Comparison of storage and lignin accumulation characteristics between two types of snow pea.

Snow pea is a very important vegetable, and its postharvest storage characteristics vary with species. Few studies on the differences in its storage characteristics are available. In this study, postharvest changes in metabolic rate (respiration rate and water loss rate), membrane permeability (relative conductivity), nutrient contents (total sugar, amino acids, starch), lignin, cellulose, β-Glucosidase (β-GC) enzyme activity, texture properties, PG enzyme activity and their relationship were analyzed in large sweet broad peas and small snow peas. On the 8th day of storage, we found that the respiration rate and water loss rate were increased, total sugars and total amino acids were decreased significantly in these two legume vegetables, and that metabolic rate was slower with less nutrients consumed in large sweet broad peas than in small snow peas. Throughout the 8-day whole storage, the lignin and cellulose contents were always lower in large sweet broad peas than in small snow peas. With the increasing storage time, small snow peas were more susceptible to lignification and fibrosis, which was observed in their texture properties. The enzyme activities related to cellulose and pectin degradation (β-GC, PG) also showed the same trend during the storage. At the late stage of storage, the taste of large sweet broad peas was better than that of small snow peas. In conclusion, the storage period of large sweet broad peas was longer than that of the small snow peas, and its lignification degree was lower than that of the small snow peas. Meanwhile, senescence and lignin accumulation led to hardening of snow pea during postharvest storage. Our findings provide a theoretical reference for improving the postharvest storage quality of snow pea and extending the shelf life.

Introduction

Snow pea (Pisum sativum L. var. macrocarpon Ser.) is grown worldwide, extensively in northern USA, Europe, Canada, Russia, and China [1, 2]. Due to the delicious flavor and abundant nutrients, its tender shoot, seed, and pod are consumed as vegetables, especially the tender pod [3]. Its pod is one of the most important and popular leguminous vegetable because of high phytonutrients including amino acids, insoluble fibers, and vitamins [4]. However, its pod is highly perishable and ripened within a few days with a relatively short shelf-life, thus restricting its market potential.

Due to the high water content, strong metabolism, and microorganism reproduction, it is easy for pods to lose water, wrinkle, and then resulting in nutrient loss even deterioration during storage [5, 6]. Kader et al. have found that precooling and storing at near 0°C could extend the shelf life of pea pod. Kumar et al. have reported that cumin essential oil is preservative to extend shelf life of Pisum sativum [7]. Nasef et al. have found that microperforated polypropylene bags with 12 microholes are suitable to preserve pea pod, thus extending its retail period [8]. However, available information has been reported on the optimization of pea storage conditions and the information on the deterioration mechanism in pea is relatively scarce. El-hamahmy et al. have suggested that decay, shriveling, and reduction in vitamin C, and sugar contents are the primary factors limiting storage quality of snow pea pods [9].

Lignification is common in plants, and lignin can guarantee the integrity of cell wall, enhance the hardness and toughness of stem, provide plants with the internal mechanical support so that plants can resist abiotic and biotic stress [10, 11]. The lignification not only affects the taste and quality, but also limits the storage and transport of postharvest horticultural products. For example, lignin content exhibits an extremely significant negative correlation with edible quality [12]. The pea pods become hard and coarse with more fiber, which affects the taste of legumes.

In the present study, the changes in metabolic rate and nutrient contents were investigated to reveal deterioration mechanism in snow peas after harvest. The metabolic rate indexes significantly increased including the respiration rate, water loss rate, and electrical conductivity. The nutrient contents indexes rapidly declined including total sugar, total amino acids, and starch. Meanwhile, lignin and cellulose contents were ascended. Further, the texture properties such as hardness and chewiness changed. The aim of this study was to evaluate the storage characteristics of legume vegetables and lay the foundation for further research on related lignification and preservation technologies.

Materials and methods

Material treatments

Two types of snow pea including large sweet broad pea and small snow pea were purchased from the vegetable supermarket on the agricultural product display platform of Agro-product Processing Research Institute, Yunnan Academy of Agricultural Sciences (Kunming, China). Freshly picked pods were selected, and they were intact, free from pests and mechanical damage with similar size, maturity degree, and plumpness. Ten samples (300g per sample) with similar maturity were selected, and one group was the observation group, and the other group was the experimental group. The samples were collected on d 0, d 2, d 4, d 6, and d 8 during storage. All samples were placed at room temperature without direct sunlight, strong wind, and suitable humidity, and the samples was spread flat to keep ventilated. All of the treatments were conducted with three biological replicates.

Assessments

To investigate the physiological and biochemical changes during storage, pods stored after 2, 4, 6, and 8 days were used as test samples. To avoid the possible error, the head and tail of selected pods were removed, and the middle part of pods was used for index measurement.

Respiration rate

Respiration rate was measured with SY-1022 Fruit and Vegetable Respiro meter. First, samples were put into the respiratory chamber for 30 min, the respiratory intensity of the sample was got according to the change of CO2 concentration in the respiratory chamber before and after sample placement.

Water loss rate

The water loss rate was determined according to the change in the sample weight after harvest and during storage [13]. The initial weight of the sample was recorded as M0, the weight of each sample at sampling time points was recorded as M. The water loss rate was computed according to the following formula:

Relative conductivity

The samples were cut into small-sized pieces, and soaked in 20 mL of distilled water for 1 h. Afterwards, the initial electronic conductivity (C0) was recorded, and then the samples were boiled for 30 minutes and cooled to record the electronic conductivity value (C1). Finally, the electronic conductivity was computed as follows:

Total sugar content

First, 2 g sample was added into 20 mL of distilled water, and then was boiled for 30min. The standard curve of glucose was constructed, y = 0.0037x+0.0928. Then it was determined according to the modified anthrone colorimetric method [8]. The absorbance value at 630 nm was measured. The total sugar content in the sample was calculated by using the following formula:

Whereas, C = sugar concentration (μg); V1 = total volume of sugar extract (mL); D = dilution factor; W = total weight of sample (g); V2 = volume of sugar used for the determination (mL).

Total amino acid content

The amino acid content was determined according to the improved ninhydrin colorimetric method [14]. A total of 5 g sample was added into 50 mL of distilled water and 5g activated carbon, and then was boiled, and filtered. Afterwards, 2 mL filtrate was added into distilled water (2 mL), 2% ninhydrin solution (1 mL), and phosphate buffer (1 mL), and boiled for 15 min. After cooling down, absorbance at 570 nm was measured.

Starch content

The starch content was determined with the plant starch content kit (Suzhou Keming Biotechnology Co., Ltd.). After measurement of the absorbance at 620 nm, starch content was calculated according to the following formula:

Where A is the absorbance value; V1 is the volume of the sample added to the reaction system; V2 is the volume of extract added; W is the sample weight.

Lignin content

The sulfuric acid method was used to determine the lignin content [15]. The initial weight of the sample was recorded as M0. The initial sample was added into 4 mL sulfuric acid (72%) and 200 mL distilled water, refluxed at 100°C for 1 h, and filtered. The filter residue was washed with hot distilled water, and dried completely to a constant weight (M). The lignin content was calculated as follows:

Cellulose content

Cellulose content was determined by the method of acidity washing. The samples were added into 20 mL of cetyl trimethyl ammonium bromide. The remaining procedures were similar to those for the determination of lignin.

Determination of enzyme activities of β-GC (β-Glucosidase) and PG (ploygalacturonase)

The 0.1 g of fresh snow pea tissue sample was added the extract and ground it in an ice bath. After high-speed centrifugation, the supernatant was taken and subjected to β-GC and PG enzyme activity determination with assay kits (Suzhou Keming Biotechnology Co., Ltd.), respectively, according to kit instruction. The absorbance value at 400 nm was measured, and 1 nmol p-nitrophenol produced per gram tissue per minute was defined as an enzyme activity unit of β-GC. The obtained supernatant was further diluted 5 times for PG determination, and the absorbance values were measured at a wavelength of 540 nm. One mg of galacturonic acid generated from pectic acid from per gram sample decomposion per hour at 40°C and pH 6.0 was defined as an enzyme activity unit of PG (U).

