The Key Regulators and Metabolic Intermediates of Lignin Response to Low Temperatures Revealed by Transcript and Targeted Metabolic Profiling Analysis in Poplar

Round 1

Reviewer 1 Report

In this manuscript, Zhao et al. studied the impact of low temperatures on lignin biosynthesis in poplar. Through metabolic profiling, the authors discovered that the amounts of several metabolic intermediates of lignin were reduced by low temperatures. Transcriptomic analyses also identified that the expression of several genes related to lignin contents was altered by cold stimulation. The effect of cold stress on lignin biosynthesis has been described previously by other groups but has never been systemically studied. Therefore, the current study has the potential to unravel unknown mechanisms by which low temperatures affect lignin contents in plants. However, I found some flaws in the results and I don’t agree with some of the conclusions in the manuscript. See my comments below.

Major point:

1. Figure 1: I don’t see statistically significant differences in lignin contents between day 0 and day 3 samples at 4°C. Even though the difference in lignin contents between samples at 4°C and 25°C on day 3 was statistically significant, it was quite small. These results suggested that cold temperatures do not have a dramatic effect on lignin contents in poplar. Actually, some studies have shown similar results in other species (e.g., Ji et al., PLoS Genetics, 2015). However, some other studies also showed that although no changes in the levels of lignin or its precursors were observed in plants maintained at low temperatures, there was an increase in related enzyme activities as well as an increase in gene expression (reviewed in Moura et al., JIPB, 2010). The authors should explain these in the Results and Discussion sections.

2. Figure 2: A and B are basically the same data. Is it necessary to show both? The statistical analyses were missing in Figure 2B. Besides, the description in the Results section did not match the data shown in Figure 2. For example, in Paragraph 1 of 3.2, the authors described “The other compounds, including sinapinaldehyde, p-coumaraldehyde, l- phenylalanine, caffeate, ferulic acid, coniferaldehyde, and cinnamic acid, that were present at comparatively higher levels were induced by low-temperature treatment at different time periods”, which is not true. Some of these compounds were not induced by low-temperature treatment. In Paragraph 2 of 3.2, the authors described “The overlapping results among the five pairwise comparisons showed that caffeate and coniferaldehyde were the only two compounds that consistently decreased following low-temperature induction”, which is also not accurate. It is difficult to make this conclusion without statistical analysis. Two other compounds, p-coumaraldehyde and ferulic acid, might also show decreased levels following low-temperature induction.

3. Figure 3: I don’t agree with some of the authors’ conclusions. In the last paragraph of 3.3, the authors stated that “With the extension in the time of low-temperature treatment, the number of genes that varied increased, indicating that the poplar tissue culture plantlets could resist cold stimulation by expressing more genes.” A higher number of genes does not necessarily mean plants could ‘resist’ cold stimulation because the changes in gene expression could be either passive or active. In the same paragraph, the authors also concluded that “It is apparent that the level of expression of most of the genes changed between -5≤log2 fold change≤5, while the level of expression of many genes changed significantly, suggesting that they may play an important role in the resistance of poplar to cold temperature stress.” Similarly, these changes could be either passive or active, and they are not necessarily involved in cold resistance.

4. Results 3.5, Paragraph 1: the authors described “The genes that positively regulated the intermediates in lignin metabolism are shown in subclasses 1 and 2, and the expression of these genes generally decreased.” There were thousands of genes in each subclass. Please provide the data showing all the genes that positively regulated the intermediates in lignin metabolism to support the statement.

5. Figure 4B: I suggest the authors use a different color range. Please set 1 as the midpoint. The current color scheme is a little misleading because values lower than 1 were also displayed in a warm color.

Minor point:

1. It would be much easier for the readers to digest the data if the authors include a biosynthetic pathway of lignin in the manuscript, indicating the key metabolites, enzymes, and potential transcription factors regulating the expression of these enzymes.

2. Page 2, Paragraph 1: the order of the metabolites used for the polymerization to form monomers of syringyl, guaiacyl, and p-hydroxyphenyl is wrong. Please double check and correct it.

3. Page 2, Paragraph 2: full name of ERF8 should be provided.

4. Page 2, Paragraph 2: what type of transgenic plants? The authors need to provide some background of P. alba x P. grandidentata as well.

5. Methods part (Page 4, Paragraphs 1 & 2; Page 5, Paragraph 1): what does “Three biological and technical replications were conducted” mean? Were biological or technical replications conducted?

