In this publication, we showcased the potential of targeted analysis of low molecular weight compounds (LMWC) through LARSI-MSI in SRM modes, directly applied to intact plant tissues. One of the main advantages of this method is atmospheric pressure ionization that allows for the direct analysis of native or frozen samples without the need for extensive sample preparation or vacuum conditions. This is particularly important for tissue imaging, as it enables researchers to study the spatial distribution of various molecules, including metabolites, lipids, and proteins, without altering their native distribution within the tissue (Wu et al. 2013b). This also makes it possible to perform real-time imaging, providing dynamic information about molecular distributions and changes over time without the need for labeling or staining (Xiao et al. 2020).
The LARESI-SRM-MSI method offers a notable advantage in terms of its straightforward and efficient sample preparation. With this technique, a fragment of the object, measuring up to 5 x 5 cm, is required to be placed directly in the instrument's chamber, eliminating the need for additional coating with MALDI matrix. This streamlined approach simplifies the process, saving time and reducing the risk of introducing artifacts that could alter the sample's original composition (Dong et al. 2016b).
The option of cooling tissue samples is of considerable importance in tissue analysis (Goodwin 2012). In the LARESI method, the tissue sample is securely mounted on the Peltier stage, which can be set to a temperature of -18°C. This cooling capability offers several advantages during the analysis process. Firstly, it helps to preserve the integrity of the biological material, minimizing any potential degradation or changes in the molecular composition that could occur at higher temperatures. Secondly, the cooling of the tissue sample can slow down metabolite diffusion and thus enhance the spatial resolution and sensitivity of the analysis, allowing for more precise and accurate imaging of the molecules within the tissue.
The key advantage of this method lies in its utilization of a mid-IR laser for the analysis of biological material. Firstly, the IR laser provides enhanced sampling depth, allowing researchers to analyze molecules deeper within the tissue, thereby providing a more comprehensive understanding of the molecular composition (Wu et al. 2013b). Mid-IR lasers with a wavelength around 2.94 µm, can effectively couple their energy into the O-H stretching mode of hydrogen-bonded water, which is present in hydrated biological materials. This results in a greater depth of sampling, allowing for analysis of molecules deeper within the tissue, up to about 10 µm for a single laser pulse. Secondly, the IR laser ablation is a non-destructive technique, preserving the integrity of the biological material during analysis and enabling researchers to study the spatial distribution of compounds without altering their native state. Additionally, the IR laser offers high sensitivity and selectivity, allowing for the targeted analysis of specific compounds of interest, while reducing chemical noise (Wu et al. 2013b).
For our study, we utilized commercially available banana tissue slices as the sample material. Figures 1 and 2 depict ion images of twenty-six selected metabolites found in slices of Cavendish banana and Red Dacca Banana tissue, respectively. Mass spectrometry parameters of LARESI SRM MSI Experiments are listed in Table 1. To further validate the MSI results, an extensive analysis of banana tissue extracts was conducted using ultra-high-performance liquid chromatography and ultra-high-resolution mass spectrometry (UHPLC-UHRMS). The untargeted results of the analysis of banana extracts are listed in Table 2.
