WO2024097724A2 - Nouvelle approche pour interférer dans la voie de biosynthèse des phénylpropanoïdes pour réduire un trouble des nervures roses chez une laitue fraîche - Google Patents

Nouvelle approche pour interférer dans la voie de biosynthèse des phénylpropanoïdes pour réduire un trouble des nervures roses chez une laitue fraîche Download PDF

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WO2024097724A2
WO2024097724A2 PCT/US2023/078310 US2023078310W WO2024097724A2 WO 2024097724 A2 WO2024097724 A2 WO 2024097724A2 US 2023078310 W US2023078310 W US 2023078310W WO 2024097724 A2 WO2024097724 A2 WO 2024097724A2
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lettuce
melatonin
discoloration
composition
pink
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WO2024097724A3 (fr
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Jeongim KIM
Steven A. SARGENT
Catherine E. BELISLE
German V. SANDOYA MIRANDA
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings

Definitions

  • L-cysteine (0.1 % or 8.3 mM) applied to ATTORNEY DOCKET NO.222112-2090 fresh-cut iceberg lettuce delayed pink rib development ( ⁇ 20 % of the midrib surface area) for 6 d at 8 °C, while untreated control midribs became discolored after 2 d (Pace et al.2015).
  • L-cysteine (0.5 % or 41.3 mM) treatment also successfully delayed pulp browning in fresh-cut cherimoya for up to 12 d at 0 °C (Campos-Vargas et al.2008).
  • L-cysteine (0.5 % or 41.3 mM) was ineffective when applied to fresh-cut fennel during 6 d at 5 °C (Capotorto et al.2018) or to fresh- cut artichoke, which developed browning or became yellow (Cabezas-Serrano et al.2013). These studies revealed variable results of L-cysteine to reduce discoloration in lettuce and other crops.
  • Natural compounds were evaluated in other crops, including serotonin (1 mM solution), which was applied to apple and pear discs for 30 min and inhibited browning for up to 24 h at 4 °C; this treatment was more effective than ascorbic acid (Bajwa et al.2015).
  • the disclosure in one aspect, relates to a method for reducing or delaying pink rib discoloration in lettuce, the method comprising contacting the lettuce with a composition including melatonin and, optionally, DMSO (dimethyl sulfoxide).
  • the lettuce can be Romaine lettuce, Butterhead lettuce, iceberg lettuce, another lettuce, or any combination thereof.
  • contacting the lettuce with the composition comprises immersing the lettuce in the composition.
  • a method for objectively quantifying the degree of pink rib discoloration in a cut lettuce surface is also disclosed.
  • FIGs.2A-2B show phenylalanine ammonia-lyase (PAL) enzyme activity after storage at 5 °C for 5 d for iceberg (FIG.2A) and Romaine (FIG.2B) lettuce midribs wounded and treated immediately with L-cysteine (4.1 mM) or melatonin (4.3 mM).
  • Mean ⁇ Standard Error (SE) (n 3). Different letters indicate significant differences among treatments within a lettuce type by Tukey’s HSD (P ⁇ 0.05).
  • FIGs.4A-4B show polyphenol oxidase (PPO) enzyme activity after storage at 5 °C for 5 d for iceberg (FIG.4A) and Romaine (FIG.4B) lettuce wounded and treated immediately with L- cysteine (4.1 mM) or melatonin (4.3 mM).
  • FIGs.5A-5D show pink rib severity images and visual ratings for (FIGs.5A-5B) iceberg and (FIGs.5C-5D) Romaine midribs over 5 d of storage at 5 °C after wounding and immediate treatment with serotonin (both) or naringenin (iceberg only). Mean ⁇ SE.
  • FIG.6 shows a positive correlation between visual pink rib rating and a* value, with P- value calculated via an F-test.
  • FIGs. 7A-7C are photographs of whole romaine lettuce leaflets wherein midribs were treated with 4.3 mM of melatonin for 1 min and stored at 10 °C for five days.
