De novo Characterization of the Platycladus orientalis Transcriptome and Analysis of Photosynthesis-Related Genes during Aging
, Xiamei Yao 3, Shuo Tang 4, Xiulian Zhao 1, Nan Deng 1, Shengqing Shi 1, Jianfeng Liu 1 and Zeping Jiang 1,5,*Round 1
Reviewer 1 Report
The review of manuscript titled "De novo characterization of the Platycladus orientalis transcriptome and analysis of photosynthesis- and senescence-related genes during aging" by Ermei Chang, Jin Zhang, Nan Deng, Shengqing Shi, Jianfeng Liu, Xiulian Zhao, Zeping Jiang and Xiamei Yao.
The manuscript by Zeping Jiang, Xiamei Yao (corresponding authors) and others aimed at investigating the relationship between age and growth rate of Platycladus orientalis at the transcriptome and physiological level. The authors used biochemical analysis and RNA-sequencing technology for functional genomics analysis to accomplish the purpose of the research. The results of experiments are interesting and touches on a rarely studied range of plant senescence process. They also show molecular basis for future research of senescence process of P. orientalis and other tree species. The manuscript however contains significant deficiencies which require attention.
The Abstract and Introduction sections of manuscript are generally well written. The abstract contains essential results and the most important conclusions. The aim and hypothesis of the work are well described. However the Introduction section contains several problems. Several times the authors use “carotenol” instead of “carotenoids” expression. I think It should be corrected. On the next pages, in the Discussion section of manuscript, they used the proper form of this expression (for example lines: 342, 343, 344). I also think that the sentence in lines 56-58 (“However the relative contributions …”) should be corrected. There are publications about chlorophyll and carotenoids metabolism (degradation) during the senescence process in woody plants, for example:
RIVAS F, FORNES F, and AGUSTÍ M. 2008. Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in citrus leaves. Environmental and Experimental Botany 64: 256–263.
RIVAS F, FORNES F, RODRIGO MJ, ZACARÍAS L, and AGUSTÍ M. 2011. Changes in carotenoids and ABA content in citrus leaves in response to girdling. Scientia Horticulturae 127: 482–487.
YOUNG AJ. 1991. The photoprotective role of carotenoids in higher plants. Physiologia Plantarum 83: 702–708.
My doubts are raised by the description of the age of P. orientalis trees. The data presented are very inaccurate. For example, 1700 ±500-years-old means that these individuals can be 1200 or 2200 years old. May be it could be presented in a different way. I also wonder what was the genetic relationships among individuals of P. orientalis which were used in this research.
The authors write that they determined total protein contents, however in referenced publication (27) soluble protein contents was determined. In the Result section in one place authors write “soluble protein” (line 160) and then “total protein” (line 167 and in Figure 2). It should be corrected in these lines and in figures 2 and 10 – use soluble protein or total soluble protein.
The sentence “Components of stilbenes” (line 106) is incomplete I think. Please correct.
Results
In Figure 2, 8 and 10 there are no description of the error bars. Are their standard deviation or standard error bars. Please add this information to the captions under these figures.
Line 363-401: This part of Discussion section contains many statements about many different reactions and metabolic pathways (genes, proteins). It seems that this fragment of the Discussion section should be remodeled and focused more specifically on a specific group or several gene groups that determine the occurrence of certain metabolic pathways associated with senescing.
Line 105: the reference Breeze et al. is missing in the reference list.
Author Response
Responses to comments from Reviewer #1:
Firstly, thank you very much for your comments and suggestions!
Comments and Suggestions for Authors
The review of manuscript titled "De novo characterization of the Platycladus orientalis transcriptome and analysis of photosynthesis- and senescence-related genes during aging" by Ermei Chang, Jin Zhang, Nan Deng, Shengqing Shi, Jianfeng Liu, Xiulian Zhao, Zeping Jiang and Xiamei Yao.