Texture properties

The hardness, cohesiveness, springiness, gumminess, and chewiness were measured by the TMS-TOUCH texture analyzer equipped with a cylindrical detection probe (35 mm diameter). The test parameters were set as follows: the load unit of 200 N (Newton), the target deformation of 50%, the test speed of 60 mm/s, and the rising height (from the sample) of 10 mm, the initial force of 0.1 N, and the residence 0 s between two extrusion cycles. The measurement was repeated 10 times.

Statistical analysis

All data were presented as the mean ± standard error (SE). The statistical difference between groups was analyzed through one-way analysis of variance (ANOVA) in SPSS software. P<0.05 was considered as statistically significant.

Results and discussion

Respiration rate and water loss

Water is essential to ensure the freshness of fruits and vegetables. When water loss exceeds 5%, fruits and vegetables usually begin to wilt and lose the freshness [16]. However, due to the strong respiration and transpiration, legumes are prone to lose water and wilt, and deteriorate. Hence, the respiration rate and water loss rate were examined in the present study (Fig 1). As shown in Fig 1A, the respiration rate was dramatically increased by 55.38% (from 66.54 to 103.39 mg·kg-1·h-1) for large sweet broad pea and by 139.75% (from 30.77 to 73.77 mg·kg-1·h-1) for small snow peas after 8-day storage, respectively. This indicated that the respiration change rate of the small snow peas was stronger. In addition, it was observed that the water loss rate of large sweet broad peas increased from 13.79% to 32.56%, and that of small snow peas increased from 15.82% to 36.03% (Fig 1B). The water loss rate of both types of peas increased during storage, while compared to large sweet broad peas, small snow peas is more likely to lose water. Above results indicated that respiration intensity of legumes increased with the extension of storage, which might be closely related with water loss.

Fig 1

Changes in respiration rate (A) and water loss rate (B) of two types of snow peas during storage. Different lower-case letters indicate significant differences in mean among different treatment groups(n = 3)(P<0.05). The following was the same.

Relative conductivity

Relative conductivity is an important indicator for estimating the degree of destruction of cell membrane structure in fruits and vegetables in the course of storage [17]. Therefore, the current study analyzed the changes in relative conductivity of two snow pea during storage. As shown in Fig 2, the relative conductivity was increased by 30.68% (from initial 37.68% to 49.24%) for large sweet broad peas and by 47.33% (from 34.73% to 51.17%) for small snow peas after storing for 8 days. This indicated that the cell membrane structure small snow peas is more drastically destructed than large sweet broad peas during postharvest storage.

Fig 2

Changes in relative conductivity of two types of snow peas during storage.

Total sugar, total amino acid, and total starch content

Sugar, amino acids, and starch are the main nutrients in legume vegetables. However, these nutrients were usually declined with the extension of storage [18]. Hence, in the current study, total sugar, total amino acids, and starch were investigated (Fig 3). As shown from Fig 3A and 3B, both the total sugar and total amino acid decreased with the extension of storage. The total sugar content declined relatively smooth from 4.42% to 3.35% in large sweet broad peas, whereas that in small snow pea declined sharply from 3.96% to 1.10% (Fig 3A). Thus, after 8-day storage, total sugar content of small snow pea was significantly lower than that of large sweet broad peas.

Fig 3

Changes in total sugar (A), total amino acid (B), and total starch (C) contents in two types of snow pea during storage.

