In-situ pulp incubation method for postharvest lignification click chemical imaging analysis of fruits

Abstract

The invention discloses an in-situ pulp incubation method for click chemical imaging analysis of postharvest lignification of fruits. The in-situ pulp incubation method comprises the following steps: (1) preparing a high-concentration hydrogel solution and a low-concentration hydrogel solution; (2) preparing a CA-Alk working solution and a CA-Alk-containing hydrogel incubation solution; (3) cutting a wound on the fruit with a knife to expose pulp, soaking the wound of the fruit in a CA-Alk working solution for 24 hours, taking out the fruit, then coating the surface of the wound with a hydrogel incubation solution containing CA-Alk, and curing to form a first gel layer; and then coating the first gel layer with the high-concentration hydrogel liquid, and coating the first gel layer with a preservative film for fixation. According to the method, the integrity of pulp cells is almost not damaged when observed under an optical microscope, and the incubation time can last from original 1-2 days to 7 days or above. Meanwhile, due to in-situ incubation, the reaction on the lignin accumulation rule is more accurate.

Description

Technical Field

[0001] This invention relates to the field of postharvest physiological research technology for fruits and vegetables, and in particular to an in-situ pulp incubation method for postharvest lignification click chemical imaging analysis of fruits. Background Technology

[0002] Loquat is a perennial woody fruit tree native to southern my country, belonging to the Rosaceae family and the Eriobotrya genus. Loquat fruits are mostly harvested in the hot and rainy early summer season. They are not suitable for storage at room temperature, and the fruit ages and spoils rapidly. Low-temperature storage can significantly inhibit the respiration rate and ethylene release of loquat fruits after harvest, extending storage time; however, low temperatures can cause chilling injury stress on red-fleshed loquat varieties, leading to chilling injury lignification. Lignification of the flesh also occurs during the natural aging process of red-fleshed loquat fruits after harvest. The most typical symptoms of post-harvest lignification in loquat fruits are the accumulation of lignin in the flesh and increased hardness, accompanied by browning of the flesh tissue, roughness, reduced juice content, bland flavor, and difficulty in separating the peel from the flesh, significantly affecting the fruit's flavor and texture. Currently, post-harvest lignification has become one of the most prominent bottleneck problems in the storage and logistics of loquat fruits.

[0003] Over the past two decades, researchers both domestically and internationally have conducted extensive studies on postharvest lignification of loquat, primarily focusing on postharvest physiology and biochemistry, regulatory techniques, molecular basis analysis of lignification, transcription factors, and transcriptional regulatory mechanisms. To further elucidate the biological and molecular mechanisms and regulatory networks of postharvest lignification in loquat, researchers have investigated the functions and enzyme activities of lignin synthesis-related structural genes in loquat fruits based on different postharvest treatments, gradually delving into the transcriptional regulatory level.

[0004] Currently, much research on postharvest lignification in loquat primarily utilizes various postharvest treatments, based on physiological and biochemical indicators, to explore the molecular mechanisms of this process. However, these physicochemical and genetic analyses are all based on pulp tissue homogenates, presenting only the average lignin content and related gene expression information at the tissue level. This information can only indicate the presence and abundance of lignin molecules and related genes at the tissue level, but it cannot provide spatial distribution information at the cellular level, and the heterogeneity between cells or even tissues cannot be reflected.

[0005] The lignin accumulated in the cells of loquat fruit before harvest and the newly synthesized lignin during the post-harvest lignification process cannot be distinguished. Imaging technology based on click chemistry, through monomer labeling, tissue "feeding," and click reaction, can achieve specific labeling of newly synthesized target molecules. It is currently applied to the specific labeling and real-time tracking imaging of biomolecules in living cells. The core of click chemistry is to achieve rapid molecular synthesis through the bonding of small chemical units, such as carbon-heteroatoms. Click chemistry has advantages such as high yield, no byproducts, readily available raw materials, and simple reaction conditions. Its reactions can be carried out in a living environment, which is of great significance for life science research. Click chemistry, applied to the study of the synthesis and metabolism of macromolecules in organisms, is also known as bioorthogonal reaction. It mainly involves introducing orthogonal groups through biometabolic labeling to specifically label target molecules; then, a fluorescein molecule with another orthogonal group is added. Under simple conditions, the two can bind, thereby achieving the localization and tracking of the target molecule.