Table 2
Metabolite Identification in Cavendish Banana Extract using UHPLC-UHRMS Method
No | Name | Molecular Formula | m/za | RT [min] | Ions | Δm/z [ppm] |
1 | 3-Hydroxybutyric acidb,d | C4H8O3 | 146.0812 | 1.10 | [M + H]+, [M + H + CH3CN]+ | 0.5 |
2 | 2,3-Butanediolb,d | C4H10O2 | 132.1016 | 18.11 | [M + H]+, [M + H + CH3CN]+ | -2.0 |
3 | 2-Hydroxyacetanilideb,d,e | C8H9NO2 | 152.0703 | 17.75 | [M + H]+ | -2.1 |
4 | 2-Octenoic acidb,c | C8H14O2 | 184.1330 | 17.88 | [M + H]+ | -1.3 |
5 | 2-Oxoarginineb,d | C6H11N3O3 | 174.0871 | 8.45 | [M + H]+ | -1.2 |
6 | 3-Hydroxy-4-methoxycinnamic aicdb,d | C10H10O4 | 236.0912 | 0.97 | [M + H]+ | -1.9 |
7 | 4-Ethylbenzoic acidb,c | C9H10O2 | 151.0750 | 3.94 | [M + H]+ | -2.4 |
8 | 4-Methylcatecholb,d | C7H8O2 | 166.0860 | 0.72 | [M + H + CH3CN]+, [M + NH4]+ | -1.7 |
9 | 5α-Androstane-3β,17β-diolb,d | C19H32O2 | 310.2736 | 8.43 | [M + H]+ | -2.1 |
10 | 5-Aminopentanoateb,d,e | C5H11NO2 | 118.0861 | 3.17 | [M + H]+ | -1.4 |
11 | 6-(Methylamino)purineb | C6H7N5 | 150.0772 | 1.64 | [M + H]+ | -1.8 |
12 | 6-Phosphogluconic acidb,c | C6H13O10P | 277.0318 | 2.28 | [M + H]+ | -0.4 |
13 | Acetic acidb,d | C2H4O2 | 102.0548 | 2.89 | [M + H]+ | -1.2 |
14 | Adenosineb,d,e | C10H13N5O4 | 268.1033 | 1.76 | [M + H]+, [M + Na]+ | -2.3 |
15 | Biotinb,d | C10H16N2O3S | 245.0948 | 0.98 | [M + H]+ | -1.7 |
16 | Butanalb,d | C4H8O | 114.0910 | 2.05 | [M + H]+ | -2.7 |
17 | Cellobioseb,c,d | C12H22O11 | 360.1495 | 5.57 | [M + NH4]+, [M + H-H2O]+, [M + H]+ | -1.5 |
18 | Cytidineb,c,d | C9H13N3O5 | 307.1020 | 6.42 | [M + H]+ | 2.1 |
19 | Alanineb,c | C3H7NO2 | 90.0549 | 6.57 | [M + H]+ | -0.8 |
20 | Galactoseb,c,d | C6H12O6 | 203.0528 | 1.17 | [M + H]+ | 0.8 |
21 | Glucoseb,c,d | C6H12O6 | 219.0262 | 7.93 | [M + H]+ | -1.9 |
22 | Dimethylglycineb,c,d | C4H9NO2 | 142.0261 | 1.63 | [M + H]+ | -0.2 |
23 | Maltoseb,c,d | C12H22O11 | 381.0788 | 1.15 | [M + H]+ | -1.8 |
24 | Dodecanoic acidb,c | C12H24O2 | 242.2108 | 1.07 | [M + H]+ | -2.8 |
25 | Epigallocatechinb,d,e | C15H14O7 | 307.0805 | 1.11 | [M + H]+ | -2.3 |
26 | Erythroseb | C4H8O4 | 162.0759 | 2.33 | [M + H]+ | -1.3 |
27 | Ethyl isopropyl ketoneb | C6H12O | 139.0517 | 1.10 | [M + H]+ | -2.1 |
28 | Formamideb | CH3NO | 125.0109 | 1.41 | [M + H]+ | -1.9 |
29 | Fructose 6-phosphateb,c | C6H13O9P | 298.9925 | 1.81 | [M + H]+ | -1.2 |
30 | Glutamylcysteineb,d | C8H14N2O5S | 251.0695 | 0.98 | [M + H]+ | -0.9 |
31 | Glycerolb,c | C3H8O3 | 134.0812 | 1.06 | [M + H]+ | 0.1 |
32 | Glycerophosphocholineb,c,d | C8H20NO6P | 296.0656 | 8.09 | [M + H]+ | -1.2 |
33 | Guanidineb,d | CH5N3 | 139.0384 | 1.84 | [M + H]+ | 2.5 |
34 | Heptadecanoic acidb | C17H34O2 | 293.2457 | 1.10 | [M + H]+ | 2.0 |
