Novel Aspects of Regulation of Nitrogen Responses in the Tea Plant (Camellia sinensis (L.))
Round 1
Reviewer 1 Report
Dear Authors
I attach a file with my comments.
The manuscript needs substantial improvement. The development of the manuscript is not understood from the stated objectives. It contributes very poorly to the knowledge of nitrogen acquisition in the tea plant. It is necessary to incorporate more diagrams that help to understand what is novel in the work.
Best regards
Comments for author File: 
Comments.pdf
Author Response
Point 1: The manuscript needs substantial improvement.
Response 1: This review had been revised according to the comments made by the reviewers in the article. (in red)
Point 2: Conculsion: This is not conclusion, move to introduction
Response 2: Conculsion made the following modifications: There are many studies on the absorption and transfer of nitrogen in tea plants at present, as well as how nitrogen levels and nitrogen forms affect the growth and development of tea plants. However, there are few in-depth studies on how nitrogen regulates the physiological metabolism of tea plants as a whole and how nitrogen affects the genetic changes of tea plants. In the future, the use of omics technology to explore the effects of nitrogen on the whole tea plant (such as metabolomics, proteomics, epigenomics, long non-coding RNA analysis, etc.) is an important direction for tea plant scientific research. In addition, in-depth study of the effect of nitrogen on the key genes of tea plant by molecular techniques will also be an important part of revealing the mechanism of nitrogen affecting tea plant. Thirdly, the picking of tea trees is mainly based on new shoots, and nitrogen is the most needed nutrient element for tea trees, but the utilization efficiency of tea trees for nitrogen is very low. At present, the utilization efficiency of nitrogen is mainly improved by reasonable cultivation measures combined with fertilization methods in tea garden production. In the future, it will be an important research direction to study the nitrogen absorption and utilization of tea trees to excavate the related genes that improve the nitrogen utilization efficiency of tea trees and screen tea varieties with high nitrogen utilization efficiency. (in red)
Author Response File: 
Author Response.docx
Reviewer 2 Report
Tea (Camellia sinensis L.) is a perennial leaf-harvested crop infused from the tender leaves and buds, holds the empyrean position of the most manufactured and inexpensive beverage in the world. Nitrogen (N) is an indispensable element in biological systems available for plants in the form of nitrate and ammonium . Like most of the crops, N is responsible for the determination of quality components of tea as reported by several studies and this particular relationship is vital in the improvement of fertilizer application technology in tea production with the incorporation of integrated nutrient management which helps in maintaining or improving the tea quality.
Application of N fertilizer should increase productivity per unit area in commercial tea plantations with recommended rates ranging from 100 kg N ha−1 year−1 to 800 kg N ha−1 year−1 for green tea production. During the summer, the amount of N should be reduced per ha. New findings suggest that incorporation of N fertilizer could play an influential role in modifying the biochemical constituents of tea shoots, while also affecting the quality of certain tea products produced.
Several studies show through the results that a higher application of N fertilizers enriches the formation and accumulation of free amino acids while increasing or decreasing the concentration of polyphenols in tea shoots. Research results show that higher nitrogen accumulation significantly increased the concentration of caffeine, while it significantly decreased the concentration of soluble sugars in tea shoots, which is not good.
A number of key transcription factors are known to coordinate plant N responses. Rapid technolog-ical advances have given rise to innovative methods for genomic characterization, in-cluding metabolomics, epigenomics, and long non-coding RNA analysis, and these ap-proaches will no doubt lead to identification of new mechanisms by which various N forms influence the growth and chemical composition of tea plants. As more relevant genes and their functions are identified, this information can be used to sustain high tea yields and address environmental problems.
I believe that you should definitely look for solutions in optimizing the application of nitrogen fertilizers, use slow-release fertilizer granules, and certainly introduce the use of biostimulators based on humic acids until the new required methods are solved.
Your presentation of the problem of nitrogen fertilizer application is rather modestly described. Nitrogen application and uptake is a special case and depends on many parameters looking for new solutions is justified, but until then the problem should be solved with good technology and understanding of the soil - plant - climate relationship. This is not stated in your manuscript. Also, the review of the literature is mostly of an older date, because there are already findings in new scientific studies. Linguistic errors are visible in the work. I suggest the authors to rearrange the manuscript with new knowledge.
Author Response
Point 1: I suggest the authors to rearrange the manuscript.
Response 1: Nitrogen plays an important role in the growth and quality formation of tea plant. Reviewers suggested that nitrogen nutrition and fertilizer should be reviewed. In this review, we discuss the recent identified tea plant genes that have an essential role in the responses of different nitrogen forms in short-term period and the long-term nitrogen effect. In addition, we also pay special attention to how nitrogen regulates the physiological processes of growth and development of tea plants. Therefore, the review direction of this paper is inconsistent with the direction suggested by the reviewer.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors provided a reasonable amount of published studies focusing on the impact of different forms of nitrogen on the acquisition and utilization (efficiency) of tea plants. However, it is difficult for readers to learn the notion of review since the authors did not make a strong connection among performed studies.