[0006] In previous click-chemical imaging analyses, our research group used CA-Alk as a substrate to culture loquat tissues, with a CA-containing culture medium serving as a control. The specific procedures were as follows: (1) Loquat pulp tissue culture (incubation): The isolated loquat pulp tissue blocks (approximately 0.4 cm × 0.4 cm × 0.4 cm) were obtained from the equatorial region of the loquat fruit. The pulp tissue was taken from different stages: the young fruit stage (S1), the color-changing stage (S2), and the ripening stage (S3). The isolated pulp tissue was stored at 20°C for 24 hours, then washed twice with distilled water, and sectioned freehand. The obtained sections were used for subsequent labeling and imaging.

[0007] Loquat pulp tissue (pulp blocks) was cultured in liquid MS medium containing 100 μM CA-Alk, while the control was culture medium containing 100 μM CA (coniferol).

[0008] (2) Click chemical imaging The click chemical imaging method was referenced in: Bukowski N, Pandey JL, Doyle L, et al. Development of a clickable designer monolignol for interrogation of lignification in plant cell walls[J]. Bioconjugate Chemistry, 2014, 25(12):2189-2196. (DOI:10.1021 / bc500411u). The obtained plant tissue sections were washed four times with liquid MS medium and then placed in 2 ml of labeling solution containing 1 μM Alexa 594-azide, 2 mM L-ascorbic acid, and 1 mM CuSO4. The sections were cultured in the dark with shaking (100 rpm) for 1.5 h at 25℃. The labeled plant tissue sections were washed twice with liquid MS medium and once with 70% methanol (1 h) to remove unbound monomers and dyes. They were then washed four times with liquid MS medium. After the obtained slides were mounted, microscopic images were acquired using a laser confocal microscope.

[0009] This method involves "incubating" the lignin monomer-modified mimic CA-Alk to image newly synthesized lignin in cells, aiming to identify patterns in its synthesis and distribution. However, due to the high water content of fruit cells, the imaging time for newly generated lignin is only 1-2 days after soaking the pulp in liquid MS culture medium containing CA-Alk. Extending the incubation time leads to severe cell breakage, with a cell breakage rate exceeding 70% after 7 days. Furthermore, the incubation target in this system is the pulp rather than the whole fruit, resulting in a biased response to the lignin accumulation pattern. Summary of the Invention

[0010] The purpose of this invention is to provide an in-situ pulp incubation method for postharvest click chemical imaging analysis of fruit lignification. This method allows for observation of the integrity of pulp cells under an optical microscope with minimal damage, and the incubation time can be extended from the original 1-2 days to over 7 days. Furthermore, because it is in-situ incubation, it provides a more accurate response to the lignin accumulation pattern.

[0011] The technical solution adopted by this invention to solve its technical problem is: An in-situ pulp incubation method for postharvest lignification click chemical imaging analysis of fruit includes the following steps: (1) Mix 0.02 g / mL pectin aqueous solution and 0.02 g / mL gelatin aqueous solution in equal volumes to form a viscous low-concentration hydrogel solution; mix 0.04 g / mL pectin aqueous solution and 0.02 g / mL gelatin aqueous solution in equal volumes to form a viscous high-concentration hydrogel solution; (2) Dissolve CA-Alk in acetone to prepare a CA-Alk stock solution with a concentration of 20 mM; Dilute the CA-Alk mother liquor to prepare the CA-Alk working solution; The CA-Alk stock solution was mixed with a low-concentration hydrogel solution to form a hydrogel incubation solution containing CA-Alk. (3) Make a cut on the fruit to expose the pulp, immerse the cut in CA-Alk working solution to ensure that the cut is fully in contact with CA-Alk working solution, and maintain it at 4℃ for 24 hours; After soaking for 24 hours, the wound was removed, and then a hydrogel incubation solution containing CA-Alk was applied to the wound surface and gelled at 4°C to form the first gel layer. Next, a high-concentration hydrogel solution was coated onto the first gel layer, and then wrapped with plastic wrap to fix it. It was then stored at 4°C.

