Coordinated Effect of Ascorbate Biosynthesis and Recycling in Maize Seed Germination and Seedling Establishment under Low Temperature

Round 1

Reviewer 1 Report

Thank you for the paper submitted for review. It presents important results in regulating the response to low-temperature stress and thus may be an important resource for maize breeding.

Keywords: antioxidative enzymes; breeding; crop species; post-germinative growth; reactive oxygen species; ROS; stress conditions; Zea mays

Lines 9-11 - please add space before "(W.S)" and "(J.Z.)", etc.

Please add space before "°C", e.g., "10 °C"; "mM"

Please change "ml" for "mL" (Lines 118)

Lines 14, 42, 96 - please add "LT" in sentence "L/low temperature (LT) ..." and "NT" in "... normal temperature (NT) ..." (line 96)

Lines 45, 72, 74, 75, 78, 80, 179, 258, 262, 265, 338, 342, 401, 425, 433, 442 - please space before references number "damage[5, 6]."; "homeostasis[11]"; "Citrullus lanatus[12]"; "activity[13]."; "tobacco[14]."; "plants[15]."; "inbred[16]", etc.

Lines 58-59 - "... APX, glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR)." As previously, see lines 51-55.

Lines 64, 65 - "... monodehydroascorbate (MDHA) recycled..."; "... dehydroascorbate (DHA)." similar as in line 65 "photosystem I (PSI)" and lines 51-55.

Line 68 - ".. to oxidized glutathione (GSSG) that ..."

Line 103 - please add space between "7day"

Line 152 - "... mM AsA))."

Line 164 - "2 μg total RNA per ...". Using oligo(dT) or hexamer primers? It could be described in methods.

Line 166 -  Please add the name of the kit used for qPCR, reactions parameters, the final volume of reactions. Expression was calculated from delta Ct (please add the efficiency of reactions) or copy number of the gene (please add data about standard curve preparation, melt, reaction efficiency)

Lines 293, 299 - please add space between "JingX201(Figure 4B)"; "JingX201(Figure 4F and 4G)."

Line 298 - please write 1.2-fold higher, because 1-fold couldn't be higher, it's the same or similar as a control. Similarly, please complete the information in line 228. In line 344, please describe "4.x-fold higher ...". Similarity in 375 and 378 lines: 2.x-fold higher" or "4.x-fold higher", respectively. Lines 445 "2.x-fold", also.

Please change "kernels" in supplementary materials for "seed" as is used in the main manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This is an interesting manuscript and looks at a very important issue of cold temperatures in maize. It is well written, clear and easy to follow. The methods are robust and the results are clear however there are some minor points with the statistical analysis detailed below.

1. The abstract is clear but there are some minor typos and grammar needs looking at again including Line 12 latitude not latitudes and Line 17 conditions not condition, Line 17- these inbred what? Maybe lines? The term maize inbred is used many times in the manuscript and would be clearer as inbred lines/ accessions or inbred maize. This would better identify the exact type of plant material being used. There are several other grammar errors throughout the manuscript so I would suggest another thorough review of the grammar and language.
2. In the methods section what temperatures constituted low and normal temperatures. Where there any other confounding factors such as day length or rainfall? The public meteorological data is referenced but not given in the text. The data for temperature is clearly given in figure 1.A but some ranges would be helpful in the methods section. Methods for chlorophyl content and proline measurement have not been included in the methods section. Where whole seedlings used for the RNA extraction and how were they treated before extraction, for example where they snap frozen in liquid N2, kept on ice or placed in a buffer? At what growth stage in days after emergence where they sampled and was it the same for both lines?
3. In section 3.1 it would be useful to include the P values for the statistics carried out.
4. Figure 1 is hard to interpret. It is not clear what the error bars signify and if all the seeds germinated during the time period indicated in the coloured zones. Some information on exactly how many days on average (with P vales) between the two treatments would make it clearer.
5. Again for section 3.2 some P values should be included in the text for figures 2B-D to compare between treatments as well as between lines. A two way ANOVA would be more appropriate here than a one way for capturing between treatment variation.
6. The data in figure 4 should be compared statically using a two way ANOVA as treatment and line rather than treating the data as 4 separate lines in a one way ANOVA.
7. Again figure 5 would be better analysed using a two way ANOVA with treatment and line.
8. Figure S2 should be labelled as relative expression instead of relative transcripts level.
9. Figure S3 presents interesting results and would add to the manuscript if it was added into the main text. These results show the physiological differences caused by the treatment and complement the rest of the data well.
10. There is a lot of data here that would benefit not only from a two way ANOVA rather than a one way but also a principal component analysis. This would allow many off the different traits that have been measured to be combined to really pull out the underlying differences between the two treatments.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf