plant materials were cultivated under controlled field conditions and were not collected from the wild. No specific collection permits or licences were required, as no wild specimens were collected. The guidelines followed for the use of plants or plant materials in the study. Consent to publication Not applicable. Competing interests The authors declare no competing interests. Funding This study was supported by the research grant to G.K.Surabhi by Rashtriya Krishi Vikash Yojana, Government of India (No. AG(RKVY)04/2017–9975/Ag.dt.22.06.2017; OR/RKVY-HORT/2017/774), Science and Technology Department, Government of Odisha (No.27552800232014/202830, STBBSR, dt.17.7.2015) and Forest, Environment and Climate Change Department, Government of Odisha, India, is gratefully acknowledged. Author Contribution SM, DP: Sample collection, lab work, formal analysis, investigation, interpretation of the data, writing - original draft; GKS: Conceptualization, writing-review and editing, critical revising, research supervision, PS: Bioinformatic analysis. All authors approved the manuscript. Acknowledgement The authors wish to acknowledge the Mass Spectrometry Facility at IIT Bombay (MASSFIITB), supported by the Department of Biotechnology (BT/PR13114/INF/22/206/2015), for their support with the mass spectrometry analysis of the samples. We thank the Chief Executive, Regional Plant Resource Centre, for extending the facilities. Data Availability The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Surabhi GK, Patnaik S, Mohanty S. A comparative method for protein extraction and proteome analysis by two-dimensional gel electrophoresis from banana fruit. 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Identification and biochemical characterization of the glutathione reductase family from Populus trichocarpa . Plant Sci. 2020;294110459. https://doi.org/10.1016/j.plantsci.2020.110459 . Chen S, Harmon AC. Advances in plant proteomics. Proteom. 2006; 6(20):5504-16. https://10.1002/pmic.200600143 . PMID: 16972296. Tables Table 1 Comparison of protein expression based on peptide coverage in different tissues at pre-climacteric and climacteric stage of banana for cell wall modification, stress response and defense, signal transduction, hormone regulation and sugar metabolism functional categories of proteins based on mass spectrophotometry analysis. Name of the protein Protein ID/stage/tissue Peptide coverage % 90-DAF 12-DAR 90-DAF 12-DAR peel pulp peel pulp peel pulp peel pulp Cell wall modification Pectinesterases M0TGN6, M0SC42, M0RPN3 M0RPM3 M0TGN6, M0SC42, M0RPN3 M0RPM3 8,4,12 2 4,8,14 10 Pectate lyase M0U687 M0U687, M0TAX1 M0U687 M0U687, M0TAX1 4 5,18 17 10,29 β-hexosaminidase M0T2F6 M0T2F6 M0T2F6 M0T2F6 2 4 6 8 Xyloglucan endotransglucosylase/hydrolase M0RMW1 - M0RMW1 - 8 - 16 - α-mannosidase M0TWG0, M0U935, M0T6Z4 - M0TWG0, M0U935, M0T6Z4 - 2,3,3 - 4,9,6 - β-galactosidase M0SQP6 M0SQP6, M0RVL3 M0SQP6 M0SQP6, M0RVL3 4 2,1 2 9,21 Stress response and defense Superoxide dismutase M0S4H9, M0SFL6, M0RG01 M0S4H9, M0S978 M0S4H9, M0SFL6, M0RG01 M0S4H9, M0S978 20,33,12 19,22 38,49,18 25,57 Peroxidase M0TCA6, M0RVD5, M0THT5, M0RUU7 M0TBJ2 M0TCA6, M0RVD5, M0THT5, M0RUU7 M0TBJ2 61,17,68,19 12 68, 3, 72, 29 20 Glutathione peroxidase M0SWM2 M0RP81 M0SWM2 M0RP81 25 7 30 21 Tyrosinase copper binding protein (Cu-bd) M0UBI2 - M0UBI2 - 43 - 6 - Germin-like proteins M0U4X1, M0S2X3, M0UCZ2, M0U0R8 - M0U4X1, M0S2X3, M0UCZ2, M0U0R8 - 25, 23,12, 5 - 16, 13, 12, 2 - Barwin protein M0T386 - M0T386 - 73 - 72 - Isocitrate dehydrogenase M0RX69 - M0RX69 - 31 - 4 - Thioredoxin M0TQW7, M0SR53 - M0TQW7, M0SR53 - 48,58 - 34,28 - Catalase M0SEK3, M0S0R3 - M0SEK3, M0S0R3 - 21, 13 - 30,23 - Eukaryotic translation initiation factor M0T7M2, M0S626 M0S626 M0T7M2, M0S626 M0S626 27,22 22 43,13 29 Peroxiredoxin M0RH93, M0TP05, M0RKE2 - M0RH93, M0TP05, M0RKE2 - 19,27, 60 - 30,27,41 - Lactoylglutathione lyase M0RNM6 - M0RNM6 - 23 - 23 - Glutaredoxins M0S2Q5 - M0S2Q5 - 9 - 9 - 26S proteasome regulatory subunit - M0RYR4 - M0RYR4 - 2 - 1 Cyclophilins M0RZQ7 - M0RZQ7 - 31 - 49 - ClP protease ATPase subunit - M0TSN5 - M0TSN5 6 - 3 - Signal transduction Nucleoside diphosphate kinases M0U940 M0SZK9 M0U940 M0SZK9 15 27 18 9 Tubulin M0SS87 - M0SS87 - 7 - 17 - Hormone regulation Aminomethyl transferase M0T8H5 M0SM28 M0T8H5 M0SM28 6 15 26 33 Sugar metabolism Sucrose synthase M0RJE1 M0RJE1 M0RJE1 M0RJE1, M0SI67 1 48 1 12, 62 Fructose-bisphosphate aldolase M0T021 M0U9P4 M0T021 M0U9P4 13 6 33 14 Additional Declarations No competing interests reported. Supplementary Files Suppl.Table1.docx Suppl.Table2.docx Suppl.Table3.docx Suppl.Table4.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 23 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers invited by journal 31 Mar, 2026 Editor invited by journal 30 Mar, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 26 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Values are mean of three replicates anderror bars represent the standard deviation (±SD).\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/3c4a68ea6dc98930e1614bd6.jpg"},{"id":105566938,"identity":"c86e2661-0da7-46cf-bbe0-0107505a7639","added_by":"auto","created_at":"2026-03-27 12:57:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":342972,"visible":true,"origin":"","legend":"\u003cp\u003eRepresents the biochemical changes in peel and pulp tissue of banana fruit during different developmental and ripening stages. (a) Starch content of banana peel and pulp tissues at different developmental and ripening stages, measured in g kg\u003csup\u003e-1\u003c/sup\u003e (b) total sugar content of banana peel and pulp tissues at different developmental and ripening stages, measured in g kg\u003csup\u003e-1\u003c/sup\u003e. Values are mean of three replicates and error bars represent the standard deviation (±SD).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/1556455bdbf2fa02b6ad4534.jpg"},{"id":105566270,"identity":"2bd7060a-87f4-43cf-81a5-8607a4c44a5b","added_by":"auto","created_at":"2026-03-27 12:55:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1257200,"visible":true,"origin":"","legend":"\u003cp\u003eRepresenting the total soluble sugar content of banana fruit (A) sucrose (mg/g FW) (B) glucose (mg/g FW) (C) maltose (mg/g FW) content. Each value represents the mean of three biological replications of three fruits analyzed at each ripening stage and vertical error bars represent the standard deviation (±SD).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/c8293e17988f5d8a536b56ac.jpg"},{"id":105553707,"identity":"a5e19a11-adc9-4302-bd35-2fea2e0226bd","added_by":"auto","created_at":"2026-03-27 10:33:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":68515,"visible":true,"origin":"","legend":"\u003cp\u003eSodium dodecylsulfate-polyacrylamide gel electrophoresis profile showing separation of proteins from banana peel and pulp tissues of pre-climacteric and climacteric stage by using phenol extraction method. Known amount of proteins (100µg) was loaded in each lane and proteins were resolved on 12% SDS-PAGE followed by Coomassie blue staining. A. 90-DAF pulp, B. 90-DAF pulp C. 12-DAR peel and D. 12-DAR pulp. M. represents the SDS-PAGE marker (KDa).\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/4455b5a6c62991b0925f81a1.jpg"},{"id":105567165,"identity":"04b771c3-0b16-43fe-9b06-e83f16985a1a","added_by":"auto","created_at":"2026-03-27 12:58:31","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229375,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional categories of the identified proteins from banana fruit tissues at different developmental and ripening stage represented as (A) 90-DAF peel (B) 90-DAF pulp (C) 12-DAR peel (D) 12-DAR pulp, through Orbitrap fusion mass spectrometry (mass spectrometer combines best of quadrupole, orbitrap and linear ion trap; tribrid) analysis. Different functional categories of proteins were distributed based on their role.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/e3a49448b48b8cf9cba57368.jpg"},{"id":105566601,"identity":"8df1cb58-0c87-46ad-a004-834775dc27c7","added_by":"auto","created_at":"2026-03-27 12:56:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":279755,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview/illustrations of the proteins and their regulation in different pathways during banana fruit ripening process. Identified proteins in the current study were represented in bold letter which were involved in cell wall modification, ROS mechanism and sugar metabolism. Cell wall modification proteins- Xyloglucan endotransglucosylase (XTH), pectate lyase (PL), pectin esterase (PE); ROS mechanism proteins- superoxide dismutase (SOD), glutathione peroxidase (GPX); sugar metabolism proteins- sucrose synthase (SUS); TCA cycle- phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate dehydrogenase (PDH), citrate synthase (CS), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). Identified proteins were present in specific stage and tissue type. Different digits/codes were assigned for pulp and peel tissues of pre-climacteric and climacteric stage (1-4).\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/60a435d87047875f102d2c45.jpg"},{"id":105553714,"identity":"bd0cb7d4-d182-493d-a02c-30a2bad23a11","added_by":"auto","created_at":"2026-03-27 10:33:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":177055,"visible":true,"origin":"","legend":"\u003cp\u003eThe image showing annotated list of all proteins interacting from co-expression based on domains in a network mode for four different tissues of banana studied in the experiment. Proteins were mapped to GeneMania \u003cem\u003eArabidopsis thaliana\u003c/em\u003e to derive the orthologous interacting pairs (interologs). (A) 90-DAF peel, (B) 12-DAR peel, (C) 90-DAF pulp, (D) 12-DAR pulp.\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/0d63873e401c647a072cad0c.jpg"},{"id":105567368,"identity":"05b55825-7193-4630-9a6e-7eb64e60b977","added_by":"auto","created_at":"2026-03-27 12:59:12","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":98152,"visible":true,"origin":"","legend":"\u003cp\u003eSub-network analysis of top ranked proteins with their expanded network analysed by cytoHubba using Arabidopsis network data base. Higher the contrast, greater is the rank (maroon being top and light yellow being the lowest among top 10 ranking clusters.