[0012] This invention first soaks the wound in a CA-Alk working solution to provide raw materials for the initial synthesis of lignin in cells, thereby imaging the newly synthesized lignin. Then, it removes the wound from the liquid environment and coats it with a CA-Alk-containing hydrogel incubation solution for curing. The hydrogel slowly releases CA-Alk to meet the subsequent lignin synthesis, effectively prolonging the imaging time of newly synthesized lignin in cells. A high-concentration hydrogel and plastic wrap are then applied to the outer layer for fixation. This effectively maintains cell moisture, ensuring cell morphology integrity and activity, as well as the continuous synthesis of lignin. Therefore, while maintaining cell integrity, the incubation time can be greatly extended, thus extending the monitoring time for lignin synthesis and better reflecting the lignin accumulation pattern. This provides a theoretical basis for preventing lignification caused by chilling injury in loquat.

[0013] This invention improves the CA-Alk working solution and soaking conditions, and, combined with in-situ hydrogel incubation, significantly extends the incubation time while maintaining cell integrity. This extends the monitoring time for lignin synthesis and better reflects the lignin accumulation pattern. Low-methyl esterified pectin does not interfere with imaging, is more suitable for pulp incubation, facilitates click chemiluminescence imaging, and yields more stable results. Low-methyl esterified pectin has a thickening effect, promoting gel formation, while gelatin dominates hydrogel solidification. Incubation with low-methyl esterified pectin does not interfere with click chemiluminescence imaging, maintains cell integrity, and allows for the slow release of CA-Alk to label newly synthesized lignin.

[0014] Preferably, in step (1), the pectin is low-methyl esterified pectin.

[0015] Preferably, in step (2), the concentration of the CA-Alk working solution is 0.2 mM.

[0016] Preferably, in step (2), the CA-Alk stock solution is added to 1 / 2 MS medium (liquid) and diluted to form the CA-Alk working solution.

[0017] Preferably, in step (2), the CA-Alk mother liquor and the low-concentration hydrogel liquid are mixed at a ratio of 10 μL: 1 mL to form a hydrogel incubation solution containing CA-Alk.

[0018] Preferably, in step (3), the wound is 1.5-2cm long and 1.5-2cm wide, with a depth of 3-5mm.

[0019] Preferably, in step (3), the amount of the hydrogel incubation solution containing CA-Alk applied to the wound surface is 500-600 μL.

[0020] In step (3), the amount of high-concentration hydrogel liquid applied is to fill the wound to the shape of the fruit before the incision, that is, the volume of the excised part minus the volume of the first gel layer, and the amount is about 700-800 μL.

[0021] Preferably, the fruit is loquat or apple.

[0022] The beneficial effects of this invention are: compared with solution immersion incubation, the hydrogel incubation method developed in this invention maintains almost no damage to the integrity of fruit pulp cells when observed under an optical microscope, and the incubation time can be extended from the original 2-3 days to more than 7 days. Furthermore, because it is in situ incubation, the response to the lignin accumulation pattern is more accurate. Attached Figure Description