35 | Homogentisic acidb,d,e | C8H8O4 | 169.0490 | 1.14 | [M + H]+ | -3.0 |
36 | Imidazoleb,c,d | C3H4N2 | 110.0711 | 1.17 | [M + H]+ | -1.7 |
37 | Indolelactic acidb,c,d | C11H11NO3 | 206.0814 | 6.31 | [M + H]+ | 0.0 |
38 | Isobutyric acidb,c | C4H8O2 | 89.0595 | 1.17 | [M + H]+ | -2.6 |
39 | Isocitric acidb,c,d | C6H8O7 | 210.0607 | 1.15 | [M + H]+ | -0.7 |
40 | Arginineb,c,d | C6H14N4O2 | 213.0746 | 1.31 | [M + H]+ | -1.0 |
41 | Asparagineb,c,d | C4H8N2O3 | 133.0607 | 17.87 | [M + H]+ | -0.7 |
42 | Aspartic acidb,c | C4H7NO4 | 134.0447 | 1.20 | [M + H]+ | -1.0 |
43 | Levoglucosanb,d | C6H10O5 | 163.0598 | 1.74 | [M + H]+, [M + H-H2O]+, [M + NH4]+ | -1.8 |
44 | Fucoseb,d | C6H12O5 | 206.1006 | 1.03 | [M + H]+ | -1.3 |
45 | Glutamineb,c,d | C5H10N2O3 | 147.0762 | 1.18 | [M + H]+ | -1.4 |
46 | Histidineb,c,d | C6H9N3O2 | 194.0326 | 5.69 | [M + H]+ | -0.3 |
47 | Linoleic acidb,d | C18H32O2 | 298.2731 | 1.03 | [M + NH4]+, [M + Na]+ | -2.6 |
48 | Lysineb,c | C6H14N2O2 | 147.1128 | 1.01 | [M + H]+ | -0.3 |
49 | Malic acidb,c | C4H6O5 | 152.0551 | 1.11 | [M + H]+ | -1.8 |
50 | LPC(16:1)b,d,e | C24H48NO7P | 494.3218 | 1.10 | [M + H]+ | -4.7 |
51 | LPC(18:2)b,d,e | C26H50NO7P | 520.3374 | 1.01 | [M + H]+, [M + Na]+ | -4.5 |
52 | LPC(18:3)b,d,e | C26H48NO7P | 518.3218 | 19.70 | [M + H]+ | -4.5 |
53 | Prolineb,c,d | C5H9NO2 | 116.0705 | 1.10 | [M + H]+ | -1.3 |
54 | Serineb,c,d | C3H7NO3 | 106.0496 | 19.09 | [M + H]+ | -2.1 |
55 | Sorboseb,c,d | C6H12O6 | 163.0598 | 17.44 | [M + H-H2O]+, [M + H]+ | -1.7 |
56 | Mannose 6-phosphateb,c,d | C6H13O9P | 261.0367 | 8.12 | [M + H]+ | -1.4 |
57 | Methionineb,d | C5H11NO2S | 191.0849 | 1.09 | [M + H]+ | 0.2 |
58 | Trimethyllysineb,d,e | C9H20N2O2 | 189.1596 | 16.38 | [M + H]+ | -1.1 |
59 | Oenanthic etherb,c,d | C9H18O2 | 200.1640 | 13.15 | [M + H]+ | -2.1 |
60 | Oxoglutaric acidb,c | C5H6O5 | 147.0287 | 12.93 | [M + H]+ | -0.9 |
61 | Pantothenic acidb,d | C9H17NO5 | 220.1174 | 7.39 | [M + H]+, [M + Na]+ | -2.5 |
62 | Coumaric acidb,d,e | C9H8O3 | 165.0542 | 1.12 | [M + H]+ | -2.4 |
63 | Pentadecanoic acidb,d | C15H30O2 | 243.2311 | 1.16 | [M + H]+ | -1.9 |
64 | Phenylacetaldehydeb,d,e | C8H8O | 121.0646 | 1.08 | [M + H]+, [M + NH4]+ | -1.6 |
65 | Phenylacetic acidb,d,e | C8H8O2 | 137.0594 | 10.51 | [M + H]+, [M + NH4]+, [M + H-H2O]+ | -2.3 |
66 | Phenylalanineb,d,e | C9H11NO2 | 166.0858 | 15.53 | [M + H]+, [M + Na]+ | -2.8 |
67 | Phenylglyoxylic acidb,c | C8H6O3 | 168.0658 | 0.90 | [M + H]+ | 1.8 |
68 | Phosphocreatineb,d | C4H10N3O5P | 253.0704 | 1.02 | [M + H]+ | 3.0 |
69 | Propyl alcoholb | C3H8O | 102.0911 | 1.10 | [M + H]+ | -2.5 |
70 | Propylene glycolb,c,d | C3H8O2 | 118.0861 | 1.12 | [M + H]+ | -1.5 |