(1) The manuscript by Zeping Jiang, Xiamei Yao (corresponding authors) and others aimed at investigating the relationship between age and growth rate of Platycladus orientalis at the transcriptome and physiological level. The authors used biochemical analysis and RNA-sequencing technology for functional genomics analysis to accomplish the purpose of the research. The results of experiments are interesting and touches on a rarely studied range of plant senescence process. They also show molecular basis for future research of senescence process of P. orientalis and other tree species. The manuscript however contains significant deficiencies which require attention.
Response: Thank you very much for your review. We have revised the manuscript about figures, results, discussion, and references.
(2) Abstract and Introduction
The Abstract and Introduction sections of manuscript are generally well written. The abstract contains essential results and the most important conclusions. The aim and hypothesis of the work are well described. However the Introduction section contains several problems. Several times the authors use “carotenol” instead of “carotenoids” expression. I think It should be corrected. On the next pages, in the Discussion section of manuscript, they used the proper form of this expression (for example lines: 342, 343, 344).
Response: Thanks for your careful review. We have revised that “carotenol” instead of “carotenoids” in line 55, 56, 102, and 314.
(3) I also think that the sentence in lines 56-58 (“However the relative contributions …”) should be corrected. There are publications about chlorophyll and carotenoids metabolism (degradation) during the senescence process in woody plants, for example:
RIVAS F, FORNES F, and AGUSTÍ M. 2008. Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in citrus leaves. Environmental and Experimental Botany 64: 256–263.
RIVAS F, FORNES F, RODRIGO MJ, ZACARÍAS L, and AGUSTÍ M. 2011. Changes in carotenoids and ABA content in citrus leaves in response to girdling. Scientia Horticulturae 127: 482–487.
YOUNG AJ. 1991. The photoprotective role of carotenoids in higher plants. Physiologia Plantarum 83: 702–708.
Response: We have revised the sentence in line 55-57.
“However, more researches of the relative contributions of the chlorophyll and carotenoids contents to oxidative stress associated with aging in woody perennials are needed [19].”
(4) Materials and Methods
My doubts are raised by the description of the age of P. orientalis trees. The data presented are very inaccurate. For example, 1700 ±500-years-old means that these individuals can be 1200 or 2200 years old. May be it could be presented in a different way. I also wonder what was the genetic relationships among individuals of P. orientalis which were used in this research.
Response: Tree age was based on historical records for Zhongshan Park and the breast-height diameter of selected trees. We take the minimum age as the age of ancient trees “20 ± 3-, 300 ± 100-, 1,000 ± 300-, and 1,700 ± 500-year-old” to “about 20-, 200-, 700-, and 1,100-year-old” in line 78, 86.
Zhongshan park is built as a temple garden in Liao dynasty (AD 907-AD 1125), which 587 P. orientalis are more than one hundred years old. There are 304 ancient P. orientalis over 300 years old which were planted artificially in different dynasties. Additionally, all selected trees were located close to each other and were exposed to similar environmental conditions.
(5) The authors write that they determined total protein contents, however in referenced publication (27) soluble protein contents was determined. In the Result section in one place authors write “soluble protein” (line 160) and then “total protein” (line 167 and in Figure 2). It should be corrected in these lines and in figures 2 and 10 – use soluble protein or total soluble protein.
Response: The soluble protein content was determined in this manuscript. We have revised “total protein” in figures 2 and 11 and line 100, 176.
(6) The sentence “Components of stilbenes” (line 106) is incomplete I think. Please correct.
Response: We deleted “Components of stilbenes” in line 106:
(7) Results
In Figure 2, 8 and 10 there are no description of the error bars. Are their standard deviation or standard error bars. Please add this information to the captions under these figures.
Response: Thanks for your careful review. I have added the information of standard deviation bars to the captions in figures 2, 8 and 10.