[0023] Figure 1 These are the initial appearance diagrams of different treatment groups after treatment in Example 3; Figure 2 These are the pulp states after different treatment times in Example 3; Figure 3 This represents the morphological changes of fruit slices over time under different incubation methods. 0d indicates a slice cut from fresh fruit that has not yet been incubated; CK indicates a fruit slice incubated using the original method; LM / Gel indicates a fruit slice incubated using the method of this invention. Figure 4 This is a confocal image of fruit pulp slices after incubation and click chemical staining according to the present invention. TD: Brightfield image; DAPI: Autofluorescence imaging; Nile red: Emission-labeled fluorescence imaging of CA-Alk combined with Alexa594-azide; All: Collection of images from all channels; Figure 5 The fluorescence intensity and fluorescence area of ​​the fruit pulp slices after click chemical staining following the incubation of this invention are shown. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0025] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0026] Example 1: Synthesis of modified analog CA-Alk Coniferol (CA) standard was purchased from Sigma-Aldrich. The synthesis method of CA-Alk was based on the following reference: Bukowski N, Pandey JL, Doyle L, et al. Development of a clickable designer monolignol for interrogation of lignification in plant cell walls[J]. Bioconjugate Chemistry, 2014, 25(12): 2189-2196. (DOI:10.1021 / bc500411u). The modified mimic CA-Alk was obtained by terminally modifying the methoxy branch of the lignin synthesis precursor CA with an alkyne group.

[0027] Example 2: Preparation of hydrogel solution Add 0.4g of low-methyl esterified pectin (LM) powder to 20ml of water and heat at 50℃ until dissolved to obtain a low-methyl esterified pectin solution with a concentration of 0.02g / mL; add 0.8g of low-methyl esterified pectin (LM) powder to 20ml of water and heat at 50℃ until dissolved to obtain a low-methyl esterified pectin solution with a concentration of 0.04g / mL.

[0028] Dissolve 0.8g of gelatin in 40ml of water and heat at 35℃ until dissolved to obtain a gelatin solution with a concentration of 0.02g / mL.

[0029] Mix an equal volume of 0.02 g / mL low-methyl esterified pectin solution with an equal volume of 0.02 g / mL gelatin solution to form a viscous, low-concentration hydrogel solution; mix an equal volume of 0.04 g / mL low-methyl esterified pectin solution with an equal volume of 0.02 g / mL gelatin solution to form a viscous, high-concentration hydrogel solution; store at 4°C for later use.

[0030] Example 3: Incubation (1) Dissolve CA-Alk in acetone to prepare a CA-Alk stock solution with a concentration of 20 mM; The CA-Alk stock solution was diluted with 1 / 2 MS medium (commercially available, Shanghai Maclean Biochemical Technology Co., Ltd.) to prepare a CA-Alk working solution with a concentration of 0.2 mM. The CA-Alk stock solution and the low-concentration hydrogel solution (prepared in Example 2) were mixed at a ratio of 10 μL: 1 mL to form a hydrogel incubation solution containing CA-Alk.

[0031] (2) Make a cut on the red loquat fruit (purchased from a fruit store) with a knife (about 1.5cm long, 1.5cm wide, and 2mm deep) to expose the pulp. Soak the cut in the fruit in CA-Alk working solution to ensure that the cut is fully in contact with the CA-Alk working solution and maintain it at 4℃ for 24 hours. After soaking for 24 hours, the wound was removed, and then a hydrogel incubation solution containing CA-Alk was applied to the wound surface. The amount of CA-Alk hydrogel incubation solution was 500 μL, and the wound was gelled at 4°C to form a first gel layer. Then, a high-concentration hydrogel solution (prepared in Example 2) was applied to the first gel layer and fixed with plastic wrap. Figure 1 The initial states of the different treatment groups (LM / Gel-1d represents the group incubated with hydrogel for 1 day, LM / Gel-3d represents the group incubated with hydrogel for 3 days, LM / Gel-5d represents the group incubated with hydrogel for 5 days, and LM / Gel-7d represents the group incubated with hydrogel for 7 days) were stored at 4°C. The amount of high-concentration hydrogel solution used was to fill the wound to the shape of the fruit before incision, approximately 700 μL.