  • FIGs.8A-8B are photographs of romaine lettuce midribs treated with 1 mM of melatonin for 1 min and stored for 4 days at 10 °C. Treated midribs showed less pink rib discoloration than control samples.
  • phenolic compounds may be associated with pink rib, as their oxidation products from polyphenol oxidase (PPO) are o-quinones (Hapiot et al. 1996; Rawel and John, 2010; Toivonen and Brummell, 2008).
  • Phenylalanine ammonia-lyase (PAL) catalyzes the deamination of phenylalanine to produce cinnamic acid, which is a precursor of various phenylpropanoids, such as chlorogenic acid, and there are several reports of correlation between PAL activity and severity of pink rib (Tanaka et al.2011; Saltveit, 2018b; Teng ATTORNEY DOCKET NO.222112-2090 et al. 2019).
  • naringenin application may inhibit discoloration of lettuce.
  • Disclosed herein is a method of using exogenously applied natural compounds (L- cysteine, naringenin, serotonin, melatonin) to wounded lettuce midribs to reduce development of pink rib discoloration during simulated commercial storage.
  • metabolite changes and enzymatic (PPO and PAL) activity following induction of pink rib discoloration via wounding and immediate application of natural compounds may provide guidance as to specific metabolites, amounts, and timing of administration of the exogenously applied compounds. Also described herein is a comparison of the severity of pink rib symptoms by subjective rating and by objective color imaging. [0023] To standardize evaluations of pink rib, Cantwell (2016) developed a subjective rating scale to mimic mechanical damage that typically occurs in lettuce midribs during harvest, handling, and fresh-cut operations. Midrib sections were wounded and discoloration at the cut surfaces was subjectively rated during storage using a five-point hedonic scale.
  • the disclosed method includes the step of contacting the lettuce with a composition including from about 1.0 to about 5.7 mM melatonin, from about 1.0 to about 4.3 mM melatonin, or about 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, or about 5.7 mM melatonin, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the composition includes about 4.3 mM of melatonin.
  • the composition also includes DMSO.
  • the DMSO can be from about 0.1 to about 1 vol%, or can be about 0.1, 0.2, 0.3, 0.4, ATTORNEY DOCKET NO.222112-2090 0.5, 0.6, 0.7, 0.8, 0.9, or about 1 vol%, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the DMSO is present at 0.5 vol%.
  • the lettuce can be Romaine lettuce, Butterhead lettuce, iceberg lettuce, or any combination thereof.
  • contacting the lettuce with the composition includes immersing the lettuce in the composition, spraying the lettuce with the composition, pouring the composition over the lettuce, or any combination thereof.
  • the lettuce is immersed in the composition for up to about 60 seconds, or for up to about 30 seconds.
  • the method reduces or delays pink rib discoloration for from about 3 days to about 5 days, or for 3, 4, or 5 days.
  • the lettuce can be iceberg lettuce and the method reduces or delays pink rib discoloration for about 5 days.
  • the lettuce can be Romaine lettuce, and the method reduces or delays pink rib discoloration for about 3 days.
  • the method further includes storing the lettuce at from about 5 °C to about 25 °C, or from about 10 °C to about 25 °C, or at about 5 °C after contacting the lettuce with the composition. In still another aspect, the method further includes storing the lettuce at about 95 to about 98% relative humidity after contacting the lettuce with the composition. In one aspect, the method is performed at from about 4 hours to about 3 days following harvest of the lettuce.
  • a method for assessing pink rib discoloration in cut lettuce surfaces including at least the steps of: (a) acquiring a digital image of a cut lettuce surface; (b) extracting an RGB color value from one or more isolated pixels in the digital image; (c) converting the RGB color value to an L*a*b* color value; and (d) extracting an a* value from the L*a*b* color value; wherein a positive a* value indicates pink rib discoloration is present.
  • a higher magnitude a* value indicates a greater degree of pink rib discoloration.
  • contacting the lettuce with the composition including melatonin can result in a magnitude of increase in a* value in L*a*b* color space in a digital image of a cut surface of the lettuce of less than about 6 when compared to a digital image of the lettuce prior to contacting the lettuce with the composition including melatonin.
  • the lettuce can be iceberg lettuce and the magnitude of increase in a* value is less than about 1 after 5 days.
  • the lettuce can be Romaine lettuce and the magnitude of increase in a* value is less than about 5.5 after about 5 days.
  • the final a* value after contacting the lettuce with the composition including melatonin is less than about 1.5.
  • each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably.
  • the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.”
  • the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y.’
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x,’ ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to ATTORNEY DOCKET NO.222112-2090 about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined.
  • an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material.
  • an “effective amount” of a small molecule refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. achieving the desired level of reduction of pink rib discoloration.
  • the specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of lettuce, storage temperature of the lettuce, and time since harvest. [0047] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • a method for reducing or delaying pink rib discoloration in lettuce comprising contacting the lettuce with a composition comprising melatonin.
  • Aspect 2. The method of aspect 1, wherein the lettuce comprises Romaine lettuce, Butterhead lettuce, or iceberg lettuce.
  • Aspect 3 The method of aspect 1 or 2, wherein the composition comprises from about 1.0 mM to about 5.7 mM of melatonin.
  • Aspect 4. The method of aspect 3, wherein the composition comprises 4.3 mM of melatonin.
  • Aspect 5. The method of any one of aspects 1-4, wherein the composition further comprises DMSO.
  • Aspect 6. The method of aspect 5, wherein the composition comprises 0.5 vol% DMSO.
  • Aspect 7 The method of any one of aspects 1-6, wherein contacting the lettuce with the composition comprises immersing the lettuce in the composition, spraying the lettuce with the composition, pouring the composition over the lettuce, or any combination thereof.
  • Aspect 8 The method of aspect 7, wherein the lettuce is immersed in the composition for up to about 60 seconds.
  • Aspect 9. The method of any one of aspects 1-8, wherein the method reduces or delays pink rib discoloration for from about 3 to about 5 days.
  • Aspect 10 The method of aspect 9, wherein the lettuce is iceberg lettuce and the method reduces or delays pink rib discoloration for about 5 days.
  • Aspect 11 The method of any one of aspects 1-6, wherein contacting the lettuce with the composition comprises immersing the lettuce in the composition, spraying the lettuce with the composition, pouring the composition over the lettuce, or any combination thereof.
  • Aspect 8. The method of aspect 7, wherein the lettuce is immersed in the composition for up to about 60 seconds.
  • Aspect 9. The method of any one of aspects 1-8, wherein
  • Aspect 12 The method of any one of aspects 1-11, wherein the method further comprises storing the lettuce at from about 5 to about 25 °C after contacting the lettuce with the composition.
  • Aspect 13 The method of any one of aspects 1-12, wherein the method further comprises storing the lettuce at 95 – 98% relative humidity after contacting the lettuce with the composition.
  • Aspect 14 The method of any one of aspects 1-13, wherein the method is performed at from about 4 hours to about 3 days following harvest of the lettuce. [0065] Aspect 15.
  • Aspect 16 The method of aspect 15, wherein the lettuce is iceberg lettuce and the magnitude of increase in a* value is less than about 1 after 5 days.
  • Aspect 17 The method of aspect 15, wherein the lettuce is Romaine lettuce and the magnitude of increase in a* value is less than about 5.5 after 5 days.
  • Aspect 18 The method of any one of aspects 15-17, wherein a final a* value after performing the method is less than about 1.5.
  • Aspect 19 The method of any one of aspects 1-18, wherein performing the method reduces formation of o-diphenols, o-quinones, or both.
  • Aspect 20 A method for assessing pink rib discoloration in cut lettuce surfaces, the method comprising: (a) acquiring a digital image of a cut lettuce surface; (b) extracting an RGB color value from one or more isolated pixels in the digital image; (c) converting the RGB color value to an L*a*b* color value; and (d) extracting an a* value from the L*a*b* color value; wherein a positive a* value indicates pink rib discoloration is present.
  • Aspect 21 Aspect 21.
