Strategies to Delay Ethylene-Mediated Ripening in Climacteric Fruits: Implications for Shelf Life Extension and Postharvest Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript mainly review the ethylene mediated ripening and shelf-life extension of climacteric fruits. Overall, manuscript summarized the ethylene biosynthesis, signaling  and management strategies to extend the fruits shelf-life with relevant examples. Highlighting the 'Ethylene inhibitors, Ethylene absorbent and Ethylene scavengers by catalytic oxidation' with relevant previous studies are the novel parts of this review. However, minor corrections are need to be made before merit the publication:

1. Line 58: Sub title 'According Ethylene' is suggested to change  by 'Ethylene - a ripening plant hormone'

2. Line 221 -230: In system I of ethylene biosynthesis showed the auto-inhibitory character where in System II - ethylene biosynthesis showed the autocatalytic character, this information need to be corrected. 

3. At least figure legends of Figure 10 and 11 need to be rephrase.

Comments on the Quality of English Language

Moderate editing required. 

Author Response

REVIEWER 1 (CORRECTIONS IN YELLOW IN REVISED MANUSCRIPT)

This manuscript mainly review the ethylene mediated ripening and shelf-life extension of climacteric fruits. Overall, manuscript summarized the ethylene biosynthesis, signaling and management strategies to extend the fruits shelf-life with relevant examples. Highlighting the 'Ethylene inhibitors, Ethylene absorbent and Ethylene scavengers by catalytic oxidation' with relevant previous studies are the novel parts of this review. However, minor corrections are need to be made before merit the publication:

Thank you to Reviewer 1 for your valuable feedback and for recognizing the novel aspects of our manuscript regarding ethylene inhibitors, absorbents, and scavengers by catalytic oxidation. We appreciate your acknowledgment of the relevance and thoroughness of our review on ethylene-mediated ripening and shelf-life extension of climacteric fruits.

We have carefully considered your suggestions for minor corrections and have implemented the necessary changes to enhance the quality and clarity of our manuscript. Below, we provide a detailed response to each of your points:

1. Line 58: Sub title 'According Ethylene' is suggested to change by 'Ethylene - a ripening plant hormone'

Thank you for your valuable suggestion. We agree that the suggested subtitle better reflects the content of the section and improves the clarity of the manuscript. We have updated the subtitle accordingly. The subtitle on line 58 has been changed from "According Ethylene" to "Ethylene - a Ripening Plant Hormone".

2. Line 221 -230: In system I of ethylene biosynthesis showed the auto-inhibitory character where in System II - ethylene biosynthesis showed the autocatalytic character, this information need to be corrected. 

Thank you for pointing out the discrepancy in our description of ethylene biosynthesis systems. We appreciate your careful review and have made the necessary corrections to accurately reflect the characteristics of System I and System II in ethylene biosynthesis. The section between lines 221-230 has been revised to correctly describe System I as exhibiting auto-inhibitory characteristics and System II as exhibiting autocatalytic characteristics.

3. At least figure legends of Figure 10 and 11 need to be rephrase.

Thank you for your suggestion regarding the rephrasing of the figure legends for Figures 10 and 11. We have reviewed and revised the legends to enhance clarity and readability. For Figure 10, We have added: Active carbon acts as an ethylene adsorbent. When this phytohormone penetrates through the structural cracks in the active carbon, it remains adhered to the walls, thus preventing its effect. For figure 11, we have added:  How two different wavelengths of UV-C radiation affect ethylene removal. The first one, at 254 nm, produces an indirect ethylene elimination by producing free radicals that act on ethylene. The second wavelength works directly on the ethylene molecules

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Alonso-Salinas Ramiro et al. reviewed the strategies controlling ethylene-induced fruit ripening to extend shelf-life and improve postharvest quality. In my opinion, the manuscript can be accepted after minor revision. Please see my comments to improve the manuscript.

-The role of ethylene in non-climacteric fruit is also important, the authors should also include some content of non-climacteric fruit.

-In the introduction, the authors did not illustrate why ethylene will induce the shortening of shelf-life and the decrease of quality. Also, the role of ethylene in fruit disease resistance was not mentioned.

Line 41-45, the "Too Good To Go" app is not an academic data source. Please change the data source or delete this paragraph directly.

-Although the authors provide comprehensive information on the in vivo production of ethylene, metabolic pathways, etc., it is still missing. The authors need to provide comprehensive information on the postharvest physiological regulation of ethylene in fruit, such as its role in disease resistance and flavor changes. The authors discuss that although ethylene scavengers/inhibitors are effective in inhibiting fruit ripening, how does it affect normal fruit ripening, disease resistance, and flavor changes?