As shown in Fig 3B, the change in amino acid content was consistent with that in total sugar content, which decreased respectively by 7.5% in large sweet broad peas and 14.1% in small snow peas. Although, there was a rise from 19.72 mg·g-1 FW at the day 0 of storage to 29.14 mg·g-1 FW at day 2 for large sweet broad pea, which might be caused by the late-maturing. The total amino acid content from 29.14 mg·g-1 FW at the 2th day to 18.24 mg·g-1 FW during the storage in large sweet broad peas. Comparatively, the total amino acid content declined smoothly in small snow peas, which decreased from 28.78 mg·g-1 FW to 24.72 mg·g-1 FW.

Also, the change in starch content was analyzed (Fig 3C). The starch content of large sweet broad peas increased from initial 14.19 mg·g-1 FW to 17.45 mg·g-1 FW on day 8, which might be attributed by after-ripening. While, the starch content of small snow peas decreased by 7.06 mg·g-1 FW from 18.47 mg·g-1 FW after harvest to 11.41mg·g-1 FW on the 8th day of storage. According to above results, nourishments including the total sugar and the total amino acid was depleted in both two varieties after harvest. And it was indicated that the nutrients of small snow peas were consumed more quickly than large sweet broad peas.

Lignin and cellulose content

Lignin and cellulose, as important components of plant secondary cell wall, participate in various physiological processes, excepically in the morphological development of plants [19]. β-PC is the key enzymes responsible for cellulolysis, and inhibits the activity of enzymes related to the degradation of cell wall substances, thus delaying the decline in fruit hardness [20]. Lignification can ensure the integrity of cell wall, enhance the hardness and toughness of stem, and provide plants with the internal mechanical support so that plants can resist abiotic and biotic stresses [10]. However, the accumulation of lignin and cellulose not only affects the taste and quality of fruits and vegetables, but also limits their postharvest storage and transportation. It has been reported that fruits and vegetables become hardened due to the increased lignin and cellulose during storage or under stress conditions [21].

In the present study, lignin content in both types of peas significantly increased with the extension of the storage (Fig 4A). Although, the lignin content in large sweet broad peas raised from 10.60 to 33.57 mg·g-1 after 8-day storage, but the lignin content in large sweet broad peas was always lower than that in small snow peas. This indicated that the small snow peas were more easily lignified with the increasing storage. The cellulose between two types of peas exhibited the similar regularity as lignin content (Fig 4B). In large sweet broad peas, the cellulose content increased from 35.65 mg·g-1 to 37.48 mg·g-1 in the first 4 days of storage, and then decreased to 31.01 mg·g-1 in the last 4 days of storage, which might be due to the degradation of protopectin in the postharvest. However, the cellulose content increased from 39.67 mg·g-1 to 46.06 mg·g-1 after storage in the small snow peas during 8-day storage. In the processing of storage, the cellulose content in large sweet broad peas was also always lower than that in small snow peas.

Fig 4

Changes in lignin content (A), cellulose content (B) and β-GC enzyme activity (C) of two types of snow peas during storage.

As shown in Fig 4C, the enzyme activity of β-PC enzyme related to cellulose degradation, of large sweet broad peas reached the highest (53.98 nmol/min/g) on day 2 during storage, and then decreased continuously, and reduced by 51.89% during the entire storage period. The β-PC enzyme activity of small sweet broad peas increased continuously until 149.61 nmol/min/g in the first 6 days, and then decreased to 30.55 nmol/min/g. During the whole storage process, the β-PC enzyme activity of small sweet broad peas was greater than that of large sweet broad peas, and the change trend of β-PC enzyme activity was consistent with that of the cellulose content. Small sweet broad peas were more prone to lignification during storage and were not resistant to storage.

Texture characteristics and PG enzyme activity

With the increase in lignin and cellulose contents during the storage, the textural properties of fruits and vegetables changed greatly. The textural properties of fruits and vegetables are also important for evaluating their quality and economic values [22]. Usually, the texture profile analysis (TPA) method was used to simulate the chewing motion of human teeth to analyse the textural properties, in which the sample was compressed twice to test its cohesiveness, springiness, gumminess, and chewiness. This texture evaluation method reduced the subjective evaluation error to a certain extent [23].

