Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, Yahya et al., analyzed the effects of light and harvest maturity on midrib pinking and related metabolites in two Romaine lettuce cultivars with contrasting discolouration sensitivities. However, the conclusions drawn are not fully supported by the presented data.

The central premise involves enzymatic oxidation leading to pinking, primarily driven by Polyphenol Oxidase (PPO) and possibly Peroxidase (POD). It is a critical flaw that the activities of these enzymes were not measured. Reporting only substrate (phenolic acid) concentrations without the corresponding enzyme kinetics provides, at best, correlative data. It cannot establish causality or elucidate the purported biochemical pathways. This omission is fundamental and severely limits the paper's contribution to understanding the mechanism of pinking.
 

A striking and unexplained result is the increase in cinnamic acid during storage for Keona under L2M2 conditions (Fig 1A). This directly contradicts the described phenylpropanoid pathway logic, where cinnamic acid is the precursor expected to be depleted. The authors must provide a rigorous technical or biological explanation for this outlier. Its presence without comment suggests a lack of critical data scrutiny and calls into question the reliability of the entire metabolic dataset.

Is the sample size sufficient to be representative?

It is suggested to supplement the explanation on whether the red and blue light ratio of the LED light source may affect phenolic metabolism to enhance the completeness of the experimental design.

The author should further explain "why Icarus does not show any effect on color change under high light conditions, while Keona is significantly affected", and it is suggested to elaborate based on the differences in metabolic pathways among the varieties.

Author Response

 

RESPONSES TO SUGGESTION AND COMMENTS BY REVIEWERS
REVIEWER 1
No	Issues/comment/suggestion	Response/action taken
1	In this study, Yahya et al., analyzed the effects of light and harvest maturity on midrib pinking and related metabolites in two Romaine lettuce cultivars with contrasting discolouration sensitivities. However, the conclusions drawn are not fully supported by the presented data.	Two sentences have been reworded so that the conclusions drawn are in-line with the results recorded.
2	The central premise involves enzymatic oxidation leading to pinking, primarily driven by Polyphenol Oxidase (PPO) and possibly Peroxidase (POD). It is a critical flaw that the activities of these enzymes were not measured. Reporting only substrate (phenolic acid) concentrations without the corresponding enzyme kinetics provides, at best, correlative data. It cannot establish causality or elucidate the purported biochemical pathways. This omission is fundamental and severely limits the paper's contribution to understanding the mechanism of pinking.	We have carried out PPO assays earlier with leaves of different positions of the lettuce head. In the study, the leaves of Romaine lettuce were divided into 4 quarters (1st quarter – the oldest, and 4th quarters – the youngest.  The results obtained shows that younger leaves had a significantly higher activity of PPO. Some discussion amendment on activity of PPO seen in leaves of different age was inserted in the discussion (Lines 533-543)
3	A striking and unexplained result is the increase in cinnamic acid during storage for Keona under L2M2 conditions (Fig 1A). This directly contradicts the described phenylpropanoid pathway logic, where cinnamic acid is the precursor expected to be depleted. The authors must provide a rigorous technical or biological explanation for this outlier. Its presence without comment suggests a lack of critical data scrutiny and calls into question the reliability of the entire metabolic dataset.	Please see Lines 559-5721. We hope the explanation given appropriate and relevant.
4	Is the sample size sufficient to be representative?	This experiment was conducted under glasshouse environment in a closed hydroculture system. The plants were uniform. With two plants per plot and three biological replications, each point of data was derived from 6 plants. We think the sample size was representative. Furthermore, the analyses were run in three technical replicates.
5	It is suggested to supplement the explanation on whether the red and blue light ratio of the LED light source may affect phenolic metabolism to enhance the completeness of the experimental design.	Some notes were included (Lines 100-105).
6	The author should further explain "why Icarus does not show any effect on color change under high light conditions, while Keona is significantly affected", and it is suggested to elaborate based on the differences in metabolic pathways among the varieties.	Icarus contained a lower concentration of caffeic acid then Keona when grown under high light condition (Lines 582-584)

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Supplementary light intensity and maturity at harvest affect the midrib oxidative pinking and related metabolites of two cultivars of Romaine lettuce with contrasting discolouration sensitivities“ investigates how supplementary light intensity and harvest maturity influence pinking development, phenolic acids, and soluble sugars in two Romaine lettuce cultivars (Keona and Icarus). The authors combine postharvest assessment, targeted metabolite quantification, and multivariate analyses to explain cultivar-specific responses. The study addresses an important horticultural problem, and the topic is highly relevant to leafy vegetable production and postharvest biology.