(8) Discussion The authors write that MDA content is negatively correlated with SOD and POD activities (line 336 and 345). However there is no correlation analysis in this manuscript. The correlation should be calculated, particularly in the case of ROS and photosynthesis relationship in (line 345). The authors write that this correlation “has not been clearly demonstrated” in senescing individuals of P. orientalis. Correlation coefficients should prove this. If the authors’ statement is true, what are the reasons for this, since chlorophyll is a commonly used senescing marker in both conifers and deciduous plants.
Response: We have revised this sentence “In this study, analysis of physiology for senescence showed that the MDA contents increases, whereas decreases in chlorophyll levels, SOD and POD activity levels in ancient P. orientalis leaves (Figure 11), which is consistent with an earlier study of the senescent leaves of ancient pine.” in line 345.
(9) The sentence in lines 360-362 does not really fit in this place. It should be moved to the conclusion section of the manuscript.
Response: As you suggested, we have moved the sentence in lines 360-362 to the conclusion section in lines 403-404.
Line 363-401: This part of Discussion section contains many statements about many different reactions and metabolic pathways (genes, proteins). It seems that this fragment of the Discussion section should be remodeled and focused more specifically on a specific group or several gene groups that determine the occurrence of certain metabolic pathways associated with senescing.
Response: As you suggested, we have revised the discussion section, which focused on photosynthesis and oxidative phosphorylation pathways.
(10) In several places in Materials and Methods section of manuscript the citing of references (in the text) are incorrect (it does not agree with the references list – please check and correct it. (It also applies to citations in other sections of the manuscript).
Response: Thanks for your careful review. I missed a reference in introduction. We have revised references list.
28. Heath, R. L.; Packer, L., Photoperoxidation in isolated chloroplasts : I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 1968, 125, 189-198.
(11) Line 105: the reference Breeze et al. is missing in the reference list.
Response: Thanks for your careful review. We have revised “Breeze” instead of “Emily”, and “references” section.
29. Breeze, E.; Harrison, E.; McHattie, S. H.; Hughes, L.; Hickman, H.; Hill, C.; Kiddle, Steven.; Kim, Y.; Penfold, C. A.; Jenkins, D., High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. Plant Cell 2011, 23, 873-94.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In the work entitled “De novo characterization of the Platycladus orientalis transcriptome and analysis of photosynthesis- and senescence-related genes during aging” the authors perform physiology and transcriptomic analysis during senescence of P. orientalis. For that, the authors used trees of four distinct ages.
In general, the manuscript is well organized and the experiments are well designed and the conclusion are in concordance with the results. Pictures are of good quality but in general, the legends need further work.
Major points to consider:
- Page 8, line 236 – It would be interesting to have the list of these 17 genes. It could be included as supplemental data.
- Figure 11 – In this schematic representation of tree aging mechanisms, the authors indicate that carotenoids increase with age but according to the values obtained from leaves and the graph in Fig. 10 b, the levels of carotenoids are higher in 20 years old trees when compared with 300 and 1000 years old trees. I don´t think it´s accurate saying that carotenoids increase with aging.
- Page 13, line 342 – the statement made in this sentence meets the problem I addressed just above. Carotenoid values are higher in 20 and 100 years old trees than in older trees.
Minor points
abstract
- Page 1, line 18 – “We analyzed” instead of “we analysis”.
- Page 1, line 19 – Add “and” after senescence and remove “,”.
- Page 1, line 30 – In addition. Not Inaddition
- Page 1, line 32 – Including not include
Introduction
- Page 2, line 53 – Remove “the” in the beginning of the sentence.
Results
- Page 6, Figure 3 legend – Please indicate what letters A to E stand for.
- Page 9, Figure 6 legend – The letters attributed to each picture are misplaced in the legend. (A) is the heat map, description that is missing.
- Page 10, line 285 – Please add at the end of this sentence something like; “in a linear fashion towards older trees”.
- Figure 9 – Please add in the x-axis the tree´s age samples.