-The depth of the article needs to be enhanced, latest research advances need to be introduced, such as research progress on cold plasma in ethylene scavenging.

Author Response

REVIEWER 2 (CORRECTIONS IN RED IN REVISED MANUSCRIPT)

The manuscript by Alonso-Salinas Ramiro et al. reviewed the strategies controlling ethylene-induced fruit ripening to extend shelf-life and improve postharvest quality. In my opinion, the manuscript can be accepted after minor revision. Please see my comments to improve the manuscript.

Thank you for your positive feedback and for considering our manuscript for publication after minor revisions. We appreciate your insightful comments and suggestions to improve our manuscript. We hope the revised manuscript meets your expectations and are happy to provide further clarifications if needed. Below, we have addressed each of your comments in detail:

-The role of ethylene in non-climacteric fruit is also important, the authors should also include some content of non-climacteric fruit.

Thank you for highlighting the importance of ethylene in non-climacteric fruits. We agree that including this aspect would provide a more comprehensive overview of ethylene's role in fruit ripening. We have added information to the manuscript that discusses the role of ethylene in non-climacteric fruits, including examples and relevant studies that demonstrate its impact on ripening and postharvest quality. We believe this addition enriches the manuscript and provides a more balanced perspective on ethylene's function in both climacteric and non-climacteric fruits. Concretely we have added in pages 6-7 and lines 304-324 the following information:

About the effect of ethylene on non-climacteric fruits, although they do not show a clear increase in ethylene production rates during ripening, in certain cases, their exposure to exogenously applied ethylene can stimulate certain ripening-related processes, such as the inhibition of senescence, the inhibition of the development of physiological disorders, and the inhibition of colour changes [44]. Exogenous ethylene can react with ethylene receptors both in the early and late stages, leading to accelerated ripening. However, in this case, once exogenous ethylene application is stopped, the fruits will return to their pre-treatment levels of respiration and ethylene production [35,45]. This process is widely applied in citrus fruits, for example, in lemons. These fruits are harvested without reaching their physiological ripeness (colour change from green to yellow). Subsequently, when they are ready for consumption, they undergo a process called "degreening" in which controlled ethylene is introduced into lemon storage chambers until they reach their optimal ripeness for consumption, and then return to their pre-treatment levels of respiration and ethylene production [46].

As an example, exposure to 1-MCP had an effect on delaying rachis browning in table grape varieties (Thompson Seedless'), thus suggesting the possible involvement of the ethylene signalling pathway in the regulation of the rachis browning process on non-climacteric fruit [47]. Another example can be found on pomegranates, where the main problems associated with their postharvest storage are related to weight loss and shrivelling, as well as peel discoloration. Previous studies indicated that exposure to ethylene blockers such as 1-MCP reduced skin shrivelling [48] and the incidence of peel browning [49].

-The authors did not illustrate why ethylene will induce the shortening of shelf-life and the decrease of quality. Also, the role of ethylene in fruit disease resistance was not mentioned.

Thank you for your insightful comments. We recognize the importance of thoroughly explaining how ethylene affects the shelf-life and quality of fruits, as well as its role in fruit disease resistance.

Concretely, we have added a new section in point 4, which is subsection 4.1: "Effect of ethylene over shelf-life, quality and disease resistance," covering pages 10-11 and lines 491-534 with the following information:

4.1. Effect of ethylene over shelf-life, quality and disease resistance

Also, this phytohormone is a key player in how long fruits and vegetables stay fresh. For example, ethylene speeds up the ripening process, which is great for achieving peak flavour and texture. However, beyond this point, ethylene can cause them to over-ripen and spoil much faster [51]. Ethylene also initiates the aging process in plants, known as senescence, and induces abscission. Consequently, this results in a decline in the overall quality of the produce, affecting both its appearance and texture [53]. Another crucial role of the ethylene is stimulating the production of enzymes that degrade cell wall components, resulting in the softening of fruit tissue. This softening increases the susceptibility of the fruit to mechanical damage and microbial decay [101]. Lastly, ethylene increases the respiration rate in fruits, leading to a faster depletion of their energy reserves. This elevated metabolic activity accelerates the loss of firmness, flavour, and nutritional value, thereby contributing to a reduced shelf-life [102].

Ethylene plays a crucial role in impacts the quality of fruits and vegetables, but its effects can be a double-edged sword if not carefully managed. Firstly, ethylene is known to induce the production of enzymes that break down cell walls, which leads to the softening of fruits. While this is essential for ripening, it can also result in a loss of texture and firmness, qualities highly valued by consumers [103]. Another effect of ethylene is the degradation of chlorophylls. This process affects the vibrant green colour of many fruits and vegetables, making them appear less fresh and less appealing. In addition, ethylene causes over-ripening, provoking a significant drop in overall quality and visual appeal. This deterioration can make fresh produce look unappetizing and less desirable [104]. Furthermore, the presence of ethylene during storage and transport is a significant issue. It can speed up the ripening process before the produce reaches consumers, resulting in a noticeable decline in quality by the time it reaches the shelves [105].