71 | Putrescineb,c | C4H12N2 | 89.1073 | 11.50 | [M + H]+ | -0.6 |
72 | Pyridoxamineb | C8H12N2O2 | 169.0969 | 0.93 | [M + H]+ | -1.7 |
73 | Pyroglutamic acidb,c,d | C5H7NO3 | 130.0497 | 1.23 | [M + H]+ | -1.6 |
74 | Pyruvaldehydeb | C3H4O2 | 114.0548 | 1.09 | [M + H]+ | -1.7 |
75 | Quinic acidb,c | C7H12O6 | 193.0705 | 1.09 | [M + H]+ | -0.7 |
76 | Quinoneb,c,d | C6H4O2 | 109.0283 | 1.21 | [M + H]+ | -0.8 |
77 | Raffinoseb,d,e | C18H32O16 | 522.2032 | 5.52 | [M + H]+ | -2.2 |
78 | Raffinoseb,c,d | C18H32O16 | 543.1313 | 6.52 | [M + NH4]+, [M + Na]+ | -1.9 |
79 | Rhamnoseb,c | C6H12O5 | 206.1021 | 1.76 | [M + H]+ | -0.8 |
80 | Salicinb | C13H18O7 | 309.0938 | 1.09 | [M + H]+ | -2.2 |
81 | Salsolinolb,d,e | C10H13NO2 | 180.1015 | 7.30 | [M + H]+ | -2.5 |
82 | Senecioic acidb,c,d | C5H8O2 | 118.0858 | 0.87 | [M + H]+ | -2.8 |
83 | Methylmethionineb,d,e | C6H13NO2S | 164.0738 | 19.73 | [M + H]+ | -1.0 |
84 | Succinic acid semialdehydeb,d | C4H6O3 | 85.0285 | 14.61 | [M + H-H2O]+, [M + NH4]+ | 1.0 |
85 | Sucroseb,d,e | C12H22O11 | 360.1494 | 1.72 | [M + NH4]+, [M + H]+, [M + H-H2O]+ | -2.0 |
86 | Trehaloseb,c,d | C12H22O11 | 365.1047 | 1.76 | [M + H]+ | -2.2 |
87 | Tyrosineb,d,e | C9H11NO3 | 182.0809 | 8.84 | [M + H]+ | -1.9 |
aExperimental monoisotopic mass; bthe metabolites identified by high precursor mass accuracy; cthe metabolites identified by matching retention time; dthe metabolites identified by matching isotopic pattern; ethe metabolites identified by matching MS/MS fragment spectra; m/z: mass-to-charge ratio; RT: retention time; |
Bananas are known for being abundant in various compounds found in the plant world, such as phenolic and polyphenolic substances like catechin, myricetin, quercetin, and phenolic acids like caffeic, protocatechuic acid, ferulic acid, and sinapic acids. Additionally, volatile compounds like isobutyl butyrate, eugenol, and pyruvic acid, representing alpha-keto acids, are also present in significant quantities. Phenolics and polyphenolics are the dominant compounds in banana fruits, and they are widespread throughout the plant kingdom. These secondary metabolites are of utmost importance due to their various health benefits, including antioxidant, antimicrobial, anti-inflammatory, anticancer, anti-mutagenic, and antidiabetic properties. The profiling of phenolic compounds in different banana varieties was carried out using HPLC–ESI-HR-MS and HPLC-DAD methods (Simirgiotis et al. 2013; Tongkaew et al. 2022). The research findings indicate that bananas serve as an excellent source of phenolic compounds, although their content can vary in different parts of the fruit, such as the pulp and peel.
