Cuticular waxes and cutin monomers were extracted in series from isolated cuticular membranes according to the previous methods with minor modifications (Huang et al., 2017 (link)). The isolated cuticular membranes were completely dipped in chloroform (Guangzhou Chemical Reagent Factory, China) with a mild temperature of around 40°C to better release the soluble waxes. The extraction time was set for 2 min. Each sample was extracted three times and combined with the extracts. Then, n-tetracosane (Sigma–Aldrich, Shanghai, China) was added as an internal standard to evaluate the cuticular contents. The solvent in the extracts was evaporated by gentle nitrogen gas until they dried for further analysis. After that, the membranes that have been removed of soluble waxes were subsequently depolymerized in boron trifluoride with methanol (BF3-methanol, 10%, ~1.3 M, Sigma–Aldrich, Shanghai, China) and incubated for 16 h at 70°C. After membranes were lysed, n-dotriacontane (Sigma–Aldrich, Shanghai, China) as an internal standard was added. Saturated aqueous sodium chloride solution and chloroform were added in series to extract the cutin monomers. The organic phase was collected and evaporated to dryness under a gentle stream of nitrogen gas for further analysis.
N tetracosane
Manufactured by Merck Group
Sourced in United States, China, Germany
N-tetracosane is a long-chain alkane with the chemical formula C24H50. It is a colorless, waxy solid at room temperature. N-tetracosane is commonly used as a reference standard in analytical chemistry and as a component in various industrial applications.
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Lab products found in correlation
N dotriacontane, by Merck Group (3 mentions)
N eicosane, by Merck Group (2 mentions)
Centrivap console, by Labconco (2 mentions)
N hexane, by Merck Group (2 mentions)
Chloroform, by Guangzhou Chemical (1 mentions)
Bf3 methanol, by Merck Group (1 mentions)
Phenylacetaldehyde, by Merck Group (1 mentions)
Benzaldehyde, by Merck Group (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Benzyl alcohol, by Merck Group (1 mentions)
Naphthalene, by Merck Group (1 mentions)
Vanillin, by Merck Group (1 mentions)
Benzyl benzoate, by Merck Group (1 mentions)
Eugenol, by Merck Group (1 mentions)
2 phenylethanol, by Merck Group (1 mentions)
Isoeugenol, by Merck Group (1 mentions)
L phenylalanine, by Merck Group (1 mentions)
6 p toluidino 2 naphthalenesulfonic acid sodium salt tns, by Merck Group (1 mentions)
Methyl benzoate, by Merck Group (1 mentions)
Ergosterol, by Merck Group (1 mentions)
14 protocols using n tetracosane
1
Cuticular Wax and Cutin Monomer Extraction
2
Chemical Reagent Preparation and Characterization
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Benzyl alcohol, benzaldehyde, benzylbenzoate, methylbenzoate, 2-phenylethanol, phenylacetaldehyde, eugenol, isoeugenol, vanillin, 6-(p-toluidino)−2-naphthalenesulfonic acid sodium salt (TNS), toluidine blue, n-tetracosane, l -phenylalanine, and naphthalene were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com ).
3
Extraction and Analysis of Banana Cuticular Waxes and Cutin Monomers
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The cuticular waxes and cutin monomers of banana fruit were extracted using the isolated cuticular membranes following the reported procedure previously (Huang, 2017 ). The dry and fine intact cuticular membranes isolated from banana fruit were immersed in chloroform completely. To better release the soluble waxes, the extraction was set with a moderate temperature at 40°C. The extraction for each sample was repeated three times, and each time for 2 min. After combining the extracted solution, n-tetracosane (Sigma-Aldrich, Shanghai, China) as an internal standard was added to help detect the accumulation of cuticular waxes. Gentle stream of nitrogen gas was used to dry the extracts for further analysis.
The above matrix membrane after removing waxes was subsequently depolymerized in boron trifluoride with methanol (BF3-methanol, 10%, ∼1.3 M) overnight at 70°C. Then, n-dotriacontane (Sigma-Aldrich, Shanghai, China) was added as an internal standard. Saturated aqueous sodium chloride solution and chloroform were added subsequently to extract the cutin monomers. The organic phase containing cutin monomers was collected and evaporated to dryness under a gentle stream of nitrogen gas for further analysis.
The above matrix membrane after removing waxes was subsequently depolymerized in boron trifluoride with methanol (BF3-methanol, 10%, ∼1.3 M) overnight at 70°C. Then, n-dotriacontane (Sigma-Aldrich, Shanghai, China) was added as an internal standard. Saturated aqueous sodium chloride solution and chloroform were added subsequently to extract the cutin monomers. The organic phase containing cutin monomers was collected and evaporated to dryness under a gentle stream of nitrogen gas for further analysis.