\u003c/p\u003e","description":"","filename":"Fig.9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/22fdae523b2ce3bc13adfa80.jpg"},{"id":105567275,"identity":"ae39da10-73c8-4150-a7c2-bd0f30c25e78","added_by":"auto","created_at":"2026-03-27 12:58:45","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":26101,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram exhibiting the commonality and uniqueness of identified proteins in different tissues and stages of banana fruit. (A) 90-DAF peel, (B) 90-DAFpulp, (C) 12-DAR pulp, (D) 12-DAR peel.\u003c/p\u003e","description":"","filename":"Fig.10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/05cb496f335313925ab7bcb1.jpg"},{"id":105553712,"identity":"c32dcdee-9b2c-4e61-bc9a-733c4bc50f2b","added_by":"auto","created_at":"2026-03-27 10:33:30","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1075591,"visible":true,"origin":"","legend":"\u003cp\u003eReal time quantitative polymerase chain reaction (RT-qPCR) analysis representing the gene expression profiles of (A) XTH4, (B) POD, (C) PEL, (D) SUS and (E) SOD, and CYP as reference gene. PC, pre-climacteric stage and C, climacteric stage. Each vertical bar represents the relative expression of gene at different developmental and ripening stages and vertical bars represent standard deviation from three replicated assays.\u003c/p\u003e","description":"","filename":"Fig.11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/418535fd101ac140981e5321.jpg"},{"id":105567413,"identity":"85d9b458-4b9c-4c3f-8c25-48765cc02f87","added_by":"auto","created_at":"2026-03-27 12:59:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80263,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/19a51c098596048d3b73f8a7.docx"},{"id":105567180,"identity":"956be4b4-63e9-4931-b574-7ec2157691cd","added_by":"auto","created_at":"2026-03-27 12:58:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":226964,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/bcf504ab738c9f884bbc7c89.docx"},{"id":105553715,"identity":"ff308674-a90d-4e01-b646-a5dd24ed6aee","added_by":"auto","created_at":"2026-03-27 10:33:30","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":78079,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/a11bbd5948fcff82c5b908cb.docx"},{"id":105751963,"identity":"2d64530b-d8fe-4efb-9a09-7badabcdd00a","added_by":"auto","created_at":"2026-03-30 15:52:04","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":69667,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9184162/v1/191dd127bd2a56e03127a73a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBanana belonging to the family Musaceae is the fourth most important food crop after rice, wheat, and corn. Banana is a fruit crop cultivated across tropical and subtropical regions with overall consumption of 21 kgs per person per year. As a climacteric fruit, the physicochemical changes that occur during banana ripening are fast, resulting in a notably perishable fruit [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although ripening is an irreversible, genetically regulated, and coordinated process, it produces the desired quality characteristics of edible fruit [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, over-softening exacerbates handling damage, reducing fruit's shelf life and making it more vulnerable to post-harvest diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. About 35\u0026ndash;40% of the produced fruit and vegetables in India are lost yearly due to excessive softening [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile a genome remains constant to a large extent, the proteins in any particular cell change substantially or drastically because of mRNA degradation, ineffective translation, or selective splicing as well as the post-translational modifications (PTMs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, in fruit ripening and related post-harvest studies, the proteomic approach has emerged as a crucial technique [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As proteins are considered effectors of gene expression, they play a central role in regulating many metabolic pathways related to the ripening process [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this context, by identifying several functional proteins impacted during the process, proteome studies can aid in elucidation of fruit ripening [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, expression levels and post-translational modifications may help to understand the complex cellular processes at the protein level in fruit biology research [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndeed, there have been several gel-based and gel-free proteomic investigations on the ripening of diverse fruits, including climacteric fruit such as apricot, mango, peach, tomato, oil palm, melon, and non-climacteric fruit such as grapes, chinese bayberry, strawberry and olive [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] However, in the case of bananas, few studies are reported on the proteome of different organs or tissues, such as meristem of the plant, during drought in leaf tissue and chilling injury mechanism in peel tissues [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Further, Dominguez-Puigjaner et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] studied the analysis of proteins from banana pulp tissue limited to separating proteins extracted from the fruit during four stages of maturation. These samples subjected to 2D gel electrophoresis resulted in only five proteins of the same molecular weight identified by immunoblotting as polygalacturonase-related proteins [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the current application of proteomic technique, investigating banana fruit ripening through proteomics is rather scanty. The accurate quantification of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The mass spectrometry-based platforms have provided significant advances in accuracy, sensitivity, and the ability to multiplex vastly complex samples through bioinformatic tools [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Toledo et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] have identified 26 proteins in the pulp tissue of both pre-climacteric and climacteric stages using nano-LC-MS/MS analysis. Another study by Yun et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] identified 94 proteins in the peel tissue of bananas at 1, 8, 15, 17, 19, and 21days after harvest by a combined 2DE and MALDI-TOF/TOF-MS based proteome study. The orbitrap fusion mass spectrometry is a high-throughput technique that allows a greater number of peptide coverage with a high resolution at a faster rate with more accuracy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The sensitivity of the mass spectrometry technology used in this investigation have demonstrated by the identification of 950\u0026ndash;1416 proteins in various banana tissue types and stages using orbitrap-fusion mass spectrometry coupled with nano-LC-MS/MS.\u003c/p\u003e \u003cp\u003ePrevious studies by Inaba et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] have clearly demonstrated that ethylene signalling was negatively controlled in banana pulp tissue, whereas positively controlled in banana peel [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There are currently few tissue-specific proteome studies, and more research is needed to understand the many plant parts and metabolic processes, such as the starch synthesis and its breakdown into sugars, which are promoter-specific in various tissues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The current proteomic study provides information on glucose, maltose and sucrose, protein expression, functional category, protein-protein interactions, and gene expression for selective proteins of diverse metabolic pathways during pre-climacteric and climacteric stages and tissues in banana. The main objectives of this study were (i) to investigate the stage and tissue-specific proteome changes, and (ii) to investigate the proteins involved in various metabolic processes, and protein-protein interactions of the identified proteins that were operational during the ripening process. Our study provides an overview of critical biological processes that was operational during banana fruit ripening. Nevertheless, this is the first comprehensive gel-free proteome analysis of banana fruit tissues that has led to the identification of proteins implicated in various metabolic processes affecting the cell wall re-modelling, sugar and starch metabolism, hormone control, signalling and stress, and defence processes that cause the changes in texture that take place when banana ripens.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials\u003c/h2\u003e \u003cp\u003eAt 90- days after flowering (DAF), the Cavendish banana fruit (\u003cem\u003eMusa acuminata\u003c/em\u003e cv. Grand Naine) was collected from OUAT banana germplasm unit, Bhubaneswar, Odisha. Fully developed mature banana fruit at 90-DAF were allowed to ripen at ambient temperature for different time points, i.e., 2, 4, 6, 8, 10, and 12 days after ripening (DAR). Fruit samples were selected for uniformity in size, shape, colour, firmness, and free from visual defects. After separating the peel and pulp tissues from the pre-climacteric and climacteric stages, samples were flash frozen in liquid N\u003csub\u003e2\u003c/sub\u003e and kept at -80\u0026deg;C until further experiment. For the biometric, biochemical, and proteomic analyses, banana fruit tissues (pulp and peel) during pre-climacteric and climacteric phases were taken into consideration. Banana fruit samples from 90-DAF and 12-DAR were considered for the proteome analysis. At the same time, banana fruit samples from different developmental stages, i.e., 20, 40, 60, 80, and 90-DAF and ripening stages, i.e., 2, 4, 6, 8, 10, and 12-DAR, were considered for the biometric and biochemical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biometric assays\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Measurement of fruit fresh weight and firmness\u003c/h2\u003e \u003cp\u003eFresh weights were recorded in triplicate for each developmental and ripening stage (g kg-1) FW and plotted in the graph after the fresh weights of the banana fruit (whole fruit) and tissues (peel and pulp) from various stages were separated. Using a penetrometer (model no. FR-5120, Lutron, USA) with a 6 mm plunger tip, the fruit was punctured to determine its firmness. After tearing off a tiny piece of banana fruit peel, three identical fruits were examined for hardness, each with five distinct points. Newtons (N) were used to represent the maximal force needed to pierce the banana fruit with the plunger tip.