Integrated Phenotypic Physiology and Transcriptome Analysis Revealed the Molecular Genetic Basis of Anthocyanin Accumulation in Purple Pak-Choi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study explores the genetic basis of anthocyanin accumulation in purple Pak-choi, a vegetable known for its high content and dual ornamental and health benefits. By comparing two Pak-choi lines, 'PQC' and 'HYYTC', the researchers found that 'PQC' had significantly more anthocyanins than 'HYYTC', but less chlorophyll. They identified key structural and regulatory genes involved in anthocyanin synthesis, with BrC4H3, BrF3H1, and BrCHS1 emerging as core genes. However, in my opinion, the work should be refined.

On page 15, the text is a bit out of sync.

Figure 3 has a very strange scale, I understand the cutoff, but after it, the x-axis is off. What is also interesting is the lack of standard deviation in the 3rd bar.

Didn't the authors wonder about the different chlorophyll content? The Materials and Methods describe that all dye measurements are spectrophotometric, if so - why wasn't the whole trinity measured? Including carotenoids? The ratio of chlorophyll a to b would also be useful.

According to Figure 2, adult green and purple plants are quite different from each other - and probably not only in the shape of the leaves but also in plant weight (there is also no information about this) - why was this stage omitted in transcription studies? Since, as noted in the introduction, leaves are often coloured differently in response to environmental factors, it is worth checking what transcription factors are responsible for regulating the anthocyanin synthesis pathway in adult individuals. This way the work would be more interesting.

The discussion lacks more detailed information regarding the groups of genes negatively regulated in the purple variety. Maybe they are related to a different shape - or to the eye (photos are not to scale) smaller growth of the plant.

The discussion mentions the observation of appropriate genes in other purple plants - however, there is no information on what stage of ontogeny the research with which the authors compare their results was carried out.

Overall, the work is quite detailed but requires several corrections, after which I think it may be of interest to readers.

Author Response

Thank you very much for taking the time to review this manuscript. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Line 46. I guess the correct abbreviation for early biosynthetic genes is EBGs, no EDGs.

Line 91. the largest leaf free from disease and insect infestation was selected and placed in the refrigerator at 4 ° C for subsequent test. (If samples were kept at 4ºC for more that two days, physiological conditions of the leaves may change drastically. Check this information to make it clear).

Line 94. triangular bottles, (???? they must be Erlenmeyer flasks, if not specify the kind and capacity of used bottles).

Lines 74-78. Starting Materials and Methods, authors indicated that the characters P1, P2, T1 etc., would be used for cultivars and growing stages. They were used in Fig. 1; however, they were not used in Figs. 2, 3 and 4 but used again in the other figures. It is recommended to homogenize the description of the material all along the manuscript. 

Lines 247-250. Although the expression of the ligase gene BraA05002651 (Br4CL4), which is used to synthesize 4-coumaryl CoA, is down-regulated in purple materials, BraA05001886 (Br4CL1), BraA05002646(Br4CL2)，BraA07003006 (Br4CL3) were expressed normally in the two material. Lines 308-312 In addition, 784 genes were newly identified that may be involved in anthocyanin biosynthesis. Such as BraA04002675 (BrPAL1), BraA09004715 (BrPAL2.1), BraA02000559 (BrCHS3), BraA05001212 (Peroxidase 19), BraA10000387 (Peroxidase 3), BraA10002815 (Cytochrome P450) and BraA07003905 (BrCOMT), these genes were up-regulated in the two comparison groups (G2 and G3).  Line 356. This indicates that the Br4CL4 gene is not the rate-limiting enzyme gene for anthocyanin accumulation in purple materials.

The number of members of each gene family must be considered to have an overall idea of the metabolism. The fact that only one 4CL(4) was down regulated, not necessarily means that this biochemical reaction is affected having other three 4CL (1,2,3) genes functioning. In this sense it seems necessary to know how many PAL, as well as the other flavonoid and lignin biosynthetic genes are in the two plants and mainly which metabolic role are each one involved in. So, Table 3 maybe quite biased to upregulated genes, although the indicated in the table may not be related to the anthocyanin metabolism, e.g. BrPAL2.2 may be related mainly to lignin biosynthesis to allow plants to grow in the assayed stages, but not to anthocyanin metabolism. Also, peroxidase, cytochrome P250 and COMT are involved mainly in lignin than in anthocyanin biosynthesis. This is the reason to consider metabolons and the recommended references. 

 

Line 227. BraA090028000035 is wrong, must be BraA09002835

 

In Table 4, What means that there are not any NAC transcripts detected:

BraA09002226	0	0	0	28.04	24.80	28.55	down	down	down	NAC69
BraA03000407	0	0	0	5.17	5.02	5.83	down	down	down	NAC82

It is intriguing why there is not any expression of NAC genes in P1-T1, P1-T2, P1-T3, which is one of the largest families of transcriptional regulators in plants. Considering that these play important roles in various biological processes, including in the regulation of flower and leaf development, secondary cell wall thickening, protein and lipid metabolism pathways, leaf senescence and fruit development, lateral root formation and seed germination, it could be expected that at least some transcripts may be detected in the indicated stages. Comments and comparison with appropriate references would be desirable.

 

Author Response

Thank you very much for taking the time to review this manuscript. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank You for the changes in the text, especially for improving the chart and adding a fragment to the discussion. 

Please add information on line 382 that the expression found was also in seedlings at the same stage of plant development.

After minor corrections, the manuscript is ready for publication.

Author Response

 Thanks for your positive comments and valuable suggestions to improve the quality of our manuscript.

Comments 1：Please add information on line 382 that the expression found was also in seedlings at the same stage of plant development.

Response 1: Thank you for pointing this out. We have added the stage of plant development on line 382.

Thank you again for your comments.

Reviewer 2 Report

Comments and Suggestions for Authors

I accept the explanations on the points indicated and with that the manuscript is accepted.

Author Response

 Thank you again for your positive comments and valuable suggestions to improve the quality of our manuscript.