6. Page 5, Paragraph 2: “one-month old” should be “one-month-old”.

7. Page 5, Paragraph 3: “green (downregulated) and red (upregulated)” should be “red (upregulated) and green (downregulated)”.

8. The Figure 4 and Figure 5 legends should be exchanged.

9. Page 6, Paragraph 6: Fig. 2B should be Fig. 3B

10. Page 6, Paragraph 6: “subclasses 3 and 6 whose expression continually decreased” should be “subclasses 3 and 6 whose expression continually increased”

11. Page 6, Paragraph 6: What do you mean by “generally increased and decreased”?

12. Page 7, Paragraphs 1 & 2: Fig. 3B should be Fig. 4B. Please check the whole manuscript because you may have made the same mistakes in other places.

Author Response

In this manuscript, Zhao et al. studied the impact of low temperatures on lignin biosynthesis in poplar. Through metabolic profiling, the authors discovered that the amounts of several metabolic intermediates of lignin were reduced by low temperatures. Transcriptomic analyses also identified that the expression of several genes related to lignin contents was altered by cold stimulation. The effect of cold stress on lignin biosynthesis has been described previously by other groups but has never been systemically studied. Therefore, the current study has the potential to unravel unknown mechanisms by which low temperatures affect lignin contents in plants. However, I found some flaws in the results and I don’t agree with some of the conclusions in the manuscript. See my comments below.

Major point:

1. Figure 1: I don’t see statistically significant differences in lignin contents between day 0 and day 3 samples at 4°C. Even though the difference in lignin contents between samples at 4°C and 25°C on day 3 was statistically significant, it was quite small. These results suggested that cold temperatures do not have a dramatic effect on lignin contents in poplar. Actually, some studies have shown similar results in other species (e.g., Ji et al., PLoS Genetics, 2015). However, some other studies also showed that although no changes in the levels of lignin or its precursors were observed in plants maintained at low temperatures, there was an increase in related enzyme activities as well as an increase in gene expression (reviewed in Moura et al., JIPB, 2010). The authors should explain these in the Results and Discussion sections.

A: According to our experimental results, after three days of growth at 25°C treatment, lignin of “84k” poplar (Populus alba×Populus glandulosa) tissue culture plantlets would increase slightly, which indicated three days of growth produced more lignin. However, lignin decreased slightly after the 4°C treatment, indicating that low temperature may inhibit the growth of poplar tissue culture plantlets, and lignin content decreased. The lignin content of poplar tissue culture seedlings after 3 days of 4-degree treatment was significantly lower than that after 3 days of 25-degree treatment. The difference in the samples we processed (“84k” poplar (Populus alba×Populus glandulosa) tissue culture plantlets) may be the reason for the difference between our results and others, because we speculate that for young tissue culture plantlets, low temperature environment mainly restricts the expression of lignin-related enzymes and inhibits lignin synthesis.

 

2. Figure 2: A and B are basically the same data. Is it necessary to show both? The statistical analyses were missing in Figure 2B. Besides, the description in the Results section did not match the data shown in Figure 2. For example, in Paragraph 1 of 3.2, the authors described “The other compounds, including sinapinaldehyde, p-coumaraldehyde, l- phenylalanine, caffeate, ferulic acid, coniferaldehyde, and cinnamic acid, that were present at comparatively higher levels were induced by low-temperature treatment at different time periods”, which is not true. Some of these compounds were not induced by low-temperature treatment. In Paragraph 2 of 3.2, the authors described “The overlapping results among the five pairwise comparisons showed that caffeate and coniferaldehyde were the only two compounds that consistently decreased following low-temperature induction”, which is also not accurate. It is difficult to make this conclusion without statistical analysis. Two other compounds, p-coumaraldehyde and ferulic acid, might also show decreased levels following low-temperature induction.

A: Figure 2: A and B are basically the same data. We use heat maps and bar graphs to make the results more visible to the reader. "The other strains, including sinapinaldehyde, p-coumaraldehyde, l-phenylalanine, Caffeate, ferulic acid, coniferaldehyde, and cinnamic acid, That were present at fraction higher levels were induced by low-temperature treatment at different time periods" The mean expression of these substances can be induced to decrease or increase in a certain period.  According to our experimental results as shown below, caffeate and coniferaldehyde were the only two compounds that consistently decreased following low-temperature induction. The p-coumaraldehyde and ferulic acid might also show decreased levels following low-temperature induction. But they don't always go down.