In this study, the TPA method was used to analyze changes in textural properties of the two types of peas during storage. The results indicated that the cohesiveness of two types of peas was increased by 21.83% from 0.20 to 0.24 for large sweet broad peas and increased by 121.63% for small snow peas from 0.17 to 0.39. This corresponded exactly to the water loss rate, and a large amount of water loss caused the internal cohesiveness to increase.

The springiness of the two types of peas showed an upward trend until the 6th day of storage, increasing from 3.023 to 4.06 mm for large sweet broad peas, and from 2.605 to 3.7 mm for small snow peas, and then springiness declined relatively smoothly in both types of peas. Overall, in 8-day storage, springiness increased by 8.9% for large sweet broad peas and by 33.05% for small snow peas.

The gumminess and chewiness of the two types of peas showed a rapid upward trend during 8-day storage. In large sweet broad peas, the gumminess increased by 57.53% from 1.00 to 2.73 N, and the chewiness increased by 209.13% from 3.021 to 9.30 N. On the 8th day, gumminess and chewiness values of large sweet broad peas were slightly decreased, which was similar to springiness in large sweet broad peas, and this decrease might be attributed to the consumption of nutrients in the late storage stage. In small snow peas, the gumminess increased by 245.90% from 1.07 to 3.70 N, and the chewiness increased by 375.53% from 2.73 to 12.97 N. Compare with the large sweet broad peas, the rising trend of chewiness in small snow peas was more obvious.

PG is related to the degradation of pectin during fruit softening. Koutsimanis et al. have showed that 1 μL/L 1-MCP treatment could effectively inhibit the decrease in PG activity and protopectin content of Tardibelle peach fruit during shelf life at 20°C, and maintain the fruit higher hardness [24]. As shown in Fig 5C, the PG enzyme activity of large sweet broad peas increased from 7.04 to 31.74 mg/h/g on day 4 during storage, which was consistent with the increase in their cellulose content. On day 8, PG enzyme activity of large sweet broad peas decreased to 9.8 mg/h/g. During the whole storage process, their PG enzyme activity increased by 39.17%. The PG enzyme activity of small sweet broad peas reached the highest on day 6 (9.27mg/h/g), and then decreased slightly, and it increased by 101.30% during the whole storage process. This result indicated that the small sweet broad peas were easier to soften during storage and were not resistant to storage, which was consistent with the analysis results of texture.

Fig 5

Changes in texture characteristics AB) and PG enzyme activity (C) of legume vegetables during storage.

Conclusion

This study revealed that small snow peas had a fast metabolism and consumed more nutrients than large sweet broad peas after harvest, resulting in increased membrane permeability and greater perishability. In addition, postharvest lignification degree of small sweet broad peas was higher than that of large ones, and their cellulose content and related enzyme activities were higher than those of large sweet broad peas, which affected their flavor and shelf life. The texture properties and PG enzyme activity related to pectin degradation also verified this. Overall, the large sweet broad peas had a longer shelf life than the small ones, and their lignification degree was lower than that of the small ones. Future research is suggested to focus on the molecular biological mechanism of pod lignification during storage and to explore preservation methods to alleviate pod lignification. Our findings provide a theoretical basis for the preservation of legume vegetables.

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Reviewer #1: In the manuscript PONE-D-21-40678, the authors investigated the senescence and lignin accumulation lead to hardening of snow pea during postharvest storage.

Overall, the methodology used by the authors about two varieties of snow pea and the targeted measurements are rather basic, and hence do not provide any additional information to what is already known about the biochemical changes in pea. There are a number of issues of concern and limited amount of new science in the manuscript. Authors are advised to find out more metabolites for comparison of the two varieties of snow pea, as the quality related measurements are very basic. Cell wall degrading enzymes, browning indexes and cell damage indexes would support more precisely the story.