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Biochemical assays\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Starch estimation\u003c/h2\u003e \u003cp\u003eThe starch content in banana peel and pulp tissues during the developmental and ripening stages was estimated according to Shafiee et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Total sugar content\u003c/h2\u003e \u003cp\u003eThe total sugar content was analysed by following Franscistt et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Sample preparation and estimation of glucose, maltose and sucrose\u003c/h2\u003e \u003cp\u003eFruit tissue samples were prepared by following the modified method of Nath et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and then 5 mL of Carrez I solution, 5 mL of Carrez II solution and 10 mL 100 mM of NaOH solution was added and mixed properly after each addition. Then the volumetric flask was filled to 50ml with distilled water, mixed properly and filtered with Whatman\u0026reg; paper no1. From the filtrate 0.1mL (100\u0026micro;l) of liquid sample was taken for sugar estimation. Free D-glucose, maltose and sucrose contents of the samples were estimated by the method described by megazyme K-MASUG assay kit (Megazyme International Ireland Ltd, Wicklow, Ireland).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Extraction of proteins\u003c/h2\u003e \u003cp\u003eOnly molecular biology-grade chemicals were used in the current proteome study. Protein extraction was done by following Carpentier et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] with slight modification, the proteins from the pulp and peel tissue of banana fruits at 90-DAF and 12-DAR were phenol extracted. One gram of pulp and peel (separately) tissue samples were ground in liquid N2 and added 5.0 mL of ice-cold extraction buffer (50 mM Tris-HCl (pH 8.5), 5 mM EDTA, 100 mM KCl, 1% w/v dithiothreitol [DTT], 30% w/v sucrose), containing a protease inhibitor cocktail (Sigma-Aldrich, USA, P9599). After adding an equivalent amount of cold Tris-HCl buffered phenol (pH 8.0), the mixture was stirred for 15 min at 4\u0026deg;C. Centrifugation at 6000\u0026times;g for 30 min at 4\u0026deg;C was used to recover the phenol phase, which was then extracted again using the aqueous buffer. The samples were kept overnight at -20\u0026deg;C to precipitate the proteins using five volumes of 100 mM ammonium acetate in methanol. The proteins were then recovered by centrifugation (16,000 \u0026times;g, 45 min, 4\u0026deg;C). The pellets were washed with 0.2% w/v DTT in acetone and air dried at RT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sample preparation\u003c/h2\u003e \u003cp\u003e300 \u0026micro;l of 1\u0026times;PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) was used to dissolve 30 mg of dry protein pellet samples. The mixtures were then vortexed briefly for five minutes. The pellet sample of dissolved proteins was centrifuged for five minutes at 4\u0026deg;C at 12,000\u0026times;g. For later usage, the clear supernatant was transferred to 1.5 mL eppendorf tube and kept at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Quantification of protein samples\u003c/h2\u003e \u003cp\u003eProtein concentration was determined by using Quick start 1X Bradford assay kit (BioRad, USA, 5000205) and BSA as a standard reference. At 595nm, the absorbance value of unidentified protein samples was determined. A standard curve was plotted based on the absorbance and BSA content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 SDS-PAGE separation of proteins\u003c/h2\u003e \u003cp\u003e100 \u0026micro;g of protein samples were dissolved in loading buffer (0.5 M tris-HCl [pH 6.8], 10% SDS, glycerol, and β-mercaptoethanol) before being incubated for four minutes at 95˚C in a water bath for SDS-PAGE analysis. Protein samples were added to the first lane, followed by the marker/protein ladder (Bio-Rad, USA, 1610317) with a molecular mass (Mw) range of 6.5\u0026ndash;200 kDa. The protein samples were then resolved on a 12% polyacrylamide gel and stained with the GelCode blue dye reagent (Pierce, USA, 24590).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 LC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eProteins were reduced, alkylated, and digested at 37\u0026deg;C in ammonium bicarbonate and with trypsin, as described in Ray et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Orbitrap fusion mass spectrometry was used to analyse the peptides after they had been desalted using spin columns. One microgram of digested peptides was placed onto a pre-analytical column (100 \u0026micro;m \u0026times; 2 cm, nanoViper C18, 5 \u0026micro;m, 100 A; Thermo Fisher Scientific) after each fraction was reconstituted in 15 \u0026micro;l of solvent A. The peptides were eluted with an isocratic gradient from 5% to 30% B for 110 min, 30\u0026ndash;95% B for 2 min, and then held at 95% B for 8 min at 300 nl/min flow rate on an analytical column (75 \u0026micro;m \u0026times; 50 cm, 3 \u0026micro;m particle, and 100 \u0026Aring; pore size; Thermo Fisher Scientific) coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA). The MS parameters were as follows: for full MS spectra, the scan range was \u003cem\u003em/z\u003c/em\u003e 375\u0026ndash;1700 with a resolution of 60000 at \u003cem\u003em/z\u003c/em\u003e100. MS/MS acquisition was carried out in full speed mode with a cycle time of three seconds. At m/z100, the resolution was 15000. The maximum injection time was 50 ms, and the intensity threshold was 5000. The isolation window was 1.2 m/z, and the AGC target was set to (4.0e5) 400000. Higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 30% successively broke apart ions having charges of 2+, 3+, and 4+. 40 seconds was chosen as the dynamic exclusion duration.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Data analysis\u003c/h2\u003e \u003cp\u003eProteome Discoverer (PD) version 2.2 (Thermo Fisher Scientific) was used to process raw instrument files. The Sequest HT and Mascot (v2.6.0) search engines were used to search MS2 spectra against \u003cem\u003eMusa acuminata\u003c/em\u003e fasta from the Uniprot database. Every search was set up with static modifications such as carbamidomethyl (+\u0026thinsp;57.021Da) on cysteine, monoisotopic masses, and trypsin cleavage (maximum two missed cleavages) and dynamic modifications on methionine residues (+\u0026thinsp;15.9949Da). The MS/MS tolerance was 0.05Da and the peptide precursor mass tolerance was 10 ppm. For proteins, peptides, and peptide spectral matching (PSMs), the false discovery rate (FDR) was maintained at 1%. Proteome Discoverer 2.2 was used to export the protein quantification values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Protein-protein interactions\u003c/h2\u003e \u003cp\u003eTo determine the functions and interactions of the identified proteins, a protein-protein interaction network (PPI) was analysed using Gene Mania.org [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The orthologous interaction pairings (interologs) were obtained by mapping the pulp/peel DAF and DAR differentially expressed proteins to GeneMania Arabidopsis thaliana. Venny (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the common and unique proteins, and additional pathway maps between them were examined. Although the \u003cem\u003eMusa acuminata\u003c/em\u003e genes were mapped to GeneMania, String-db.org (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.string.embl.de\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.string.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to directly verify these interologs in the Musa database, which was mapped a relatively few.\u003c/p\u003e \u003cp\u003eWe use cytoHubba [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] to find the top-ranking genes based on the clustering coefficient and the coherent pathways and clusters associated with these distinct protein-coding genes. While betweenness, closeness, and centralities could form the other ranking coefficients, we felt that the clustering coefficient would yield more distinct interaction partners.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3 Functional categorization\u003c/h2\u003e \u003cp\u003eProteins identified through mass spectrometry were functional categorized based on the literature available on different climacteric fruit in connection with the ripening process.