[0032] Example 4: Incubation (1) Dissolve CA-Alk in acetone to prepare a CA-Alk stock solution with a concentration of 20 mM; Dilute the CA-Alk stock solution with 1 / 2 MS medium (commercially available) to prepare a CA-Alk working solution with a concentration of 0.2 mM; The CA-Alk stock solution and the low-concentration hydrogel solution (prepared in Example 2) were mixed at a ratio of 10 μL: 1 mL to form a hydrogel incubation solution containing CA-Alk.

[0033] (2) Make a cut (about 2cm long, 2cm wide, and 3mm deep) on the red loquat fruit (purchased from a fruit store) with a knife to expose the pulp. Soak the cut in the fruit in CA-Alk working solution to ensure that the cut is fully in contact with the CA-Alk working solution and maintain it at 4℃ for 24 hours. After soaking for 24 hours, the wound was removed and then coated with a hydrogel incubation solution containing CA-Alk. The amount of the hydrogel incubation solution containing CA-Alk was 600 μL, and the wound was gelled at 4°C to form the first gel layer. Next, a high-concentration hydrogel solution (prepared in Example 2) was coated onto the first gel layer, and then wrapped with plastic wrap for fixation and stored at 4°C. The amount of high-concentration hydrogel solution used was enough to fill the wound to the shape of the fruit before incision, specifically about 800 μL.

[0034] Example 5: Click-through chemical staining imaging After incubation, the pulp on the wound surface was sliced ​​with a knife; for specific imaging operations, refer to the literature: Bukowski N, Pandey JL, Doyle L, et al. Development of a clickable designer monolignol for interrogation of lignification in plant cell walls[J]. Bioconjugate Chemistry, 2014, 25(12): 2189-2196. (DOI:10.1021 / bc500411u): The obtained plant tissue slices were washed 4 times with liquid MS medium, and then placed in 2 mL of labeling solution containing 1 μM Alexa594-azide, 2 mL of ascorbic acid and 1 mM CuSO4, and cultured in the dark with shaking (100 rpm) for 1.5 h at 25 °C; the labeled plant tissue slices were washed twice in liquid MS medium and once in 70% methanol (1 h) to remove unbound monomeric substances and dyes, and then washed 4 times with liquid MS medium. After the obtained slides were mounted, microscopic images were acquired using a laser confocal microscope.

[0035] Experimental results: Figure 2 It shows the state of the pulp after incubation for different times, and... Figure 1 Compared to the previous period, there was no significant change.

[0036] Different methods were used to incubate fruit lignin monomers. The original method (CK) (described in the background art) resulted in fruit slices showing obvious fragmentation within 1-5 days. Images from CK-5 days show obvious pulp fragments around the slices, and the pulp slices darkened significantly in color by day 7. However, when the fruit wounds were incubated using the present invention (Example 3), relatively intact slices could still be easily cut from the fruit by hand within 0-7 days, and the appearance of the pulp slices showed no significant changes. Figure 3 ).

[0037] Click chemical staining was performed on fruit slices incubated according to the present invention (Example 3), and the images were imaged under a Nikon A1R confocal microscope. Figure 4 This section shows a representative field of view under a confocal microscope. The cell wall boundaries are clear and the cell shape is intact under the microscope. After adjusting the focus, clear lignin autofluorescence and labeled fluorescence can be seen on the cell wall. As time progresses, the average intensity of the labeled fluorescence does not significantly increase; however, the area of ​​labeled fluorescence expands, almost occupying the entire field of view by day 5. Figure 5 The figure illustrates the patterns of lignin synthesis and accumulation.

[0038] Comparative Example 1 The difference between this example and Example 3 is that sodium alginate is used instead of low-methyl esterified pectin. Compared to sodium alginate, pectin hydrogel has greater viscosity and is a major component of the extracellular matrix (ECM) of plant cells, making it suitable for use as a dressing for fruit pulp. Sodium alginate hydrogel has high fluidity, making it inconvenient to apply to the fruit pulp, difficult to clean during incubation, and challenging to obtain fruit pulp slices subsequently.

