MdSGR2 Negatively Regulates Chlorophyll Degradation in Apple

Round 1

Reviewer 1 Report

The manuscript by Beveridge and co-authors deals with the mechanism of chlorophyll degradation in mature green and yellow apple (Malus domestica Borkh.) peels. Based on the transcriptomic analysis, the authors identified the DEGs associated with chlorophyll degradation, including MdSGR2, MdNYC1 and MdHCAR. The results obtained are important for understanding the regulatory mechanism governing apple fruit color, which is important for the marketability of the fruit. Overall, the scientific background of the manuscript is good, but there are some points that need to be improved.

Abstract: Please detail the description of the object.

Introduction:

“Apple fruit emerges from the interplay of chlorophyll, carotenoid, anthocyanin, and flavonoid distribution in specific proportions”. Do you mean color of the apple fruit?

“As a result, consumers are largely reluctant to buy green, low anthocyanin or carotenoid accumulation apples”. Green color does not mean the low content of carotenoids. The yellow color of carotenoids is masked by the presence of chlorophyll and manifests itself when chlorophylls are destroyed. However, this is only a visual manifestation of the color.

“Chlorophyll degradation products have antioxidant effects and can maintain cell activity [7]”. I did not find this statement in [7]. But this is an important issue considering the nutritional value of apple fruit. Could you discuss it in more detail and provide further references?

Methods (2.3): Provide a reference to the method and formulas used to determine chlorophyll content. Ct - is Chl?

Methods (2.4): “…differentially expressed genes (DEGs) based on the criteria: |log2 (fold-change) | > 1”. For a significant difference, it should be at least 1.5.

Figure 1: What does Y, G, L*, a*, b*, h mean? It should be encoded in the caption.

Figure 2: The captions are very small. They are difficult to read. (B) Venn diagram of DEGs? I see the number of DEGs. (D) GO enrichment analysis of DEGs. What do BP, CC and MF mean?

Figure 3. Are the error bars the standard deviation or the error of the mean? Do the different lowercase letters indicate significant differences? Axis Y - left and right: please indicate where the relative expression of qRT-PCR and RNA-seq are?

Results (3.5): “To validate the accuracy of the RNA-seq findings, nine DEGs associated with chlorophyll degradation were randomly chosen from the transcriptomic data for qRT-PCR verification”. So, out of several thousand DEGs, you randomly selected the 6 genes from the chlorophyll degradation pathway? This is a really nice surprise.

Discussion: The transcriptome data is poorly described. I would expect the GO and KEGG enrichment analysis to be detailed. Is there any difference between the green and yellow apple peels other than pigment content?

Author Response

请参阅附件。

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper may be of interest for apple horticulturalists or breeders as they identify a gene involved in chlorophyl degradation that encodes a significative agronomic trait (green peel). The approach to identify is correct, but the way the results are presented requires major improvement.

Figure 1: Please describe the parameters in the Y axis in pannels B, C, D and E, Ehich is the number of replicates for each bar? Which statistical analisys has been perfomed in the samples? Please include this information in the legend.

Figure 2A: enlarge the lettering. 

Figure 3: I do not really understand what the auhors are representing. They are comparing the DEG and the qRT results in the same figure? this is not correct, unless ypu explain how have you normalized data from very different sources. Usually q-RT PCR using the data of calculation only allows relative expression, given that you cannot have absolute data as each pair of primers is different. So you normaliza on of the values to 1. But none of the values is 1. In addition authors only used un pair of primers as control, this usually gives unreliable data as the consesus is two use at least two pairs of control primers.

Figure 4B: nothing can be seen. Please enlarge and enhance the resolution

Figure 5: Include the n number for each bar, and the statistical analysys performed. For pannels D, G and J I have the same criticism than before. Only one set of primers has been used as a control and the values are not normalized, so the calculations do not seem to be correct. 

English needs some revision.

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The paper has a serious flaw. Student's t test represents a comparison with a control in a pair wise manner. In the figures you see different "a", "b" and "c" upon the different bars. This cannot be calculated with a student's test but in a two-way anova or similar.

Paper cannot be accepted with this major mistake.

Author Response

please see the attachment

Author Response File: Author Response.pdf