- Page 12, line 330 – Replace “in” by “of”.
Discussion
- Page 14, line 402 – Please add full stop after “P orientalis leaves”.
- Page 14, line 412 – In addition
- Page 14, line 413 – Please write “shows that” instead of “contains”.
Comments for author File: 
Comments.pdf
Author Response
Responses to comments from Reviewer #2:
Firstly, thank you very much for your comments and suggestions!
In the work entitled “De novo characterization of the Platycladus orientalis transcriptome and analysis of photosynthesis- and senescence-related genes during aging” the authors perform physiology and transcriptomic analysis during senescence of P. orientalis. For that, the authors used trees of four distinct ages.
In generahese 17 l, the manuscript is well organized and the experiments are well designed and the conclusion are in concordance with the results. Pictures are of good quality but in general, the legends need further work.
Response: Thank you very much for your review.
Major points to consider:
(1) Page 8, line 236 – It would be interesting to have the list of these 17 genes. It could be included as supplemental data.
Response: As you suggested, we listed the 17 genes in Table S3 in line 222-223.
(2) Figure 11 – In this schematic representation of tree aging mechanisms, the authors indicate that carotenoids increase with age but according to the values obtained from leaves and the graph in Fig. 10 b, the levels of carotenoids are higher in 20 years old trees when compared with 300 and 1000 years old trees. I don´t think it´s accurate saying that carotenoids increase with aging.
Response: As you suggested, we added the sentence “The carotenoids were highly accumulated in 20-year-old samples and had lowest concentration in 200-year-old samples.” in line 334-335.
(3) Page 13, line 342 – the statement made in this sentence meets the problem I addressed just above. Carotenoid values are higher in 20 and 1000 years old trees than in older trees.
Response: We revised “In contrast” to “In three ages of ancient trees” in line 366.
Minor points
abstract
(4) Page 1, line 18 – “We analyzed” instead of “we analysis”.
Response: Thanks for your careful review. Revised. Line 20.
(5) Page 1, line 19 – Add “and” after senescence and remove “,”.
Response: Revised. Line 396.
(6) Page 1, line 30 – In addition. Not Inaddition
Response: Revised. Line 30.
(7) Page 1, line 32 – Including not include
Response: Revised. Line 32.
Introduction
(8) Page 2, line 53 – Remove “the” in the beginning of the sentence.
Response: As you suggested, We removed “the” in the beginning of the sentence.
Results
(9) Page 6, Figure 5 legend – Please indicate what letters A to E stand for.
Response: As you suggested, we added “(A) Cellular processes, (B) Environmental information processing, (C) Genetic information processing, (D) Metabolism, and (E) Organismal systems.” in Figure 5 legend.
(10) Page 9, Figure 6 legend – The letters attributed to each picture are misplaced in the legend. (A) is the heat map, description that is missing.
Response: Thanks for your careful review, We revised the Figure 6 legend:
(11) Page 10, line 285 – Please add at the end of this sentence something like; “in a linear fashion towards older trees”.
Response: Revised. Line 281.
(12) Figure 9 – Please add in the x-axis the tree´s age samples.
Response: We added the x-axis of the tree´s age samples in Figure 9.
(13) Page 12, line 330 – Replace “in” by “of”.
Response: Revised. Line 332.
Discussion
(14) Page 14, line 402 – Please add full stop after “P orientalis leaves”.
Response: Revised. Line 392.
(15) Page 14, line 412 – In addition
Response: Revised. Line 398.
(16) Page 14, line 413 – Please write “shows that” instead of “contains”.
Response: Revised. Line 398.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The manuscript has been significantly improved. The Authors have clarified and change some sentences or parts of the previous version of the manuscript. The Results and Discussion sections of manuscript have been enriched with fragments more accurately describing the changes in senescing leaves of Platycladus orientalis. The figures were also changed as suggested in the first review which greatly influenced the level of the manuscript.