Among the compounds under analysis, phenolic acids played a prominent role, including protocatechuic acid, caffeic acid, ferulic acid, and sinapic acid. Protocatechuic acid, classified as a dihydroxybenzoic acid, is a naturally occurring phenolic acid widely distributed across the plant kingdom, alongside other plant phenols. In previous studies, both protocatechuic acid and caffeic acid were successfully identified in acetone banana pseudostem extracts using reverse-phase HPLC and ESI-MS, indicating their presence in this plant tissue (Saravanan and Aradhya 2011). Furthermore, quantification of these compounds was performed in banana rhizome extracts from eight commercial banana cultivars, with protocatechuic acid identified as the primary phenolic compound in acetone extracts of banana rhizomes(Kandasamy and Aradhya 2014) Caffeic acid, a common cinnamic acid derivative, is renowned for its antioxidant and antimicrobial properties and was also detected in banana peel extracts using paper spray-mass spectrometry in separate studies (Vu et al. 2018; Silva et al. 2020).
Bananas are renowned for being rich in compounds with valuable medicinal properties, including ferulic acid. As a phenolic acid and derivative of cinnamic acid, ferulic acid is widely distributed throughout the plant kingdom, naturally occurring in plant cell walls, notably in conifers' leaves, seeds, and bark, as well as in grains like wheat, rice, corn, and rye. The significance of ferulic acid extends to various beneficial biological activities, especially for human health, such as its role as an antioxidant, antimicrobial, anti-inflammatory, anticancer, anti-mutagenic, and antidiabetic agent. Its widespread applicability is reflected in its use across the food, cosmetics, and pharmaceutical industries, owing to its low toxicity and numerous health benefits (Zduńska et al. 2018) Notably, ferulic acid's potent antioxidant properties surpass even those of vitamins C and E. Previous studies have involved the mechanical extraction of ferulic acid from banana stem waste, aiming to determine kinetic parameters in the production of this compound using HPLC. In our study ferulic acid is primarily found in the outer part of the banana peel (Fig. 1J). The presence of ferulic acid in the peel of a banana can explain its structural role in cross-linking and strengthening of plant cell wall polysaccharides (Russell et al. 2009). The presence of ferulic acid in banana fruit and stem extracts has been consistently confirmed in numerous publications, with HPLC being the chosen method for its identification (Zainol et al. 2018). Furthermore, ferulic acid was successfully determined among other bioactive compounds in stem juices from banana plants using a tandem high-resolution mass spectrometry (UHPL-HRMS/MS) method. An investigation into variations in the occurrence of potentially antidiabetic compounds revealed significant differences in the concentration of ferulic acid based on the origin of the bananas, underscoring the influence of growth conditions on its presence (Nguyen et al. 2017). Phenolic compound profiling in various banana varieties was also conducted through two analytical methods, HPLC–ESI-HR-MS and HPLC-DAD. The research findings demonstrated that bananas serve as a rich source of phenolic compounds; however, their content exhibits variations between the pulp and peel of this fruit (Passo Tsamo et al. 2015).
The remarkable antioxidant properties of bananas can be attributed to the presence of various phenolic compounds, including sinapic acid. Similar to ferulic acid, sinapic acid is a derivative of cinnamic acid and is predominantly found in the outer region of the pulp near the peel (Fig. 1K). Sinapic acid has been identified through LCMS/MS analysis in banana roots (Sebatian Fenolik dan Komposisinya dalam Tindak Balas Pertahanan Pokok Pisang Berangan yang et al. 2021). Furthermore, it has been observed that sinapic acid levels increase in bananas treated with artificial ripeners (Tallapally et al. 2020). The LARESI method revealed pyruvic acid to be the compound with the lowest molecular weight in this study. Previous research on extracts from different morphological parts of bananas indicated that ferulic acid is more abundant in leaf sheaths and floral stalks, whereas rachis contains it in smaller quantities (Oliveira et al. 2006).