4
Tocopherol Homologs Analytical Standards
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Standards of α, β, γ, and δ homologs of tocopherol (T) (purity >95%) were purchased from Merck Millipore (Darmstadt, Germany). Methyl heptadecanoic acid (analytical standard, purity ≥ 99%), γ-linoleic acid (analytical standard, purity ≥ 99%), dodecanal (analytical standard, purity ≥ 98%), 1-octadecanol (ReagentPlus, purity ≥ 99%), n-tetracosane (analytical standard, purity ≥ 99.5%), and ergosterol (pharmaceutical secondary standard, purity 95%) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), while n-paraffin analytical standard (C8–C40 alkanes calibration standard) was obtained from Supelco Analytical (Bellefonte, PA, USA). HPLC grade pyridine was purchased from Honeywell Riedel-de Haën GmbH, (Seelze, Germany), HPLC grade methanol, n-hexane, petroleum ether (puriss. p.a., ≥99.9%, boiling point 50–70 °C), bis(trimethylsilyl) trifluoroacetamide (BSTFA) and boron trifluoride–methanol solution (BF3/MeOH) were purchased from Sigma-Aldrich Chemie Ltd., (St. Louis, MO, USA). The 2.2-diphenyl-1-picrylhydrazyl, 2-propanol (HPLC grade), resazurin sodium salt were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Deionized water was prepared using an Elix Advantage 3 water purification system (Millipore S.A.S., Molsheim, France).
5
Synthesis of Alkanes and Polymers
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All chemicals were used as purchased without further purification. n-Octadecane (99%), n-eicosane (99%), n-tetracosane (99%), MMA (99%), sodium dodecylbenzenesulfonate (SDBS, technical grade), and ammonium persulfate (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized (DI) water was produced using a Millipore Milli-Q system.
6
Extraction and Isolation of Leaf Surface Waxes
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The shoots with concurrent bud break were selected, and the fourth leaf from each shoot was harvested. Twenty leaves were randomly selected and pooled together as one biological replicate; four biological replicates were used. EWs were removed by gum arabic; the film was collected into a glass tube containing 21 ml of chloroform:water (2:1, v/v), and 75 μg of n-tetracosane (Sigma-Aldrich, St. Louis, United States) was added as internal standard. After vigorous agitation and phase separation, the organic phase was transferred into a new glass tube. Extraction was repeated with another 4.5 ml of extraction buffer. The organic phases were combined and evaporated under CentriVap Console (Labconco, KS, United States). The adaxial EWs were firstly removed and then abaxial EWs were isolated from the same batch of leaves.
After EW removal from both leaf surfaces by gum arabic, the leaves were still physically intact, and were used to extract IWs. The adaxial IWs were rinsed five times by chloroform; the elution was collected into a glass beaker. Abaxial IWs were isolated in the same manner. The elution was dried down by CentriVap Console to recover IWs.
After EW removal from both leaf surfaces by gum arabic, the leaves were still physically intact, and were used to extract IWs. The adaxial IWs were rinsed five times by chloroform; the elution was collected into a glass beaker. Abaxial IWs were isolated in the same manner. The elution was dried down by CentriVap Console to recover IWs.
7
Isolation and Characterization of Epicuticular and Intracuticular Waxes
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The epicuticular waxes were isolated by the method described in the study by Zhang et al. (2020) (link). Delipidated gum arabic in 90% (w/w) aqueous solution was evenly applied to the leaf surface by a soft paintbrush; dry polymer film was peeled off and collected into a glass tube containing 21 ml of chloroform: water (2:1, v/v) and 75 μg of internal standard n-tetracosane (Sigma-Aldrich, St. Louis, United States). After vigorous vortexing and phase separation, the organic phase was transferred into a new glass tube. The extraction was repeated once, and the organic phases were combined and evaporated under the CentriVap Console (Labconco, KS, United States) to obtain epicuticular waxes. The adaxial epicuticular waxes were isolated first, followed by the isolation of abaxial epicuticular waxes.
After the removal of epicuticular waxes, the leaves were used to extract the intracuticular waxes by rinsing with chloroform (Zhang et al., 2020 (link)). The adaxial intracuticular waxes were rinsed first, followed by rinsing with the abaxial intracuticular waxes. The collected chloroform solution was evaporated dry to get the adaxial or the abaxial intracuticular waxes, respectively.
After the removal of epicuticular waxes, the leaves were used to extract the intracuticular waxes by rinsing with chloroform (Zhang et al., 2020 (link)). The adaxial intracuticular waxes were rinsed first, followed by rinsing with the abaxial intracuticular waxes. The collected chloroform solution was evaporated dry to get the adaxial or the abaxial intracuticular waxes, respectively.
8
Profiling of Wax and Cutin Composition
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Wax profiling was carried out as described previously [38 (link)]. Briefly, fresh or frozen samples (100 mg) were immersed in chloroform for 30 s to extract the wax, and 20–50 µL (10 mg/50 mL) of n-Tetracosane (Sigma) was added as an internal standard. The extracted solution was dried with nitrogen and derived with 20 μL pyridine and 20 μL N,O-bis(trimethylsilyl) fluoroacetamide (BSTFA) (40 min at 70 °C). The derivate samples were then analyzed using gas chromatography–mass spectrometry (GC-MS).