It is hard for me to make my comments point by point because line numbers are missing in the manuscript. Here I make few comments:
1) Page 2: Authors need to be more specific on literature review, they sould express the results by fold change, percent reduction rather than using "higher" or "lower" terms.
2) Many time Nitrogen was introduced as a signaling molecule. I am wasking on what signal transduction, what responses . Auothors shold be more specific about function of Nitrogen as a signaling hormones.
3) Page 4 first paragraph: Reported studied must be more specific. what conditions, how long exposure, what culture systems?
4) Fig 1: The legend did not explained green rectangular, green rectangular is duplicated! Why green oval shape ballon (CsCILK23) is among CsNRT genes?
5) Page 9: GADs gene standing for glutamate decarboxylate not glutamte synthase as appeared in first papragraph.
6) Shelp et al. lab was center for GADs and GABA shunt studies in past 15 years, non of their work were cited in this review.
7) Fig 2: Green box was duplicated, the full name of genes must be written in the legend.
8) Conculsion has to deliver a stronger message for the readers.
Author Response
Point 1: Page 2: Authors need to be more specific on literature review, they sould express the results by fold change, percent reduction rather than using "higher" or "lower" terms.
Response 1: This part was modified as: Nonetheless, tea plant roots obtain most N from the soil as nitrate, which is at least partially reduced in the roots before transport to the leaves, and nitrate concentrations in tea leaves are thus quite low (< 0.06 g NO3- kg-1 DW) . (in red)
Point 2: Page 4 first paragraph: Reported studied must be more specific. what conditions, how long exposure, what culture systems?
Response 2: This part was modified as: A number of recent studies have also examined NRT genes in C. sinenesis. Expression of the primary nitrate response genes CsNRT1.1 and CsNRT1.2 was strongly induced as the tea plants were cultured with NO3- starvation for 10 days under the hydroponic system. (in red)
Point 3: Fig 1: The legend did not explained green rectangular, green rectangular is duplicated! Why green oval shape ballon (CsCILK23) is among CsNRT genes?
Response 3: The green box indicates a transcription factor; the light green dashed box indicates CsNRT genes family (CsNRTs). CsCILK23 does not belong to CsNRT genes. As shown in the figure1, the combination of CsCILK23 and CsNRT1 negatively regulates nitrate uptake.
Point 4: Page 9: GADs gene standing for glutamate decarboxylate not glutamte synthase as appeared in first papragraph.
Response 4: This article hads modified glutamte synthase to glutamate decarboxylate. (in red)
Point 5: Shelp et al. lab was center for GADs and GABA shunt studies in past 15 years, non of their work were cited in this review.
Response 5: This article had supplemented the research on GADs and GABA by Shelp et al. (in red)
Shelp, B. J.; Bozzo, G. G.; Zarei, A.; Simpson, J. P.; Trobacher, C. P.; Allan, W. L. Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. II. Integrated analysis. Botany 2012, 90, 781-793.
Point 6: Fig 2: Green box was duplicated, the full name of genes must be written in the legend.
Response 6: We carefully looked at the reviewers ' opinions. In Figure 2, the green box describes the physiological state of the tea plant, while the blue box describes the related genes. If the reviewer proposed the gene in the blue box, we made corresponding modifications in text. (in red)
Point 7: Conculsion has to deliver a stronger message for the readers.
Response 7: Conculsion made the following modifications: There are many studies on the absorption and transfer of nitrogen in tea plants at present, as well as how nitrogen levels and nitrogen forms affect the growth and development of tea plants. However, there are few in-depth studies on how nitrogen regulates the physiological metabolism of tea plants as a whole and how nitrogen affects the genetic changes of tea plants. In the future, the use of omics technology to explore the effects of nitrogen on the whole tea plant (such as metabolomics, proteomics, epigenomics, long non-coding RNA analysis, etc.) is an important direction for tea plant scientific research. In addition, in-depth study of the effect of nitrogen on the key genes of tea plant by molecular techniques will also be an important part of revealing the mechanism of nitrogen affecting tea plant. Thirdly, the picking of tea trees is mainly based on new shoots, and nitrogen is the most needed nutrient element for tea trees, but the utilization efficiency of tea trees for nitrogen is very low. At present, the utilization efficiency of nitrogen is mainly improved by reasonable cultivation measures combined with fertilization methods in tea garden production. In the future, it will be an important research direction to study the nitrogen absorption and utilization of tea trees to excavate the related genes that improve the nitrogen utilization efficiency of tea trees and screen tea varieties with high nitrogen utilization efficiency.(in red)
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear authors,
Thanks for taking the suggestions into consideration and making the appropriate changes. The manuscript has improved.
Regards
Reviewer 3 Report
Changes made by authors to address comments were accepted.