Quercetin, catechin, and myricetin are prominent members of the flavonoid family, renowned for their robust antioxidant activity. These naturally occurring phenols and secondary metabolites are widespread among various plants. Structurally, they share similarities and belong to the flavonol class of flavonoids, exhibiting numerous common functions. In our study, all three compounds were detected in close proximity to the fibrovascular bundles (Fig. 1M, 1N, 1O). Flavonoids serve vital roles as secondary metabolites synthesized by plants, contributing to various functions, including stress defense, pigmentation, and attraction of pollinators or seed dispersers (Buer et al. 2010). In the context of fruits, flavonoids play a crucial role in pigmentation and safeguarding against environmental factors (Harborne and Williams 2000). The strategic location of flavonoids like quercetin, catechin, and myricetin near fibrovascular bundles can be attributed to their specific transport and localization patterns. Fibrovascular bundles play a pivotal role in facilitating substance movement within fruits, ensuring the efficient distribution of flavonoids to regions where they are needed (Quattrocchio et al. 2006). This localization in proximity to fibrovascular bundles ensures the availability of flavonoids for protective and pigmentation purposes, contributing to the overall health and resilience of the banana fruit. The antioxidant properties of polyphenols such as quercetin, catechin, and myricetin in bananas have been extensively researched. Notably, in a study comparing 15 commonly consumed fruits and vegetables, bananas were found to have the highest phenolic content (JP et al. 2016). Catechin has been identified as the primary constituent in both banana peel (Someya et al. 2002) and pulp (Bennett et al. 2010) extracts. Additionally, quercetin has been detected in bananas using UPLC-MS analysis (Septembre-Malaterre et al. 2016). These findings highlight the significant presence of these beneficial polyphenols in bananas and underscore their potential health-promoting effects.
Flavonoids, such as flavanols like quercetin and myricetin, have been successfully identified in the sap of various dessert banana species using HPLC-ESI-MS (Pothavorn et al. 2010)Both quercetin and catechin have been detected in the banana pulp at all stages of fruit development. Among the flavonoids, quercetin emerged as the predominant metabolite in its free soluble form in bananas (Dong et al. 2016a). Furthermore, myricetin has been previously reported in extracts from the banana peel (Zhang et al. 2019).
In this study, pyruvic acid was identified as the compound with the lowest molecular weight, using the LARESI method, and was found in the pulp close to the center of the banana fruit (Fig. 1D, 1E). Pyruvic acid is a three-carbon carboxylic acid containing both a ketone and a carboxyl functional group. Its significance lies in its crucial role as an intermediate in various metabolic pathways within the cell, including those involving lipids, amino acids, and carbohydrates.In the cell's environment, pyruvic acid dissociates into pyruvate ions (pyruvate). It is produced during glycolysis, where glucose molecules are broken down into two pyruvate ions. Subsequently, in the mitochondria's matrix, pyruvate is converted into acetyl Coenzyme A (acetyl CoA), which further participates in the energy-supplying citric acid cycle (Krebs cycle) when oxygen is present. In the absence of oxygen, pyruvate ferments into lactate (lactic acid). Moreover, pyruvate can be converted into alanine via transamination or into oxaloacetate through carboxylation. Previous studies have quantified pyruvic acid in banana pulp extracts using gas-liquid chromatography (GLC) (J Colloid et al. 1987) and high-performance liquid chromatography (HPLC) (KOZUKUE 1981). These studies also revealed that the level of pyruvic acid slightly decreased during ripening after five months of storing bananas at -20°C. However, the isolation, separation, and identification of pyruvic acid, like other α-keto acids in biological materials, pose challenges due to their instability.