For cutin analysis, samples were immersed in chloroform and methanol (1:1, v/v) in a glass vial for 2 weeks to remove phospholipids. Samples were then air-dried and reacted with methanol/HCl at 80 °C for 2 h. Saturated NaCl was used to end the reaction. Cutin was extracted with hexane, the extracted solution was dried with nitrogen, and the samples were derived with 20 μL pyridine and 20 μL BSTFA (40 min at 70 °C). Then, 20–50 μL (10 mg/50 mL) of dotriacontane (Sigma) was added to each sample as an internal standard. The derivate samples were then analyzed using GC-MS.
For cutin analysis, samples were immersed in chloroform and methanol (1:1, v/v) in a glass vial for 2 weeks to remove phospholipids. Samples were then air-dried and reacted with methanol/HCl at 80 °C for 2 h. Saturated NaCl was used to end the reaction. Cutin was extracted with hexane, the extracted solution was dried with nitrogen, and the samples were derived with 20 μL pyridine and 20 μL BSTFA (40 min at 70 °C). Then, 20–50 μL (10 mg/50 mL) of dotriacontane (Sigma) was added to each sample as an internal standard. The derivate samples were then analyzed using GC-MS.
9
Extraction of Epicuticular Waxes and Internal Waxes
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Epicuticular waxes and IWs were isolated from the 5th leaf of WW control, day 8 of WD, and day 10 of RE as Zhang et al. (2020) (link). Delipidated gum arabic was dissolved in water to a final concentration of 90% (w/v), and aqueous solution was evenly applied to leaf surface to form a film. Dry polymer film was peeled off and collected, then extracted in 21 ml of chloroform/water (2:1, v/v) containing 75 μg of internal standard n-tetracosane (Sigma-Aldrich, St. Louis, MO, United States). After vigorous agitation and phase separation, the organic phase was collected into a new glass tube. The residue was extracted one more time in 4.5 ml extraction buffer; the organic phase was combined and evaporated in a rotary evaporator (Labconco, Kansas, MO, United States) to obtain EWs. The adaxial EW was removed first, followed by abaxial EW.
After EW removal from both surfaces, IWs were extracted with chloroform rinsing. The adaxial IWs and the abaxial IWs were subsequently extracted. The collected chloroform solution was evaporated dry to obtain adaxial and abaxial IWs.
After EW removal from both surfaces, IWs were extracted with chloroform rinsing. The adaxial IWs and the abaxial IWs were subsequently extracted. The collected chloroform solution was evaporated dry to obtain adaxial and abaxial IWs.
10
Screening Alkane Utilization in Strain SJTD-1
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n-Decane (>99% pure) was purchased from Alfa Aesar Organic Co., Inc (Tianjing); n-dodecane, n-tetradecane, n-hexadecane, and n-octadecane (all >99% pure) from Sangon (Shanghai, China); and n-pentadecane, n-eicosane, n-docosane, n-tetracosane, n-triacontane, and n-hexane (of HPLC gradient grade) from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used in this study were of analytical reagent grade.
Luria-Bertani (LB) medium (tryptone 10.0 g, yeast extract 5.0 g, NaCl 10 g/L) and basal salt medium (BSM) (K2HPO4 3.815 g, KH2PO4 0.5 g, (NH4)2HPO4 0.825 g, KNO3 1.2625 g, Na2SO4 0.2 g, CaCl2 0.02 g, FeCl3 0.002 g, and MgCl2 0.02 g/L) were used in this study. To examine the utilization of alkanes by strain SJTD-1, both liquid and solid alkanes, maintained at room temperature, were first dissolved in n-hexane to form 500 mg/mL alkane-hexane solutions. These solutions were then added into the BSM medium to attain various concentrations. The n-hexane was neither toxic to the strain, nor was it utilized by the strain.
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n-Decane (>99% pure) was purchased from Alfa Aesar Organic Co., Inc (Tianjing); n-dodecane, n-tetradecane, n-hexadecane, and n-octadecane (all >99% pure) from Sangon (Shanghai, China); and n-pentadecane, n-eicosane, n-docosane, n-tetracosane, n-triacontane, and n-hexane (of HPLC gradient grade) from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used in this study were of analytical reagent grade.
Luria-Bertani (LB) medium (tryptone 10.0 g, yeast extract 5.0 g, NaCl 10 g/L) and basal salt medium (BSM) (K2HPO4 3.815 g, KH2PO4 0.5 g, (NH4)2HPO4 0.825 g, KNO3 1.2625 g, Na2SO4 0.2 g, CaCl2 0.02 g, FeCl3 0.002 g, and MgCl2 0.02 g/L) were used in this study. To examine the utilization of alkanes by strain SJTD-1, both liquid and solid alkanes, maintained at room temperature, were first dissolved in n-hexane to form 500 mg/mL alkane-hexane solutions. These solutions were then added into the BSM medium to attain various concentrations. The n-hexane was neither toxic to the strain, nor was it utilized by the strain.
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