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.9 RNA isolation and cDNA synthesis\u003c/h2\u003e \u003cp\u003eThe RNA isolation from both the tissue of banana was carried out by following the method of Meisel et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. First-strand cDNA was synthesized using total RNA by using Superscript-II reverse transcriptase first strand cDNA synthesis Kit (Invitrogen, USA) and Oligo dT primer (Sigma) for random amplification of unknown RNA according to the manufacturer\u0026rsquo;s recommendation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Quantitative real-time PCR (RTqPCR) validation\u003c/h2\u003e \u003cp\u003eThe RTq-PCR study was implemented with Invitrogen Quantstudio-5 (Applied Biosystems, USA) under the following condition: step (1) 50˚C 2 min, step (2) 95˚C 2 min, step (3) (95˚C 0.15 min, 50\u0026ndash;60˚C 0.15 min, 70 ˚C 1min) \u0026times; 40 cycles, followed by the thermal dissociation curve [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The relative expression level was analyzed using the 2-ΔΔCt method and normalized with reference housekeeping gene cyclophilin (CYP) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eThe present study considered banana fruit from pre-climacteric and climacteric stage for the proteome study. The fruit was selected based on its size, shape, texture, colour, and visual appearance. The fruit size was larger and has slight ridges on the pedicel parts in the 90-DAF (pre-climacteric stage) stage and appears to be green with faint yellow traces. In contrast, fruit in the 12-DAR (climacteric stage) had no discernible ridges, the pedicle portion turned black and dried, the fruit was brown with spots, and it had a softer texture than fruit in the 90-DAF stage (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFor the biometric and biochemical analysis, banana fruit tissues (pulp and peel) during pre-climacteric and climacteric phases were taken into consideration. A graph was created by measuring the fresh weights of the banana fruit (whole fruit) and tissues (peel and pulp) from various developmental and ripening stages and fresh weights were recorded in triplicate for each stage (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Weight loss of banana fruit increased with the progress of ripening and the maximum weight loss recorded after 6-DAR. The maximum increase in the weight of pulp tissue was recorded at 90-DAF. The fruit firmness results are depicted in Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e\n\u003cp\u003eIn the current study, the starch content of banana fruit tissue increased during developmental stages (40, 60, and 90-DAF) and reduced during various ripening stages (2, 4, 6, 8, 10, and 12-DAR) (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), describing the process of starch hydrolysis and sugar synthesis. Notably, the current study revealed an ascending trend in total sugar content from the developmental to the ripening stages of banana, signifying that sugar accumulation peaks at the commencement of ripening (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Further, the total sugar content pattern mirrored the expression patterns of sugar metabolism proteins, indicating that starch degradation into simple sugars caused textural alterations during banana fruit ripening. In consistence with the total sugar content, monosaccharides (D-glucose), disaccharides (maltose), and polysaccharides (sucrose) in banana peel and pulp tissues dramatically increased at the climacteric stage compared to the pre-climacteric stage (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003ePhenol-based extraction method was followed to extract proteins from banana pulp and peel tissues in both the pre-climacteric and climacteric stages. Samples were considered for the experiment from three independent biological replicates. Further, extracted proteins were quantified by Bradford assay using BSA as a standard. All protein samples extracted with phenol from the pulp and peel tissues from different stages yielded high concentrations. For quality check, the known amount of protein (100\u0026micro;g) was separated on a 12% SDS-PAGE and visualized using gel code blue stain reagent staining. Proteins were well separated with distinct bands, reflecting protein\u0026rsquo;s excellent quality without any degradation. Most of the protein bands fell between 14\u0026ndash;200 kDa (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eA gel-free proteome analysis of banana peel and pulp tissues using Orbitrap Fusion mass spectrometry resulted 950 to 1416 proteins in the pre-climacteric and climacteric stages, respectively. Proteins participated in metabolic processes including signal transduction, cell wall modification, carbohydrate metabolism, stress response and defence, hormone regulation, and redox homeostasis. The identified proteins were catalogued together with their corresponding accession numbers, total peptide scores, peptide coverage percentages, amino acid, molecular weights in kilo-daltons (kDa), unique peptide counts, and peptide sequences. (Supplementary Table\u0026nbsp;2\u0026ndash;5).\u003c/p\u003e\n\u003cp\u003eThe functional classification was done for the mass spectrometry-identified proteins, which showed the abundance of the peel and pulp tissues of pre-climacteric and climacteric stages (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Proteome analysis revealed that the proteins involved in transport and stress/defense mechanisms were found to be abundant in the pulp tissue of pre-climacteric and peel tissue of climacteric stage, respectively. At the same time, the same proteins were low in abundance in peel tissue of pre-climacteric and pulp tissue of climacteric stage. Cell wall degrading proteins displayed a high abundant in peel and pulp tissues of climacteric stage, whereas low in peel tissue of pre-climacteric stage. However, compared to the pre-climacteric stage, it was revealed that the pulp and peel tissues of the climacteric stage had higher protein abundant linked to sugar metabolism (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D).\u003c/p\u003e\n\u003cp\u003eTable \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e represents the proteins with different accession numbers (ID) identified through mass spectrometry which was commonly found in banana tissues at pre-climacteric and climacteric stages with varied peptide coverage (%). Changes in protein quantity or modulation were noted in the pre-climacteric and climacteric banana peel and pulp tissues based on the peptide coverage (%). Cell wall modification proteins such as \u0026beta;-hex and \u0026beta;-gal were higher in the pulp tissues of pre-climacteric and climacteric stages than in peel tissue. This study identified one number of \u0026beta;-hex (#M0T2F6) in the peel tissue of climacteric stage. One \u0026beta;-hex protein in pre-climacteric (# M0T2F6) and 3 \u0026beta;-hex proteins in climacteric stage (# M0T2F6, M0RQ03, M0S3Q7) were identified in the banana pulp. Further, 4 (#M0S9W4, M0RVL3, M0SQP6, M0SX47) and 1 \u0026beta;-gal enzyme (M0SQP6) was identified in peel tissues of banana fruit at the pre-climacteric and climacteric stage, respectively.\u003c/p\u003e\n\u003cp\u003eLikewise, other cell wall degrading proteins such as PE and POD were found to be high in peel tissue in the climacteric stage compared with pulp tissue of both stages. Interestingly, in our study 1 number of PE (#M0RPM3) in pre-climacteric and 4 numbers of PE (#M0SC42, M0RPM3, M0TGN6, M0SC43) in the climacteric stage were identified in the peel. One PE (# M0RPM3) protein was commonly identified in the banana pulp during pre-climacteric and climacteric stages.\u003c/p\u003e\n\u003cp\u003eIn the current study, one (#M0TE13) and four numbers of \u0026alpha;-man (#M0TWG0, M0TLF4, M0U935, M0TWG0) were identified in the pulp tissue of pre-climacteric and climacteric stages, respectively. Three \u0026alpha;-man proteins in the pre-climacteric stage (# M0TWG0, M0U935, M0T6Z4) and four \u0026alpha;-man proteins in the climacteric stage (# M0TWG0, M0T6Z4, M0TLF4, M0U935) were identified in the banana peel. Further, one number of XTH proteins (accession no-M0U6S4) was placed in pulp tissue at the climacteric stages of banana fruit. Two numbers of XTH proteins in the pre-climacteric stage (# M0TTV0, M0U6S4, M0TZC7, M0RMW1) and four XTH in the climacteric stage (# M0RMW1, M0TQQ9, M0TGI2, M0TTV0) were identified in the banana peel. Overall, proteins such as XTH and \u0026alpha;-man were present more in peel tissue of the climacteric stage than in pulp tissues of both stages.\u003c/p\u003e\n\u003cp\u003ePL proteins were found in peel tissue of the pre-climacteric stage and pulp tissue of the climacteric stage. It is modulating more in peel tissue of the pre-climacteric stage than the climacteric stage. Overall, PL may play an active part in the ripening process because there is a higher peptide coverage in the climacteric stage than in the pre-climacteric stage. Two PL (# M0U687, M0TAX1) were identified in peel tissues of the climacteric and pre-climacteric stages and one (M0TAX1) in pulp tissues of both the stages, respectively.\u003c/p\u003e\n\u003cp\u003eIn the present study, one S-adenosylmethionine synthase (SAM-synthase) protein in peel and pulp tissue of pre-climacteric (#M0T0C8) and climacteric stage (#M0SCW4) was identified. A total of five (#M0S638, M0TN23, M0SE45, M0T0C8, M0S0A4) SAM-synthase proteins were identified in peel tissue of the climacteric stage.\u003c/p\u003e\n\u003cp\u003eSugar metabolism-related protein such as SUS was exclusively present in the peel tissues of the pre-climacteric and climacteric stage. A high peptide coverage in case of SUS during the ripening stage is clear evidence of its role in starch hydrolysis to form simple sugars during ripening. SUS proteins were found in banana pulp at the pre-climacteric (M0RJE1) and climacteric (M0RJE1, M0TSQ0) stages, respectively. Further, a total of seven numbers of fructose-bisphosphate aldolase in pre-climacteric and five number in climacteric stage were identified in banana peel tissue. Likewise, four fructose-bisphosphate aldolase proteins in pre-climacteric and seven in climacteric stage were identified in banana pulp tissue.\u003c/p\u003e\n\u003cp\u003eIdentified proteins of cell wall modification, ROS mechanism, and sugar metabolism (represented in bold) were interpreted in metabolic pathways to understand their possible role in fruit ripening (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Tissue types were shown in the legend, where proteins were abundant and present at particular stages. For pulp and peel tissues of the pre-climacteric and climacteric stages, distinct numerals or codes were assigned: 1-pre-climacteric peel; 2-climacteric peel; 3-pre-climacteric pulp; and 4-climacteric pulp (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Proteins such as XTH, PL, and PE were involved in cell wall modification. XTH acts as an intermediate to convert xyloglucan to xylose. At the same time, PE serves as an intermediate for converting pectin into PL. SUS is involved in sugar metabolism in pre-climacteric and climacteric stages of pulp tissues. SUS acts as intermediates for the conversion of sucrose into fructose. SOD and GPX were involved in the ROS mechanism and present in both the tissues (peel and pulp) and stages.\u003c/p\u003e\n\u003cp\u003ePyruvate dehydrogenase (PDH) is a convergence point in regulating the fine metabolic tuning between glucose and fatty acid oxidation. Pyruvate kinase (PK) catalyzes the final step in glycolysis, in which phosphoenolpyruvate is converted to pyruvate. The product pyruvate is then served to prime the TCA cycle.