Ethylene plays a crucial role in impacts the quality of fruits and vegetables, but its effects can be a double-edged sword if not carefully managed. Firstly, ethylene is known to induce the production of enzymes that break down cell walls, which leads to the softening of fruits. While this is essential for ripening, it can also result in a loss of texture and firmness, qualities highly valued by consumers [103]. Another effect of ethylene is the degradation of chlorophylls. This process affects the vibrant green colour of many fruits and vegetables, making them appear less fresh and less appealing. In addition, ethylene causes over-ripening, provoking a significant drop in overall quality and visual appeal. This deterioration can make fresh produce look unappetizing and less desirable [104]. Furthermore, the presence of ethylene during storage and transport is a significant issue. It can speed up the ripening process before the produce reaches consumers, resulting in a noticeable decline in quality by the time it reaches the shelves [105].

Ethylene also plays a multifaceted role in fruit disease resistance, acting as both a promoter and a suppressor depending on the context. Its influence is nuanced and varies with different factors. On one hand, ethylene can boost the fruit's defence mechanisms against pathogens. For example, research on ‘Kyoho’ grapes shows that treating them with ethephon, a compound that releases ethylene, before ripening can enhance the expression of genes related to both fruit coloration and disease resistance. This treatment led to improved resistance against Botrytis cinerea, a common fungal pathogen [106].

Line 41-45, the "Too Good To Go" app is not an academic data source. Please change the data source or delete this paragraph directly.

Thank you for your suggestion regarding the paragraph in lines 41-45. We have decided to remove the paragraph mentioning the "Too Good To Go" app to ensure the use of academic data sources throughout the manuscript.

-Although the authors provide comprehensive information on the in vivo production of ethylene, metabolic pathways, etc., it is still missing. The authors need to provide comprehensive information on the postharvest physiological regulation of ethylene in fruit, such as its role in disease resistance and flavor changes. The authors discuss that although ethylene scavengers/inhibitors are effective in inhibiting fruit ripening, how does it affect normal fruit ripening, disease resistance, and flavor changes?

We appreciate Reviewer 2's insightful comments and suggestions. In response to the concerns raised, we have expanded our discussion to address the postharvest physiological regulation of ethylene in fruit, focusing on its roles in disease resistance and flavor changes.

Concretely, we have added a paragraph in subsection 4.1: "Effect of ethylene over shelf-life, quality and disease resistance," covering page 11 and lines 535-550 with the following information:

Firstly, the ethylene response involves a variety of genes that are differentially expressed when plants are challenged by pathogens. For instance, in a study on potato, a total of 1226 ethylene-specific differentially expressed genes (DEGs) were identified, including those encoding for transcription factors, kinases, defence enzymes, and disease resistance-related genes [107]. These genes are part of the plant’s immune system, helping to activate defence responses. Secondly, transcription factors as the APETALA2/ethylene response factor (AP2/ERF) family plays a pivotal role in plant disease resistance. They act downstream of mitogen-activated protein kinase (MAPK) cascades and regulate the expression of genes associated with hormonal signalling pathways, biosynthesis of secondary metabolites, and formation of physical barriers [108,109]. For example, the ERF gene from tomato is known to confer resistance to Pseudomonas syringae pv. tomato by activating genes encoding antifungal proteins and proteins involved in oxidative burst. Ethylene’s role in disease resistance is closely linked to its biosynthesis and signalling pathways. In terms of disease resistance, the glutathione metabolism pathway, which includes key enzymes like glutathione S-transferase (GST), plays an important role in response to ethylene stimulus [110].

In addition, we have added a new subsection in point 4 named: 4.4. Effect of ethylene on the organoleptic characteristics of fruits during ripening covering pages 13-14 and lines 654-682 with the following information:

4.4. ffect of ethylene on the organoleptic characteristics of fruits during ripening

Fruit ripening brings out the best taste and scent, which influences the fruit's flavour. Fruits go through biochemical changes when they ripen, such as colour breakdown, starch hydrolysis, sugar and acid metabolism, volatile production, and cell wall disintegration, all of which affect flavour. These modifications add to the typical aroma of ripe fruits as well as their sweet and sour taste. Furthermore, when fruit ripens, its texture improves, becoming crisper, juicier, or melting, it intensifies the taste release in the mouth. Ethylene gas regulates the ripening process and contributes to the softening and flavouring of fruit. Fruit ripening is an intricate process that involves a range of physiological and biochemical changes that have a big impact on the fruit's flavour [140–142].