The introduction provides a strong scientific context for enzymatic discolouration, pinking reactions, and the role of phenolics. The experimental design appropriately includes contrasting cultivars, two light regimes, and two maturity stages. Analytical methods for phenolic acids and sugars are clearly described. The results are presented in detail, with well-labelled figures and tables, and PCA adds interpretative value. The discussion demonstrates good integration of findings with existing literature, especially regarding phenylpropanoid metabolism .

However, some issues still need to be addressed:

1. Lines 30-78 (Introduction): There are several overlapping explanations of discolouration pathways, light effects, and maturity effects. Some repetition can be removed without losing scientific context. Shorten and reorganize the introduction for better fluency.
2. Lines 165-172: Although ANOVA and Tukey tests are stated, p-values and interaction effects are not always clearly reported in the figures or text. Provide more explicit statistical interpretation for complex interactions among cultivar- light – maturity
3. Lines 368-434: PCA patterns are well described, but their biological meaning is mostly lacking. For example, why the separation across PC1 for day-0 versus day-8 is expected, or how loading vectors reflect biochemical processes.
4. Figures 1 and 2: Phenolic and sugar concentration graphs use multiple letters for significance but lack consistent formatting and clearer labeling of axes (e.g., units, cultivar grouping).
5. Lines 519-524: The suggestion that Icarus’ low pinking may be linked to “higher physiological activity during cold storage” due to sugar use is interesting but not directly supported by experimental measurements. Avoid too speculative explanations in the Discussion section.
6. Lines 435-536: Although maturity is a key treatment factor, the discussion does not sufficiently compare M1 vs. M2 physiologically, especially regarding cell wall structure, enzymatic activity, or tissue susceptibility.
7. Consider adding a schematic model summarizing how light intensity, maturity, and phenolics jointly affect pinking.
8. Add a paragraph discussing limitations, such as absence of enzyme activity measurements (PPO, PAL) that could further support conclusions.

Author Response

REVIEWER 2
1	Lines 30-78 (Introduction): There are several overlapping explanations of discolouration pathways, light effects, and maturity effects. Some repetition can be removed without losing scientific context. Shorten and reorganize the introduction for better fluency.	Some overlapping sentences were deleted.
2	Lines 165-172: Although ANOVA and Tukey tests are stated, p-values and interaction effects are not always clearly reported in the figures or text. Provide more explicit statistical interpretation for complex interactions among cultivar- light – maturity	Some statements in the results section has been improved to increase clarity.
3	Lines 368-434: PCA patterns are well described, but their biological meaning is mostly lacking. For example, why the separation across PC1 for day-0 versus day-8 is expected, or how loading vectors reflect biochemical processes.	Some relevant explanation was given (Lines 617-624).
4	Figures 1 and 2: Phenolic and sugar concentration graphs use multiple letters for significance but lack consistent formatting and clearer labelling of axes (e.g., units, cultivar grouping).	We have double checked on these matters and we feel that the labelling all are appropriate. The unit for phenolic acids and sugars are different.
5	Lines 519-524: The suggestion that Icarus’ low pinking may be linked to “higher physiological activity during cold storage” due to sugar use is interesting but not directly supported by experimental measurements. Avoid too speculative explanations in the Discussion section.	We totally agree with you. The statement was too speculative. We have deleted the sentence.
6	Lines 435-536: Although maturity is a key treatment factor, the discussion does not sufficiently compare M1 vs. M2 physiologically, especially regarding cell wall structure, enzymatic activity, or tissue susceptibility.	Some relevant explanation was included in the discussion (Lines 601-604.
7	Consider adding a schematic model summarizing how light intensity, maturity, and phenolics jointly affect pinking.	A schematic diagram is shown as Figure 5 as suggested (Line 655/656). Thank you very much for the kind suggestions.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version addresses the main issues raised in the first review round. The manuscript in its present form is acceptable for publication.

Author Response

Dear Reviewer 2,

I have responded to the issues raised by the head editor as reflected in the text. 

Thank you for reviewing my manuscript, I greatly appreciate it.