\u003c/p\u003e\n\u003cp\u003eFrom the protein-protein interaction (PPI) networks, we observe that there are unique proteins specific to particular tissue type and stage, which we assume many of the sequences are novel. However, XTH protein 8-related (PTHR31062) is the ideal candidate that is known to be associated with unique pathways in peel at the climacteric stage. We argue that, while our results suggested that ROS-related proteins possibly cross-talk with cell wall metabolism by promoting the cell wall loosening (Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), one of the striking features associated with the pre-climacteric stage of peel and pulp is that the climacteric stage peel has a distinct set of genes mapped with XTH protein 8-related (PTHR31062) or XTH family members with XTH4 forming a central node of interacting partners. This could be because of its prominent ripening role in the peel instead of the pulp, which includes later. A host of other proteins, including CYTC-2 family members and APX3, are known to primarily shown to be interacting in this milieu. To check this, we sought to ask which among these genes form the top-ranking genes, and we used cytoHubba. Our results indicate that the \u003cem\u003eMusa acuminata\u003c/em\u003e network has no well-annotated interactants.\u003c/p\u003e\n\u003cp\u003eIn contrast, the ones with \u003cem\u003eArabidopsis\u003c/em\u003e have distinct candidates that are well annotated, and the network is more robust as averse to the former (Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D). From Fig. \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the higher the contrast, the greater the rank (maroon being the top and light yellow being the lowest among the top 10 ranking clusters). Among them, we observed that cytochrome-c oxidase sub unit Vb. Cytochrome-c oxidase (CcO), the terminal oxidase, is among the top as it is known to be a multi-chain transmembrane protein in mitochondria that aids oxidation. In addition, ascorbate peroxidases and rubredoxin are the other top candidates (AT1G80230 and AT3G15640; Fig. \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This could be because PEs aid the xylem translocation during ripening in pedicels, void of water content.\u003c/p\u003e\n\u003cp\u003eThe Venn diagram (Fig. \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) depicts the detected number of common and distinct differentially expressed proteins identified from different banana tissue of 90-DAF and 12-DAR. In the case of 90-DAF peel and pulp, a total of 175 (14.6%) and 36 (3%) unique proteins, sharing 5 (0.4%) common for both tissues. This suggests that the presence of a specific set of proteins may have distinct functions in two tissues. Twenty-six (2.2%) proteins were common between pulp tissues of 90-DAF and 12-DAR. Thirty-six (3%) and 203 (16.9%) proteins were unique for 90-DAF pulp and 12-DAR pulp, respectively. 108 (9%) common proteins were identified between pulp (#203) and peel tissues (#131) of 12-DAR. 91 (7.6%) proteins are commonly studied for tissues and ripening stages. They reveal a complex nature and active metabolic pathways that are operational during ripening in pulp tissues of 12-DAR.\u003c/p\u003e\n\u003cp\u003ePeel and pulp tissues at the pre-climacteric stage had higher levels of XTH4 expression than at the climacteric stage (Fig. \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA). An averse, another cell wall modification gene, PEL expression was increased in the peel during the climacteric stage (Fig. \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC). Peroxidase (POD), superoxide dismutase (SOD) and SUS expression was decreased at climacteric stage, compared to pre-climacteric stage in the pulp tissue (Fig. \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB, E, D).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe present study revealed that the phenol-based method effectively extracted a high yield of proteins over the TCA/acetone method and resulted in good protein separation on SDS-PAGE. Fruit softening is dependent on changes to the structural characteristics of the cell wall, including the enormous depolymerisation and solubilisation of proteins, lignin, and polysaccharides (pectins, cellulose, and hemicelluloses) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe starch content decreased with an increase in sugar content during the climacteric stage, suggesting the ripening phase starch hydrolysis and sugar synthesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, the banana pulp presented significantly higher sugar content than the peel during the climacteric stage. Our results are inconsistent with the previous reports, where the total amount of mono-(D-glucose), di- (maltose), and polysaccharides (sucrose) in banana peel and pulp tissues increased significantly at the climacteric stage compared to pre-climacteric stage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Proteins associated with cell wall metabolism\u003c/h2\u003e \u003cp\u003eCell wall disassembly, which is aided by several cell wall-degrading proteins such as PE, PL, PG, PME, and others, breaks down many polysaccharide networks and promotes banana ripening. The PE functions primarily by altering the localized pH of the cell wall resulting in alterations in cell wall integrity. This enzyme is known to extensively decrease the rigidity of cell wall structure and solubilization of pectins during fruit softening [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePE proteins were previously found in pulp tissues at two phases of ripening, mesocarp tissues at ripe and unripe stages, and pulp tissues at four ripening stages in peach [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Further, the high number of PE proteins in the present study during the climacteric stage suggests that these proteins may have key role in fruit softening at this phase.\u003c/p\u003e \u003cp\u003eLikewise, PL is involved in the maceration and soft-rotting of fruit tissue [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At the climacteric stage of banana fruit, two PL proteins were up-regulated in the peel [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and pulp tissue [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which was consistent with the breakdown of cell wall components [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the current investigation, peel tissues at both phases showed a high abundance of PL proteins. In addition, the peel and pulp tissues exhibited increased expression of the PL gene, indicating PL could play essential role in breakdown of pectin in banana peel tissues during ripening, and our findings are in consistent with the earlier reports on different fruits [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFruit softening is facilitated by the ripening-specific N-glycan processing enzyme called β-hex [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Earlier, β-hex was identified in pericarp tissues of capsicum at four developmental and three ripening stages [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This protein was also identified in the pericarp tissues of tomatoes at four ripening stages [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Further, the RNAi mediated suppression of β-hex genes, resulted in extended shelf-life for 30-days in tomato and 7-days in capsicum [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, β-hex proteins were identified in high abundance during the climacteric stage, indicating that they may have an active role in banana ripening.\u003c/p\u003e \u003cp\u003eAnother class of cell wall-modifying proteins called XTH, helps in maintaining the integrity of the cell wall through endotransglucosylase and weaken it during fruit ripening through hydrolase activity. XTHs involved in the cross talk regulatory mechanism of auxin and ethylene signalling to promote fruit ripening [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this regard, XTH proteins were identified in other fruit tissues during ripening stages, such as pericarp tissues of tomatoes, kiwi, and mesocarp tissue of apricot [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similarly, Kok et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] revealed that oil palm had five different XTH proteins, all of which were up-regulated during the ripening stages. In consistent with earlier reports, in this study four XTH proteins were identified in the peel during ripening.\u003c/p\u003e \u003cp\u003eA cell wall modifying protein called β-gal aids in the debranching of pectin and enhances the depolymerisation process as fruit softens [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Previously, the β-gal during fruit softening was reported in diverse fruit such as mango [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], chocolate vine [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], tomato [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and strawberries [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this regard, β-gal proteins showed a high abundance in banana peel and pulp tissues during the climacteric stage, which suggests that β-gal could play a role in degrading the pectin in the cell wall and breakdown of polysaccharides in banana leading to a softening of fruit during ripening.\u003c/p\u003e \u003cp\u003eThe changes in the activity of the cell wall-bound PODs during the fruit ripening determine the firmness of the fruit. Also, it inhibits cell wall tightening by breaking the cell wall bonds [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Escalante-Minakata et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] have identified the PODs in both pulp and peel tissues of banana fruit during the developmental and ripening stages. In the present study, a high number of POD proteins were identified in banana peel at the climacteric stage, suggesting that POD may modulate the banana ripening process by removal of ROS, there by tightening the cell wall.