Isobutyl butyrate, a carboxylic acid ester, is a volatile compound that is produced during the ripening of banana fruits. Its identification and quantification in bananas have been extensively studied due to its significant impact on fruit and flavor quality, employing various analytical methods. In our experiment, isobutyl butyrate was detected in the banana pulp, with the highest intensity of the signal observed near the fibrovascular bundles (Fig. 1F). The quantification of isobutyl butyrate in commercial banana fruit and banana essence was achieved using GC-MS (Jordán et al. 2001). This aromatic compound was identified at two stages of banana ripening: during the color-turning and full-ripening stages, using the solid-phase micro-extraction (SPME) coupled with the GS-MS method (Zhu et al. 2018). Moreover, studies on bananas harvested in different seasons demonstrated that only fruits harvested in March contained isobutyl butyrate and exhibited a higher abundance of volatile aroma compounds (Zhang et al. 2020).
Another notable volatile compound in bananas is eugenol, which belongs to the naturally occurring phenylpropanoid class of organic aromatic compounds, featuring a propylbenzene skeleton. Similar to other secondary metabolites, eugenol was also found in close proximity to the fibrovascular bundles (Fig. 1H). It is associated with a phenolic, sweet flavor, contributing to the overall sensory profile of bananas. Eugenol, a significant flavor compound, has been identified in high concentrations in fresh banana fruit paste, banana extracts, and dehydrated banana powder, utilizing various methods coupled with GC-MS (Miranda et al. 2001; Wang et al. 2007; Pino and Febles 2013). Intriguingly, eugenol was not detected in commercial banana essence, highlighting the distinct aromatic profiles between fresh fruit and banana essence (Jordán et al. 2001). It is noteworthy that the flavor compounds in banana extracts are not formed during the growth and time of harvest, but rather during the ripening process of the fruit (Tressl et al. 1969). This implies that the development of characteristic flavors in bananas is a dynamic process, occurring primarily during the ripening stage, which significantly influences the overall sensory experience of consuming this fruit.
In the second experiment, the spatial distribution of 16 amino acids (AAs) in Red Dacca Banana tissue was examined. AAs play several important roles in banana fruit. As essential nutrients, AAs are essential building blocks for protein synthesis, which is crucial for the growth, development, and maintenance of the fruit. Proteins are essential components of various cellular structures and enzymes that regulate biochemical processes within the fruit. Additionally, amino acids are involved in various metabolic pathways within the banana fruit. They participate in energy production through processes like glycolysis and the citric acid cycle (TCA cycle), providing the necessary energy for cellular activities during fruit ripening and development (Wiskich and Dry 1985). AAs also contribute to the flavor and aroma of banana fruit. Certain amino acids are precursors to volatile compounds, which are responsible for the characteristic scent and taste of ripe bananas (Maoz et al. 2022). Furthermore, AAs play a role in stress responses and defense mechanisms in the fruit. During postharvest senescence, amino acids may be utilized to protect the fruit from oxidative damage and maintain cellular integrity (He et al. 2013). The majority of the tested AAs were found in the pulp of the fruit, with the exception of three amino acids: threonine, methionine, and cysteine, which were also present in the peel (Fig. 2G, 2L, 2R). Notably, cysteine accumulated mainly in the outer part of the banana peel, with only a negligible amount detected in the pulp. Furthermore, MS imaging revealed a high content of exogenous amino acids on the entire surface of the banana, such as valine, tryptophan, histidine, and phenylalanine, with their accumulation predominantly occurring between the peel and the pulp. In contrast, the lowest intensities in ionic images were observed for asparagine, lysine, and arginine. These amino acids possess additional amino groups in their side chains, which may contribute to their distribution pattern within the banana tissues. Our findings align with prior studies that shed light on the distribution of threonine, glutamine, and arginine within banana pulps during postharvest senescence (Yin et al. 2022) These studies revealed that these AAs exclusively accumulated in the middle region near the seed zone.