[0039] Comparative Example 2 (1) Dissolve CA-Alk in acetone to prepare a CA-Alk stock solution with a concentration of 20 mM; The CA-Alk stock solution and the low-concentration hydrogel solution (prepared in Example 2) were mixed at a ratio of 10 μL: 1 mL to form a hydrogel incubation solution containing CA-Alk.

[0040] (2) Make a cut (about 1.5 cm long, 1.5 cm wide, and 2 mm deep) on a red loquat fruit (purchased from a fruit store) with a knife to expose the pulp. Then, apply a hydrogel incubation solution containing CA-Alk to the surface of the cut. The amount of the hydrogel incubation solution containing CA-Alk is 500 μL. Gel solidify at 4°C to form the first gel layer. Then, apply a high-concentration hydrogel solution (prepared in Example 2) to the first gel layer. The amount of the high-concentration hydrogel solution applied is to fill the cut to the shape of the fruit before the cut, specifically about 700 μL. Cover with plastic wrap to fix and store in an environment of 4°C.

[0041] This incubation method suffers from poor staining stability and low reproducibility due to the initial inability of CA-Alk to rapidly penetrate the fruit pulp tissue and provide synthetic raw materials, resulting in inaccurate representation of lignin synthesis changes. In contrast, this invention first soaks the fruit in a working solution, allowing the lignin monomer solution to penetrate the intercellular spaces of the fruit pulp, providing an initial source of lignin monomers. Simultaneously, a low-concentration hydrogel loaded with lignin monomers is applied, continuously releasing lignin monomers into the loquat pulp. Therefore, click chemistry staining experiments performed using this incubation method exhibit greater stability.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. An in-situ pulp incubation method for postharvest lignification click chemical imaging analysis of fruit, characterized in that, Includes the following steps: (1) Mix 0.02 g / mL pectin aqueous solution and 0.02 g / mL gelatin aqueous solution in equal volumes to form a viscous low-concentration hydrogel solution; mix 0.04 g / mL pectin aqueous solution and 0.02 g / mL gelatin aqueous solution in equal volumes to form a viscous high-concentration hydrogel solution; (2) Dissolve CA-Alk in acetone to prepare a CA-Alk stock solution with a concentration of 20 mM; Dilute the CA-Alk mother liquor to prepare the CA-Alk working solution; The CA-Alk stock solution was mixed with a low-concentration hydrogel solution to form a hydrogel incubation solution containing CA-Alk. (3) Make a cut on the fruit to expose the pulp, immerse the cut in CA-Alk working solution to ensure that the cut is fully in contact with CA-Alk working solution, and maintain it at 4℃ for 24 hours; After soaking for 24 hours, the wound was removed, and then the hydrogel incubation solution containing CA-Alk was applied to the wound surface and gelled at 4°C to form the first gel layer. Next, a high-concentration hydrogel solution was coated onto the first gel layer, and then wrapped with plastic wrap to fix it. It was then stored at 4°C.

2. The in-situ pulp incubation method according to claim 1, characterized in that, In step (1), the pectin is low-methyl esterified pectin.

3. The in-situ pulp incubation method according to claim 1, characterized in that, In step (2), the concentration of CA-Alk working solution is 0.2 mM.

4. The in-situ pulp incubation method according to claim 1, characterized in that, In step (2), the CA-Alk stock solution is added to 1 / 2 MS medium and diluted to form the CA-Alk working solution.

5. The in-situ pulp incubation method according to claim 1, characterized in that, In step (2), the CA-Alk stock solution and the low-concentration hydrogel solution are mixed at a ratio of 10 μL: 1 mL to form a hydrogel incubation solution containing CA-Alk.

6. The in-situ pulp incubation method according to claim 1, characterized in that, In step (3), the wound is 1.5-2cm long and 1.5-2cm wide.

7. The in-situ pulp incubation method according to claim 1, characterized in that, In step (3), the amount of the hydrogel incubation solution containing CA-Alk applied to the wound surface is 500-600 microliters.

8. The in-situ pulp incubation method according to claim 1, characterized in that, The fruit in question is either loquat or apple.

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