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Protein associated with sugar metabolism\u003c/h2\u003e \u003cp\u003eThe fruit goes through a number of physiological and biochemical changes as it ripens, which breaks down the starch and causes an accumulation of sugar or sucrose [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Once sucrose reaches the sink cells, it hydrolysed by SUS or invertases into glucose and fructose. SUS is mainly involved in synthesizing carbohydrate polymers, i.e., starch or cellulose, or in the generation of active compounds which help in fruit development [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In this regard, Tian et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] have identified SUS proteins in kiwi fruit tissues (exocarp) at different developmental and ripening stages. In the present investigation, a high number of SUS proteins in the climacteric stage of banana pulp suggests that sugar synthesis is quite active during the ripening process. In contrast, reduced SUS transcript level was recorded at climacteric stage in both peel and pulp tissues, it could be due to the post translational modification, such as selective pre-mRNA splicing, methylation and de-methylation process that might occured at transcript level [\u003cspan additionalcitationids=\"CR61 CR62 CR63\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn essential component of sugar metabolism, fructose-bisphosphate aldolase (FBA) also controls the sink metabolism of fruit tissue during ripening [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. A total of four up-regulated proteins of FBA were identified in watermelon fruit at three developmental stages. The FBA proteins in melon have significantly increased, which implies that this protein may play a critical role in sucrose metabolism during fruit ripening [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Further, using TMT labelling coupled with LC-MS/MS analysis, Li et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] have revealed that FBA proteins actively participate in the sugar metabolism and are found to be increased in abundance during blueberry ripening [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this investigation, a total of four and seven FBA proteins were identified in pulp tissues of pre-climacteric and climacteric stages, respectively. Whereas, in the case of peel tissues, three and five FBA proteins were identified in both the stages of banana, respectively. Overall, our study concluded that sugar metabolism-related proteins were highly modulated during the banana fruit ripening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Proteins associated with hormonal regulation\u003c/h2\u003e \u003cp\u003eVarious plant hormones regulate fruit development and ripening process in the cell. Several studies have attempted to identify receptor proteins and signaling components in connection with multiple plant hormones. The CS protein is involved in the formation of methionine which is generally involved in ethylene biosynthesis through the intermediary\u0026rsquo;s cystathionine and homocysteine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CS enzyme was down-regulated during the ripening stages of tomato pericarp and also in mango pulp [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In the present study, one CS protein in the pre-climacteric and six CS proteins in the climacteric stage were identified in the banana pulp. Likewise, six CS proteins in the pre-climacteric and four CS proteins at the climacteric stage were identified in peel tissue. However, based on our result, it is speculated that by participating in the ethylene biosynthesis pathway, CS proteins may play a significant role in the ripening process.\u003c/p\u003e \u003cp\u003eDuring fruit ripening, S-adenosylmethionine synthase (SAM-synthase) proteins participate in various polyamine processes as well as the synthesis of ethylene [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. SAM serves as a precursor of polyamine and ethylene, which are known to regulate fruit ripening [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Proteomic investigations in the pericarp of cherry tomatoes also revealed an increase in the quantity of SAM- synthase proteins during fruit ripening [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] and peach mesocarp [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. According to Choi et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], there was a comparatively high expression of SAM-synthase in the early stages of tomato ripening as opposed to the later stages. In the present study, SAM-synthase proteins (#6) showed a high abundance in the climacteric stage peel tissue. Our findings are inconsistent with earlier reports on other fruits during ripening. In this scenario, SAM-synthase may be essential for ethylene production.\u003c/p\u003e \u003cp\u003eSignalling by auxin and ABA is mediated by tetratricopeptide repeat (TPR) domains proteins. The ETO1 (ETHYLENE-OVERPRODUCER1) protein in Arabidopsis has been shown to directly interact with a 1-aminocyclopropane-1-carboxylate synthase isoform through its TPR domains, thereby negatively regulating ethylene production in seedlings [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. TPR proteins precise function in fruit ripening is still unknown. Based on previous reports, it was observed that ABA regulates ethylene biosynthesis, and ABA-ethylene interaction triggers the fruit ripening process [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. ABA governs the transformation of 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene during fruit ripening by ethylene-dependent or -independent mechanisms [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Nevertheless, no molecular mechanism examining the relationship between ABA and ethylene during fruit ripening not been published yet. In the present study, interestingly, TPR proteins showed high abundance in peel tissue of climacteric stage (#7), and we speculate that it may have some role in hormonal regulation during ripening process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Proteins associated with stress, defense, ROS mechanism\u003c/h2\u003e \u003cp\u003eThe climacteric fruit ripening process involves an increase in the respiration and, consequently, alteration of redox homeostasis in the cell, with reactive oxygen species (ROS) build-up, which in turn determines lipid peroxidation, protein denaturation, and metabolism deterioration to achieve a final degradation state functional to seed release [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSOD is known to be involved in many biological processes, such as oxidizing the lipids and denaturation of DNA fragments [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. In higher plants, SODs act as antioxidants and protect cellular components from being oxidized by ROS [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The compartmentalization of different forms of SOD throughout the plant makes them counteract stress very effectively [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. SOD enzyme was identified in the pulp tissues of peach during two developmental and two ripening stages, and their results showed high O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents in the middle stage of peach fruit development. In another study, SOD was identified in the pulp tissues of peach at 125-DAF. The results revealed that Mn-SOD plays a key role in mitochondria for regulating the ripening and senescence process in peach fruit [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Using multiple reaction monitoring (MRM), Song et al. [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] investigated the strawberry antioxidant defense system during ripening. They have identified three SODs through QTRAP-LC-MS/MS that were down-regulated during later stages of strawberry ripening. In the current investigation, banana peel tissues at the pre-climacteric stage had a significant number of SOD proteins (#7), inconsistent with the observation, gene expression studies further revealed the reduced gene expression in the pulp tissue at climacteric stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). In this regard, our results agree with previous reports on different fruit, where SOD proteins may play a role in scavenging reactive oxygen species and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content that are arising due to high respiration rates during banana developmental and ripening stages. It may also be a signaling molecule driving different metabolic processes, promoting fruit rapid development.\u003c/p\u003e \u003cp\u003eDuring three ripening stages, three classes of GPX enzymes were identified in peach skin. The study revealed that GPX enzymes were up-regulated during later stages of peach ripening [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. The proteome study has shown that GPX proteins were involved in ROS mechanism and, exhibited higher defense anti-oxidative capacities and protected the fruit from deterioration in chilli and strawberry fruit during ripening [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In this regard, we speculate that the presence of more GPX proteins in this study may play a protecting role in banana peel tissue during the climacteric stage.