  • Example 1 Materials and Methods Plant material and experimental setup [0073] Six preliminary experiments were performed to determine treatment conditions for the three final experiments reported here. It was determined that the method developed by Cantwell (2016) induced pink rib discoloration in lettuce midribs during storage at 5 °C via mimicking mechanical damage and was, therefore, selected for use in all preliminary and final tests. Solution concentrations and submersion times were tested for four compounds to determine the most effective treatment that reduced pink rib discoloration during storage at 5 °C for 5 d.
  • ATTORNEY DOCKET NO.222112-2090 the evaluations for pink rib development, protein quantification, and enzyme activity were conducted with Romaine and iceberg lettuce heads (36 heads per type; cultivars unknown) purchased (February 2020) upon arrival at a local grocery store in Gainesville, FL, and immediately transported to the Postharvest Horticulture Laboratory at the University of Florida in Gainesville, FL.
  • metabolite extraction and detection were conducted with commercially grown, harvested, and cooled Romaine (’Tall Guzmaine’) and iceberg (’Chosen’) lettuce (December 2020) grown in Belle Glade, FL.
  • deionized water wounded control
  • L-cysteine 4.1 mM
  • naringenin 1.8 mM
  • DMSO dimethyl sulfoxide
  • serotonin 2.8 or 5.7 mM
  • melatonin 4.3 mM
  • RH relative humidity
  • L-cysteine (4.1 mM) solution was prepared by dissolving 500 mg of L-cysteine in 100 mL ATTORNEY DOCKET NO.222112-2090 deionized water.
  • Naringenin (1.8 mM) solution was prepared by dissolving 500 mg of naringenin in 20 mL of DMSO and diluting to a 100 mL volume with deionized water.
  • a stock solution of serotonin (5.7 mM) was prepared by dissolving 100 mg of serotonin in 100 mL of deionized water; a second solution of serotonin (2.8 mM) was prepared by diluting the stock solution two-fold with deionized water.
  • a 4.3-mM solution of melatonin was prepared by dissolving 100 mg of melatonin in 0.5 mL of DMSO and diluting to a 100 mL volume with deionized water. Ranges of concentrations of each stock solution were then prepared for each compound. Evaluation of pink rib development [0077] Midrib samples were evaluated for pink rib severity after 1, 3, and 5 d of storage in Tests 1, 2, and 3 (Table 1).
  • Isolated pixels were converted from the RGB to the L*a*b* color space (Commission Internationale de l'Elcairage) with the "convertColor" function in R version 4.0.0 (R Core Team, 2020), using Illuminant D65 as the white reference.
  • the mean a* value was calculated for each group of isolated cut site pixels at each time point.
  • r represents the degree of redness (+) or greenness (-); thus, the mean a* at each cut site quantified the severity of pink rib discoloration.
  • R was also applied to subset the data, to ensure that the cut sites chosen ATTORNEY DOCKET NO.222112-2090 for further analysis had similar initial color.
  • a* values ranged from -3 to 0 for iceberg lettuce and -6 to -3 for Romaine.
  • Frozen samples were extracted with a 50% methanol extraction solution containing a 1:1 (v:v) of methanol with deionized water. Samples were completely submerged in a 1:1.5 (w:v) of the sample weight (mg) to extraction solution volume ( ⁇ L), respectively, and incubated at 65 °C for 90 min and then centrifuged at 9660 x g for 10 min (MC-12 High Speed Micro Centrifuge, Benchmark Scientific, Edison, NJ). The supernatant was transferred to a new tube and filtered with a 0.45 ⁇ m filter (Choice, Thermo Scientific, Wilmington, DE). An aliquot of 400 ⁇ L was vacuum-dried.
  • the mobile phase consisted of solvent A (0.1% formic acid (v/v) in water) and solvent B (100% acetonitrile), and the gradient as follows: 5% to 20% increase of solvent B at 0.3 to 0.4 min, 20% to 25% increase solvent B at 5.25 to 12.5 min, 25% to 95% increase solvent B at 12.5 to 16.5 min, and 95% solvent B at 16.5 to 20 min.