Taking all the factors into account, I believe the manuscript is not suitable for publication in its present state in Journal of Plos One.

Some specific comments are given below:

Title: The title not complete reflects the content of the manuscript.

Line No. 66-75: In the present study, the changes in metabolic rate and nutrient contents were investigated to reveal deterioration mechanism in super snap peas after harvest. The targeted pea is not super snap peas in this study. The aim of this study is very general. In the introduction, no needs to write the results of this study.

Line No. 89-90: First, samples were respectively put into the respiratory chamber, after standing for 0.5 h. What did the authors mean by 0.5h?

Line No. 145-148: Pease, write the experimental design used in this study.

Line No. 155-159: As shown in Fig 1A, the respiration rate was dramatically increased by 55.38% (from 66.54 to 103.39 mg•kg-1•h-1) for large sweet broad pea and by 139.75% (from 30.77 to 73.77 mg•kg-1•h-1) for small snow peas after 8-day storage, respectively. This indicated that the respiration rate of the small snow peas was stronger. There is contradiction in you results and Fig. 1A. The Fig. 1A, indicates that the respiration rate was higher in Large sweet broad peas as compared to Small snow peas. However, the authors claimed in the results that the respiration rate was higher in Small snow peas. If the respiration rate was higher in the Large sweet broad peas as indicted in the Fig. 1A, then how the authors concluded that the shelf life of Large sweet broad was longer compared to Small snow peas?

The authors stated simple concepts and common knowledge over and over again. The results should be properly discussed in view of literature data.

Fig. 1, 2 and 3 should be constructed like Fig. 4.

Please, improve the conclusion by adding some salient and new findings of the present study.

Reviewer #2: The manuscript no PONE-D-21-40678 entitled 'Senescence and lignin accumulation lead to hardening of snow pea during postharvest storage' addressed nutritional, organoleptic and lignification changes in two varieties of snow pea after harvest. The study was very interesting and of economic importance for stakeholders, retailers, processors and exporters of pea industry. However, the manuscript needs some minor revisions in methodology section and overall the manuscript should be checked for grammatically mistakes and language improvement. I have suggested some changes in the manuscript pasted in 'Reviewer attachment'. After incorporation of these changes the manuscript can be considered for publication.

Reviewer #3: Research presented in the manuscript investigates changes in respiration, water loss, membrane permeability, nutrient contents, lignin contents, cellulose contents, and textural attributes in two snow pea varieties during storage. Findings suggest that lignin accumulation leads to hardening of snow pea during postharvest storage. Though, data presented provide adequate evidence to support the claim, however, few basic elements of research are missing and must be included in the manuscript. Storage conditions, replication size (sample weight), number of replications allocated to each removal and experimental design are missing in the "Materials and Methods" section. Figure captions must also state description of standard error bars used in graphs? Overall, manuscript is written well and needs only few corrections highlighted in the reviewed version of manuscript.

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Reviewer #1: Yes: Ghulam Khaliq

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6 Apr 2022

-RESPONSE To REVIEWERS

> Dear Sajid Ali,

> Thank you very much for your kind consideration. Thanks a lot for the reviewers’ comments and their kind suggestions on our manuscript (PONE-D-21-40678). We provide this cover letter to explain, point by point, the details of our revisions in the manuscript and our responses to the reviewers’ comments as follows. In order to make the changes easily viewable for you and the reviewers, in the revised paper, we marked the revision with red color. Besides, we have carefully proof-read the manuscript to minimize typographical, grammatical and bibliographical errors.

> We are looking forward to hearing from you soon.

> Best regards,

> Xuerui Li

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(The present research got supportion from the Yunnan Li Puwang Expert Workstation (202005AF50007). Yu Lijuan and Li Xuerui are the main accomplisher of this project, they design the study, collect and analyse dates,prepare the manuscripts; it also got supportion from Cultivation of Agricultural Product Processing Team (202002AE320007-03). Li Hong is the project director, he designed this study.)