\u003c/p\u003e \u003cp\u003eGlutathione reductase (GR) plays a key role in maintaining the cellular control of ROS. It acts as an antioxidative mechanism in the fruit ripening process, which requires a turnover of active oxygen species (AOS) such as hydrogen peroxide and superoxide anion [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. GR protein levels were elevated in the tomato's pericarp during ripening [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, two GR proteins were identified during the pre-climacteric stage, suggesting that GR protein may mediate the biochemical and physicochemical changes occurring during ripening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Protein-protein interaction studies\u003c/h2\u003e \u003cp\u003eAdditionally, a study on the protein-protein interactions has shown that the peel tissue of the climacteric stage has a distinct set of proteins mapped with XTH ​protein 8-related (PTHR31062) or XTH family members with XTH4 forming a central node of interacting partners. In addition, our results speculate that the banana peel ripening may be significantly influenced by XTH proteins by disassembling the cell wall and degradation of hemicelluloses, leading to a softening of the peel tissue, thereby reducing the shelf-life of the fruit. In contrast, XTH4 gene expression was reduced in the peel tissues at later stage of ripening, compared to pre-climacteric stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). It could be due to the fact that mRNA expression is not always correlated to the quantity of expressed proteins due to varied regions [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe functional classification of identified proteins showed differential abundance between the pre-climacteric and climacteric stages of the banana. Functional categorization of proteins from banana peel and pulp tissues at pre-climacteric and climacteric stages revealed that proteins were involved in various metabolic pathways and biological processes such as amino acid metabolism, sugar metabolism, starch biosynthesis, hormonal regulation, stress, and defense mechanism, cell wall modification, signaling, transport, protein folding, energy, and carbohydrate metabolism, etc. Moreover, the presence of numerous cell wall modification proteins in the climacteric stage compared to the pre-climacteric stage of banana fruit indicates that cell wall modification proteins actively participate in the cell wall softening process. Identification of high numbers of sugar metabolism-related proteins in the climacteric stage exhibited the phenomena of starch breakdown to form simple sugars during fruit ripening.\u003c/p\u003e \u003cp\u003eA large number of unique tissue specific protein sets (based on Venn) indicates the necessity of conducting tissue-specific proteome investigations. Interestingly, some of the proteins were identified in the form of β-adaptin, ferritin, 2-Hacid_dh_domain, PAP-fibrillin, ADK-lid domain, why domain, GLP, and clathrin protein, whose specific role in fruit ripening is still unexplored. Further, some uncharacterized proteins were also identified in our study, which could be considered as novel proteins (whose functional validation is not carried out) as sequence information is not available in the protein database. Some of the critical cell wall modification proteins identified through this investigation, such as α-man and β-hex, were identified in other fruit crops, and conformed enhancement in the fruit shelf-life, when corresponding genes were supressed using RNAi. However, further studies seek to validate and assign a functional role for these proteins in bananas.\u003c/p\u003e \u003cp\u003eThe protein-protein interaction study revealed that the XTH family members with XTH4 forming a central node of interacting partners and XTH ​protein 8-related (PTHR31062) are the ideal candidate associated with unique pathways in peel at the climacteric stage. XTH proteins were revealed to play a significant role in the post-harvest softening of fruit, auxin and ethylene-mediated signalling. It necessitates further investigation to unlock the functional role of XTH protein in banana fruit ripening. This study will shed light on the regulatory process of the ripening mechanism in bananas. Some of the key candidates identified can be further validated through the genome editing tools to assign functional roles in controlling ripening and to enhance fruit shelf-life in banana.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe fruit samples were collected from OUAT banana germplasm unit, Bhubaneswar, Odisha with the consent of the competent authority of the institute. All plant materials were cultivated under controlled field conditions and were not collected from the wild. No specific collection permits or licences were required, as no wild specimens were collected. The guidelines followed for the use of plants or plant materials in the study.\u003c/p\u003e\n\u003ch2\u003eConsent to publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was supported by the research grant to G.K.Surabhi by Rashtriya Krishi Vikash Yojana, Government of India (No. AG(RKVY)04/2017\u0026ndash;9975/Ag.dt.22.06.2017; OR/RKVY-HORT/2017/774), Science and Technology Department, Government of Odisha (No.27552800232014/202830, STBBSR, dt.17.7.2015) and Forest, Environment and Climate Change Department, Government of Odisha, India, is gratefully acknowledged.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSM, DP: Sample collection, lab work, formal analysis, investigation, interpretation of the data, writing - original draft; GKS: Conceptualization, writing-review and editing, critical revising, research supervision, PS: Bioinformatic analysis. All authors approved the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors wish to acknowledge the Mass Spectrometry Facility at IIT Bombay (MASSFIITB), supported by the Department of Biotechnology (BT/PR13114/INF/22/206/2015), for their support with the mass spectrometry analysis of the samples. We thank the Chief Executive, Regional Plant Resource Centre, for extending the facilities.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSurabhi GK, Patnaik S, Mohanty S. A comparative method for protein extraction and proteome analysis by two-dimensional gel electrophoresis from banana fruit. 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Identification and biochemical characterization of the glutathione reductase family from \u003cem\u003ePopulus trichocarpa\u003c/em\u003e. Plant Sci. 2020;294110459. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plantsci.2020.110459\u003c/span\u003e\u003cspan address=\"10.1016/j.plantsci.2020.110459\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Harmon AC. Advances in plant proteomics. Proteom. 2006; 6(20):5504-16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://10.1002/pmic.200600143\u003c/span\u003e\u003cspan address=\"https://10.1002/pmic.200600143\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 16972296.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eComparison of protein expression based on peptide coverage in different tissues at pre-climacteric and climacteric stage of banana for cell wall modification, stress response and defense, signal transduction, hormone regulation and sugar metabolism functional categories of proteins based on mass spectrophotometry analysis.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"650\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of the protein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein ID/stage/tissue \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeptide coverage %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"32\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e90-DAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12-DAR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e90-DAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12-DAR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"28\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"28\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epeel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epulp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epeel\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epulp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epeel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epulp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epeel\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epulp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 650px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell wall modification\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003ePectinesterases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TGN6, M0SC42,\u003c/p\u003e\n \u003cp\u003eM0RPN3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0RPM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TGN6, M0SC42,\u003c/p\u003e\n \u003cp\u003eM0RPN3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0RPM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e8,4,12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4,8,14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003ePectate lyase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0U687, M0TAX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0U687, M0TAX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5,18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e10,29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026beta;-hexosaminidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T2F6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0T2F6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T2F6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0T2F6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eXyloglucan endotransglucosylase/hydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RMW1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RMW1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026alpha;-mannosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TWG0, M0U935, M0T6Z4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TWG0, M0U935, M0T6Z4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2,3,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4,9,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026beta;-galactosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SQP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0SQP6, M0RVL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SQP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0SQP6, M0RVL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e9,21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 650px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStress response and defense\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eSuperoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0S4H9, M0SFL6, M0RG01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0S4H9, M0S978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0S4H9, M0SFL6, M0RG01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0S4H9, M0S978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e20,33,12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e19,22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e38,49,18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e25,57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003ePeroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TCA6, M0RVD5, M0THT5, M0RUU7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0TBJ2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TCA6, M0RVD5, M0THT5, M0RUU7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0TBJ2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e61,17,68,19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e68, 