Transcriptomic Analysis of Three Differentially Senescing Maize (Zea mays L.) Inbred Lines upon Heat Stress

Round 1

Reviewer 1 Report

RNA-Seq requires at least three to six biological replicates per treatment

For most part, it was fine

Author Response

Reply to reviewer 1

Thank you very much for your time and effort in reviewing our manuscript, and particularly for your comment and suggestion. We have improved our manuscript according to your suggestion. As for the number of RNA-seq biological replicates, certainly it is statistically sound to use three or more biological replicates. We did consult an expert in bioinformatics before conducting RNA-seq experiment, and were informed of that as long as the repeatability between the two biological replicates is high, data are reliable. We combined tissues collected from five maize plants to make a biological replicate, and before sent to sequencing, all samples were examined for the expressions of marker genes, i.e. CGGs and SAGs, to demonstrate that they are representative of particular treatments. Indeed, our spearman correlation coefficient analysis and principal component analysis showed that the sequencing quality/sample repeatability of the two biological replicates were very good/high, confirming that our data are valid.

Reviewer 2 Report

The aim of this work was to gain a better understanding of the molecular mechanisms underlying leaf senescence in maize under heat stress, in particular, during the seedling stage. The authors screened the transcriptome profile of three inbred maize lines (PH4CV, B73, and SH19B) with different senescing phenotypes under heat stress, finding that differentially expressed genes (DEGs) in response to heat stress, reactive oxygen species (ROS), and photosynthesis were enriched in all three lines. The study also revealed that silencing the gene ZmbHLH51 inhibited heat stress-induced senescence. This research is a very good contribution to the better understanding of the molecular mechanisms involved in heat stress-induced leaf senescence in maize during the seedling stage, and the silencing data point to potential avenues for applications to prevent premature heat stress triggered senescence in maize, a key food crop worldwide, particularly relevant considering the pressures posed on food production by a warming climate. My sole recommendation is to proofread the text for minor inconsistencies, such as the use or the omission of hyphenation in some words (e.g. “senescence-induced”, line 213, etc.).

Author Response

Reply to reviewer 2

Thank you very much for your time and effort in reviewing our manuscript, and particularly for your encouragement.  We have amended the manuscript as suggested. In the revised version, changes to the previous manuscript are highlighted.