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The present research got supportion from the Yunnan Li Puwang Expert Workstation (202005AF50007). Yu Lijuan and Li Xuerui are the main accomplisher of this project, they design the study, collect and analyse dates, prepare the manuscripts; it also got supportion from Cultivation of Agricultural Product Processing Team (202002AE320007-03) and Green Food Brand Construction (Intensive Processing). Li Hong is the project leader, he designs this study. There was no additional external funding received for this study.

We have added the funding “Green Food Brand Construction (Intensive Processing)” and statement “There was no additional external funding received for this study.” in my updated Funding Statement.

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Reviewer #1: In the manuscript PONE-D-21-40678, the authors investigated the senescence and lignin accumulation lead to hardening of snow pea during postharvest storage.

Overall, the methodology used by the authors about two varieties of snow pea and the targeted measurements are rather basic, and hence do not provide any additional information to what is already known about the biochemical changes in pea. There are a number of issues of concern and limited amount of new science in the manuscript. Authors are advised to find out more metabolites for comparison of the two varieties of snow pea, as the quality related measurements are very basic. Cell wall degrading enzymes, browning indexes and cell damage indexes would support more precisely the story.

Thank you for your excellent suggestion and supporting. We have added the dates and discusses about cell wall degrading enzyme activities (β-GC, PG) in Fig 4.C and Fig 5.C.

Some specific comments are given below:

Title: The title not complete reflects the content of the manuscript.

Thank you for your excellent suggestion. The title was revised to “Comparison of storage and lignin accumulation characteristics between two types of snow pea”.

Line No. 66-75: In the present study, the changes in metabolic rate and nutrient contents were investigated to reveal deterioration mechanism in super snap peas after harvest. The targeted pea is not super snap peas in this study. The aim of this study is very general. In the introduction, no needs to write the results of this study.

In the present study, the changes in metabolic rate and nutrient contents were investigated to reveal deterioration mechanism in snap peas after harvest.

We have deleted “super”. The aim of this study is Analysis of storage characteristics and lignin accumulation of two snow pea (Large sweet broad peas and small snow peas).

Meanwhile, we have deleted the sentence “These results imply that lignification in snow pea may cause the change in texture properties, thus affecting its taste and quality” in the introduction.

Line No. 89-90: First, samples were respectively put into the respiratory chamber, after standing for 0.5 h. What did the authors mean by 0.5h?

It means 30 min.

Line No. 145-148: Pease, write the experimental design used in this study.

Thank you for your suggestion, we have revised it in the chapter 2 (Materials and methods).

Material treatments

Two types of snow pea including large sweet broad pea and small snow pea were purchased from the vegetable supermarket on the agricultural product display platform of Agro-product Processing Research Institute, Yunnan Academy of Agricultural Sciences (Kunming, China). Freshly picked pods were selected, and they were intact, free from pests and mechanical damage with similar size, maturity degree, and plumpness. Ten samples (300g per sample) with similar maturity were selected, and one group was the observation group, and the other group was the experimental group. The samples were collected on d 0, d 2, d 4, d 6, and d 8 during storage. All samples were placed at room temperature without direct sunlight, strong wind, and suitable humidity, and the samples was spread flat to keep ventilated. All of the treatments were conducted with three biological replicates.

Line No. 155-159: As shown in Fig 1A, the respiration rate was dramatically increased by 55.38% (from 66.54 to 103.39 mg•kg-1•h-1) for large sweet broad pea and by 139.75% (from 30.77 to 73.77 mg•kg-1•h-1) for small snow peas after 8-day storage, respectively. This indicated that the respiration rate of the small snow peas was stronger. There is contradiction in you results and Fig. 1A. The Fig. 1A, indicates that the respiration rate was higher in Large sweet broad peas as compared to Small snow peas. However, the authors claimed in the results that the respiration rate was higher in Small snow peas. If the respiration rate was higher in the Large sweet broad peas as indicted in the Fig. 1A, then how the authors concluded that the shelf life of Large sweet broad was longer compared to Small snow peas?

The authors stated simple concepts and common knowledge over and over again. The results should be properly discussed in view of literature data.

Thank you for your suggestion. What we want to express was the rate of respiration change, which is the characterization index of postharvest aging. The faster the respiration rate changes, the more vigorous the metabolism, the more susceptible to decay.

So, we revised the sentence“This indicated that the respiration change rate of the small snow peas was stronger. ”

There are no studies comparing the storage characteristics of these two snow peas.

Fig. 1, 2 and 3 should be constructed like Fig. 4.

Thank you for your suggestion. We have revised them. The texture properties are shown more tellingly in Fig.5 AB.

Please, improve the conclusion by adding some salient and new findings of the present study.

Thank you for your suggestion. Some Chinese researchers have looked the storage characteristics of legumes, but there are no studies comparing the storage characteristics of these two snow peas. We found that the large sweet broad peas had a longer shelf life and a lower lignification degree than small snow peas. The enzyme activities related to cellulose and pectin degradation (β-GC, PG) also showed the same trend during the storage.

Reviewer #2: The manuscript no PONE-D-21-40678 entitled 'Senescence and lignin accumulation lead to hardening of snow pea during postharvest storage' addressed nutritional, organoleptic and lignification changes in two varieties of snow pea after harvest. The study was very interesting and of economic importance for stakeholders, retailers, processors and exporters of pea industry. However, the manuscript needs some minor revisions in methodology section and overall the manuscript should be checked for grammatically mistakes and language improvement. I have suggested some changes in the manuscript pasted in 'Reviewer attachment'. After incorporation of these changes the manuscript can be considered for publication.

Thank you for your excellent suggestion and supporting. We have revised grammatically mistakes according to your suggestion in 'Reviewer attachment'. Meanwhile, language was edited and polished by linguistics professor Ping Liu from Huazhong Agriculture University, Wuhan, China.

Reviewer #3: Research presented in the manuscript investigates changes in respiration, water loss, membrane permeability, nutrient contents, lignin contents, cellulose contents, and textural attributes in two snow pea varieties during storage. Findings suggest that lignin accumulation leads to hardening of snow pea during postharvest storage. Though, data presented provide adequate evidence to support the claim, however, few basic elements of research are missing and must be included in the manuscript. Storage conditions, replication size (sample weight), number of replications allocated to each removal and experimental design are missing in the "Materials and Methods" section. Figure captions must also state description of standard error bars used in graphs? Overall, manuscript is written well and needs only few corrections highlighted in the reviewed version of manuscript.

Thank you for your excellent suggestion and supporting. we have revised it in the chapter 2 (Materials and methods). It concluded storage conditions, replication size (sample weight), number of replications allocated to each removal and experimental design.

Material treatments

Two types of snow pea including large sweet broad pea and small snow pea were purchased from the vegetable supermarket on the agricultural product display platform of Agro-product Processing Research Institute, Yunnan Academy of Agricultural Sciences (Kunming, China). Freshly picked pods were selected, and they were intact, free from pests and mechanical damage with similar size, maturity degree, and plumpness. Ten samples (300g per sample) with similar maturity were selected, and one group was the observation group, and the other group was the experimental group. The samples were collected on d 0, d 2, d 4, d 6, and d 8 during storage. All samples were placed at room temperature without direct sunlight, strong wind, and suitable humidity, and the samples was spread flat to keep ventilated. All of the treatments were conducted with three biological replicates.

Figure captions have also stated description of standard error bars used in graphs.

Different lowercase letters indicate significant differences in mean among different treatment groups(n=3) (P<0.05), The following was the same.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

9 May 2022

Comparison of storage and lignin accumulation characteristics between two types of snow pea

PONE-D-21-40678R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

22 Jun 2022

PONE-D-21-40678R1

Comparison of storage and lignin accumulation characteristics between two types of snow pea

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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