3, 72, 29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eGlutathione peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SWM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0RP81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SWM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0RP81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eTyrosinase copper binding protein (Cu-bd)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0UBI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0UBI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eGermin-like proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U4X1, M0S2X3, M0UCZ2, M0U0R8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U4X1, M0S2X3, M0UCZ2, M0U0R8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e25, 23,12, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e16, 13, 12, 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eBarwin protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T386\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T386\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eIsocitrate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RX69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RX69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eThioredoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TQW7, M0SR53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0TQW7, M0SR53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e48,58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e34,28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SEK3, M0S0R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SEK3, M0S0R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e21, 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e30,23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eEukaryotic translation initiation factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T7M2, M0S626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0S626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T7M2, M0S626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0S626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e27,22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e43,13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003ePeroxiredoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RH93, M0TP05, M0RKE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RH93, M0TP05, M0RKE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e19,27, 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e30,27,41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eLactoylglutathione lyase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RNM6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RNM6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eGlutaredoxins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0S2Q5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0S2Q5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e26S proteasome regulatory subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0RYR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0RYR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eCyclophilins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RZQ7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RZQ7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eClP protease ATPase subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0TSN5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0TSN5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 650px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignal transduction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eNucleoside diphosphate kinases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0SZK9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0U940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0SZK9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eTubulin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SS87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0SS87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 650px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHormone regulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eAminomethyl transferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T8H5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0SM28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T8H5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0SM28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 650px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSugar metabolism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eSucrose synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RJE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0RJE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0RJE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0RJE1, M0SI67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e12, 62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eFructose-bisphosphate aldolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eM0U9P4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eM0T021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eM0U9P4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Post-harvest, Proteomics, Pulp softening, Ripening, Cell-wall modification, Sugar metabolism, RNAi, Shelf-life","lastPublishedDoi":"10.21203/rs.3.rs-9184162/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9184162/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlobally, bananas are a major staple food with significant nutritional and commercial potentials and are extremely perishable with a very short shelf life. Understanding the proteins expressed in different tissues during ripening can help in improving postharvest quality and shelf life. A gel-free comparative proteomic analysis was followed to investigate protein alterations in peel and pulp tissues during pre-climacteric and climacteric phases of banana. Total starch content was decreased when ripening progresses. In contrast, total sugar, sucrose, glucose and maltose content was increased several folds during the climacteric phase, in both peel and pulp tissues. The protein samples were subjected to orbitrap fusion mass spectrometry coupled with nano LC-MS/MS resulted in the identification of 950 and 1300 proteins in pulp and 1416 and 1279 proteins in peel tissues at pre-climacteric and climacteric stages, respectively. Mass-spectrometry identified proteins were categorised and they were involved in starch and sugar metabolism, cell wall modification, hormonal regulation and detoxification of reactive oxygen species (ROS), which were more prevalent in the pulp tissue during the climacteric period as compared to the pre-climacteric stage. Proteins such as α-1,4-glucan (α-gluc) phosphorylase, pectin esterase (PE), β-galactosidase (β-gal), α-mannosidase (α-man), pectate lyase (PEL), xyloglucan endotransglucosylase/hydrolase (XTH), pectin acetylesterase and β-hexoaminidase (β-hex) were found to be more in number in climacteric stage, and could be responsible for pulp softening, de-greening of the peel, alterations in texture and fruit quality. The study found that proteins related to sugar metabolism, such as fructose bisphosphate aldolase and sucrose synthase (SUS), were more abundant during the climacteric than pre-climacteric stage. This implies that during fruit ripening, these proteins contribute to the synthesis of sugars and the disintegration of starch. Interestingly, some of the proteins that play a crucial role in hormonal regulation were identified in the form of cysteine synthase (CS) (#17), amino methyltransferase (#10), and a more significant number of CS proteins were identified in pulp tissue at the climacteric stage. Venn analysis of the proteins from different tissues and stages suggests the presence of unique set of proteins in tissue specific manner, and could have a specialized role. Further, the protein-protein interaction study confirms that the XTH could be an ideal candidate known to be associated with unique pathways in peel at the climacteric stage. RTqPCR analysis revealed greater transcript levels of XTH4 and SOD in pulp, and PEL in peel tissue at climacteric stage indicates cell wall disintegration and loosening at climacteric stage. The potential candidate proteins identified in this investigation could be of immense help to gain insights of the regulatory mechanism of banana ripening process. Identified proteins can be further validated using genome editing technology to assign individual functional roles.\u003c/p\u003e","manuscriptTitle":"Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-27 10:33:12","doi":"10.21203/rs.3.rs-9184162/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T07:27:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T10:21:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146034557220151622471593941394455396558","date":"2026-04-04T19:58:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T09:08:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42655187013526292887231284890276030270","date":"2026-04-02T08:53:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-31T08:42:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-30T17:06:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-28T01:42:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T06:29:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Plants","date":"2026-03-26T06:23:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53229acc-443c-4aa9-9d1a-3fe93c813ff5","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T07:39:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 10:33:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9184162","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9184162","identity":"rs-9184162","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}