  • the flow rate was 0.4 mL min -1 and the column temperature was 40 °C.
  • the level of chlorogenic acid was quantified based on the peak area at 328 nm and the standard curve using authentic standard (Sigma Aldrich; St. Louis, MO, USA).
  • Liquid chromatography/tandem mass spectrometry LC-MS/MS analysis was performed in both negative and positive mode on a Bruker maXis impact quadrupole-time- ATTORNEY DOCKET NO.222112-2090 of-flight mass spectrometer coupled to a Waters ACQUITY ultrahigh performance liquid chromatography (UPLC) system (UPLC-Q-TOF-MS/MS). Separation was achieved on a Waters C18 column (2.1x 150 mm, BEH C18 column with 1.7- ⁇ m particles) using a linear gradient and mobile phase A (0.1% formic acid) and B (B: acetonitrile).
  • UPLC Ultrahigh performance liquid chromatography
  • MS full scan 100 to 1500 m/z
  • number of precursors for MS/MS 3
  • threshold 10 counts
  • active exclusion 3 spectra, released after 0.15 min
  • collision energy dependent on mass, 10 eV at 50 Da, 20 eV at 200 Da, 30 at 500 Da, 40 eV at 1000 Da, and 50 eV at 1500 Da.
  • the MS and MS/MS data were auto-calibrated using sodium formate, which was introduced at the end of the gradient after data acquisition. Data were processed using MetaboScape (Bruker).
  • Metabolites were putatively identified by matching their tandem spectral data against a custom tandem spectral library (Lei et al, 2018), RIKEN spectral library, and NIST20 high resolution accurate mass library (hr_msms_nist). Multivariate analysis was performed using MetaboAnalyst.
  • Crude protein preparation and PAL activity [0083] PAL activity was measured using a crude protein extraction following a method reported by Kim et al. (2020) with some modifications. Fresh lettuce samples (300 mg) were ground with liquid nitrogen in a pre-chilled mortar and pestle. A 0.1 mM Tris-HCl buffer (pH 8.3) was added at a 1:1 (w:v).
  • the solution was incubated at 4 °C for 1 h and then centrifuged at 4 °C, 9660 x g for 15 min. Supernatants were collected in chilled tubes. Total protein content was measured using Quick Start Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) and Bovine Serum Albumin (BSA) as a standard (10 mg mL -1 in deionized water). Protein concentration was calculated based on the absorbance at 595 nm measured with a NanoDrop (One Microvolume UV-Vis Spectrophotometer, Thermo Scientific, Wilmington, DE, USA) and the standard curve.
  • BSA Bovine Serum Albumin
  • PAL activity test 150 ⁇ L aliquots of the protein extract were incubated in a reaction buffer containing 0.1 mM Tris-HCl buffer (pH 8.3), 5 mM dithiothreitol (DTT), and 5 mM phenylalanine. Reaction mixtures were incubated at 37 °C for 90 min and stopped with the addition of 40 ⁇ L of 30 % acetic acid. Reaction products were extracted with 600 ⁇ L of ethyl acetate and then centrifuged at 22 °C, 9660 x g for 5 min.
  • PPO activity was prepared and determined as reported by Teng et al. (2019) with slight modifications. Lettuce samples (300 mg) were homogenized in 150 % (w/v) chilled phosphate buffer (pH 6.2, 0.05 M). Following chilling of test tubes with samples in an ice bath, samples were centrifuged at 9660 x g for 10 min at 4 °C. The increase in absorbance was measured every 3 s for 1 min.
  • the slope of the linear portion of the time curve was calculated.
  • One unit (U) of PPO activity was defined as an increase in OD 410 of 0.1 unit within 1 min.
  • Statistical analysis [0085] The imaging data (a* value), enzyme activity (PAL and PPO), and metabolite (chlorogenic acid) results were expressed as mean ⁇ standard error and analyzed by lettuce type (iceberg or Romaine). For imaging data, an unpaired t-test was used to identify differences within a treatment throughout storage and among treatment groups at each time point. Treatment groups were defined as wounded, unwounded, L-cysteine, and melatonin. Naringenin and serotonin were not evaluated for imaging due to an unsuccessful reduction of pink rib severity.