The flavor changes associated with ethylene involve a network of genes, transcription factors, and metabolic pathways. Firstly, specific genes responsible for the synthesis of flavor compounds as genes encoding enzymes like lipoxygenases, hydroperoxide lyases, and alcohol dehydrogenases are involved in the formation of volatile compounds that contribute to fruit aroma [143]. Additionally, genes related to the biosynthesis of sugars, acids, and secondary metabolites like carotenoids and flavonoids are also ethylene-responsive and contribute to flavor [144]. Secondly, transcription factors such as the RIN (RIPENING INHIBITOR), NOR (NON-RIPENING), and CNR (COLORLESS NON-RIPENING) play crucial roles in the regulation of ripening-related genes. These factors can directly or indirectly influence the expression of genes involved in flavor compound biosynthesis [143]. For instance, the transcription factor HY5 has been shown to bind to the promoter of SWEET12c, a gene involved in sugar transport, thereby modulating sugar content and influencing sweetness in tomato fruit [144]. Finally, ethylene influences several metabolic pathways that are directly related to flavor development. The Lox pathway is responsible for the production of volatile compounds that contribute to aroma [145]. The glycolytic pathway and tricarboxylic acid (TCA) cycle are involved in sugar and acid metabolism, affecting sweetness and sourness [146]. Ethylene also affects the isoprenoid pathway, which includes the biosynthesis of carotenoids, contributing to both color and flavor [144].

-The depth of the article needs to be enhanced, latest research advances need to be introduced, such as research progress on cold plasma in ethylene scavenging.

We thank Reviewer 2 for their valuable feedback. In response to your suggestion, we have significantly expanded the depth of our article by incorporating the latest research advances related to cold plasma and other technologies in their roles in ethylene scavenging.

Specifically, we have created a new section within "Ethylene Scavengers by Catalytic Oxidation" and titled it "Cold Plasma and Other Technologies." Subsequently, we have added the following information, covering the pages 26-27 and lines 1253-1289:

5.3.6. Cold plasma and other technologies

Cold plasma technology has emerged as a promising method for ethylene scavenging, offering a non-thermal and eco-friendly approach to extend the shelf-life and maintain the quality of fresh produce. This promising and novel method stands out: 1) for being a non-thermal technology, which means it does not rely on heat to achieve its effects. This is particularly beneficial for ethylene scavenging as it avoids thermal damage to the produce, maintaining its nutritional and sensory properties; 2) as favouring microbial decontamination where cold plasma has shown effectiveness in microbial decontamination, enzyme denaturation, and pesticide degradation, which are all crucial for preserving the quality and safety of food products; 3) by its versatility and applications where cold plasma technology allows for its application in various forms, such as direct treatment, plasma-treated water, and functional coatings. This flexibility makes it suitable for a wide range of uses in the food industry [243]. In addition, cold plasma is considered an eco-friendly technology due to its low energy requirements and minimal environmental impact. It represents a sustainable option for ethylene scavenging and food preservation [244]. The potential of cold plasma technology in food processing is vast, with ongoing research exploring its scalability and commercial viability. Future advancements are expected to further enhance its effectiveness and applicability in the food industry [243].

Other novel advances and developments in ethylene scavenging beyond cold plasma technology are: 1) Nanotechnology where the incorporation of nanoparticles into polymer matrices has been shown to play a major role in reducing the permeability of gases and the absorption of ethylene. This approach enhances the effectiveness of packaging materials in controlling ethylene levels around fresh produce [104].For this reason nanotechnology is at the forefront of advancing ethylene scavenging techniques to maintain the quality and extend the shelf-life of fruits and vegetables. For example, nano-silica has been isolated from rice straw and used in a polyvinyl alcohol (PVA) formulation to coat paperboard, which showed improved barrier, mechanical, and surface properties, along with higher ethylene scavenging activity compared to uncoated paperboard [245]. Also, palladium encapsulated nanofibers have been developed for ethylene scavenging. Encapsulation of 1–2% PdCl2 in nanomats increased the ethylene scavenging capacity significantly, proving to be effective in the presence of fruits like sapota [246]. 2) Mechanochemical synthesis where recent studies have investigated the use of mechanochemically synthesized absorbents for ethylene removal. These materials have shown promising ethylene scavenging activity and offer a safer, innovative, and eco-friendlier approach to active packaging [247]. This process involves the use of mechanical force to induce chemical reactions, which can be used to synthesize metal–organic frameworks (MOFs) that are effective in ethylene scavenging [248].

Author Response File: Author Response.pdf