  • the a* values of wounded control midribs significantly increased (from green to red) throughout storage from an initial of -0.89 to 7.46 in iceberg type and from -4.84 to 1.49 in Romaine type (FIGs.1C and 1F, respectively).
  • PAL activity increased to 7.44 pkat mg -1 in melatonin-treated midrib tissue and 5.43 pkat mg -1 in L-cysteine-treated tissue.
  • PAL activity reached 6.06 pkat mg -1 in melatonin-treated midrib tissue and 11.07 pkat mg -1 in L-cysteine-treated tissue.
  • PPO activity was 2.6 U in melatonin-treated and 2.6 U in L-cysteine-treated tissues; in Romaine, PPO activity was 6.2 U in melatonin-treated ATTORNEY DOCKET NO.222112-2090 and 6.4 U in L-cysteine-treated tissues.
  • ATTORNEY DOCKET NO.222112-2090 ATTORNEY DOCKET NO.222112-2090 a Ion modes defined as positive (+) or negative (-).
  • b W wounded sample
  • U unwound sample
  • M melatonin-treated wounded sample.
  • c log2 represents the peak area of wounded samples by the peak area of unwounded samples. Fold changes >0 are listed.
  • ( ⁇ ) indicates increased compounds and
  • ( ⁇ ) indicates decreased compounds in melatonin-treated wounded samples compared to wounded samples.
  • d P-values ( ⁇ 0.05) were calculated using Student’s T-test.
  • e Chlorogenic acid is listed for comparison. Chlorogenic acid only increased in wounded samples compared with unwound samples.
  • ATTORNEY DOCKET NO.222112-2090 a Ion modes defined as positive (+) or negative (-).
  • b W wounded sample
  • U unwound sample
  • M melatonin-treated wounded sample.
  • c log 2 represents the peak area of wounded samples by the peak area of unwounded samples. Fold changes ⁇ 0 are listed. ( ⁇ ) indicates increased compounds and ( ⁇ ) indicates decreased compounds in melatonin-treated wounded samples compared to wounded samples.
  • d P-values ( ⁇ 0.05) were calculated using Student’s T-test.
  • Example 3 Discussion Evaluation of the severity of the pink rib using RGB images
  • a visual observation-based system Monaghan et al., 2016; Paillart et al., 2017; Saltveit, 2004.
  • an observer makes a judgement of the severity of the pink rib response based on their perception of discoloration on the midrib.
  • Visible light imaging has been applied extensively in plant phenomics to measure a variety of phenotypes, including plant color (Yang et al., 2020).
  • naringenin a flavonoid, and as an antioxidant, a free radical scavenger
  • FRAP ferric reducing antioxidant power
  • immersion in up to 3.7 mM of naringenin in the present study did not successfully reduce pink rib as naringenin-treated midribs developed “moderately severe” pink rib in iceberg lettuce after 5 d at 5 °C (FIG.6).
  • L-cysteine is an antioxidant.
  • L-cysteine (8.3 mM) treatment reduced discoloration of midrib surface area by greater than 80 % compared with a control after 6 d at 8 °C (Pace et al.2015).
  • a concentration as high as 0.5 % (41.3 mM) had no effect on fresh-cut brown discoloration in fennel (Capotorto et al. 2018) nor artichoke (Cabezas-Serrano et al.2013).
  • L-cysteine (4.1 mM) was ineffective in reducing pink rib in iceberg or Romaine lettuce (FIGs.1A and 1D).
  • melatonin (4.3 mM) was the singular treatment that effectively inhibited the pink rib development for 5 d in wounded iceberg midribs and for 3 d in Romaine ribs as compared with wounded control midribs (FIGs.1A-1F). Similar findings have been reported for melatonin applied to cassava roots (Ma et al.2016), peach fruit (Gao et al.2016), and litchi fruit (Zhang et ATTORNEY DOCKET NO.222112-2090 al.2018). Exogenous application of melatonin (0.1 mM) to cassava pulp delayed development of discoloration (Hu et al.
  • Melatonin has many biological functions in plants, including root architecture, photoprotection, flower development, seed germination, leaf senescence, fruit ripening, vegetative growth, and response to stress (Hu et al. 2016; Shi et al. 2016). It enhances nonenzymatic antioxidants, such as phenolics, flavonoids, and anthocyanins (Boccalandro et al. 2011).
  • PAL is a key enzyme associated with pink rib discoloration in lettuce (Hunter et al.2017); it functions at the entry step of the phenylpropanoid pathway, and its induction leads to formation of phenylpropanoids, with chlorogenic acid as a major phenylpropanoid produced (Tosammlung- Barberán et al. 1997).
  • ROS reactive oxygen species
  • Romaine lettuce midribs treated with 4.3 mM of melatonin for 1 min showed less discoloration when stored at 10 °C for five days compared to control samples.
  • 4.3 mM of melatonin solution was prepared in water containing 0.5% DMSO (dimethyl sulfoxide), and control samples were treated with water containing 0.5% DMSO. This result further confirmed that melatonin reduced pink rib discoloration in romaine lettuce.
  • ATTORNEY DOCKET NO.222112-2090 A lower concentration of melatonin is effective [0111] Treatment with a lower melatonin concentration (1 mM) showed similar effectiveness in delaying pink rib discoloration in lettuce.
  • romaine lettuce midribs dipped with 1 mM of melatonin for 1 min showed less discoloration than control samples after 4 days at 10 °C.
  • 1 mM of melatonin solution was prepared in water containing 0.5% DMSO (dimethyl sulfoxide) and control samples were treated with water containing 0.5% DMSO.
  • Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL.
  • ATTORNEY DOCKET NO.222112-2090 Lei, Z., et al.2018. Construction of an ultrahigh pressure liquid chromatography-tandem mass spectral library of plant natural products and comparative spectral analyses.
  • Cinnamaldehyde inhibits enzymatic browning of cut lettuce by repressing the induction of phenylalanine ammonia-lyase without promotion of microbial growth. J. Agric. Food Chem.59, 6705–6709.
  • Crop Phenomics and High-Throughput Phenotyping Past Decades, Current Challenges, and Future Perspectives. Mol. Plant 13, 187-214. Yin, R., et al. 2012. Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation. J Expt. Botany 63(7): 2465-2478. Zhang, Y., et al. 2018. Delay of postharvest browning in litchi fruit by melatonin via the enhancing of antioxidative processes and oxidation repair. J. Agr. Food Chem.66(28), 7475- 7484. Zheng, H., et al. 2019. Effects of melatonin treatment on the enzymatic browning and nutritional quality of fresh-cut pear fruit. Food Chem.299, 125116.

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Abstract

Selon un aspect, la divulgation concerne un procédé de réduction ou de retardement de la décoloration en nervures roses chez les laitues, le procédé comprenant la mise en contact de la laitue avec une composition comprenant de la mélatonine et, éventuellement, du DMSO (diméthylsulfoxyde). Selon un aspect, la laitue peut être une laitue romaine, une laitue pommée, une laitue iceberg, une autre laitue, ou toute combinaison de celles-ci. Dans certains aspects, la mise en contact de la laitue avec la composition comprend l'immersion de la laitue dans la composition. La divulgation concerne également un procédé de quantification objective du degré de décoloration en nervures roses dans une surface de laitue coupée. Le présent abrégé est destiné à être utilisé comme outil d'exploration à des fins de recherche dans ce domaine technique particulier, et ne se limite pas à la présente divulgation.
PCT/US2023/078310 2022-11-04 2023-11-01 Nouvelle approche pour interférer dans la voie de biosynthèse des phénylpropanoïdes pour réduire un trouble des nervures roses chez une laitue fraîche Ceased WO2024097724A2 (fr)

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