 

 

3. Figure 3: I don’t agree with some of the authors’ conclusions. In the last paragraph of 3.3, the authors stated that “With the extension in the time of low-temperature treatment, the number of genes that varied increased, indicating that the poplar tissue culture plantlets could resist cold stimulation by expressing more genes.” A higher number of genes does not necessarily mean plants could ‘resist’ cold stimulation because the changes in gene expression could be either passive or active. In the same paragraph, the authors also concluded that “It is apparent that the level of expression of most of the genes changed between -5≤log2 fold change≤5, while the level of expression of many genes changed significantly, suggesting that they may play an important role in the resistance of poplar to cold temperature stress.” Similarly, these changes could be either passive or active, and they are not necessarily involved in cold resistance.

A:We modified the sentence with “With the extension in the time of low-temperature treatment, the number of genes that varied increased, indicating these altered genes may be involved in poplar tissue culture plantlets to cold stimuli.” in line 285-287. In line 287-290, we modified the sentence with “ “It is apparent that the level of expression of most of the genes changed between -5≤log2 fold change≤5, while the level of expression of many genes changed significantly, suggesting that they may participate the resistance of poplar to cold temperature stress.”

4. Results 3.5, Paragraph 1: the authors described “The genes that positively regulated the intermediates in lignin metabolism are shown in subclasses 1 and 2, and the expression of these genes generally decreased.” There were thousands of genes in each subclass. Please provide the data showing all the genes that positively regulated the intermediates in lignin metabolism to support the statement.

A:Although many genes are regulated the intermediates in lignin metabolism, we analyzed only those genes involved in lignin synthesis, as illustrated in Supplemental Table 7. XLSX

5. Figure 4B: I suggest the authors use a different color range. Please set 1 as the midpoint. The current color scheme is a little misleading because values lower than 1 were also displayed in a warm color.

 A:Thank you for your suggestion 

Minor point:

1. It would be much easier for the readers to digest the data if the authors include a biosynthetic pathway of lignin in the manuscript, indicating the key metabolites, enzymes, and potential transcription factors regulating the expression of these enzymes.

A:In the introduction we describe it.

2. Page 2, Paragraph 1: the order of the metabolites used for the polymerization to form monomers of syringyl, guaiacyl, and p-hydroxyphenyl is wrong. Please double check and correct it.

A: We modified it in line 48-50.

3. Page 2, Paragraph 2: full name of ERF8 should be provided.

A: We modified it with “REDUCED EPIDERMAL FLUORESCENCE 8, REF8” in line 72-73.

4. Page 2, Paragraph 2: what type of transgenic plants? The authors need to provide some background of  alba x P. grandidentata as well.

A: Transgenic plants means C3’H gene was knocked down and its expression was inhibited.

5. Methods part (Page 4, Paragraphs 1 & 2; Page 5, Paragraph 1): what does “Three biological and technical replications were conducted” mean? Were biological or technical replications conducted?

A: Biological and technical replications were all conducted.

6. Page 5, Paragraph 2: “one-month old” should be “one-month-old”.

A: We modified it in line 230.

7. Page 5, Paragraph 3: “green (downregulated) and red (upregulated)” should be “red (upregulated) and green (downregulated)”.

A: We modified it in line 253.

8. The Figure 4 and Figure 5 legends should be exchanged.

A: We exchanged it.  

9. Page 6, Paragraph 6: Fig. 2B should be Fig. 3B

A: We modified it in line 310.

10. Page 6, Paragraph 6: “subclasses 3 and 6 whose expression continually decreased” should be “subclasses 3 and 6 whose expression continually increased”

A: We modified it in line 316.

11. Page 6, Paragraph 6: What do you mean by “generally increased and decreased”?

A: We mean the level of expression of the genes in subclasses 4 and 5 goes up and down.

12. 12. Page 7, Paragraphs 1 & 2: Fig. 3B should be Fig. 4B. Please check the whole manuscript because you may have made the same mistakes in other places.

A: We modified it.

Reviewer 2 Report

The authors omitted the all the figures in the submission. The figures are not provided neither in the pdf file nor as supplementary material. Without figures I can not compare the results. Please, also provide the captions of the supplementary figures.

Author Response

Comments and Suggestions for Authors

The authors omitted the all the figures in the submission. The figures are not provided neither in the pdf file nor as supplementary material. Without figures I can not compare the results. Please, also provide the captions of the supplementary figures.

 

A: I am sorry to confuse you. We added the figures.

Round 2

Reviewer 1 Report

The authors answered most of my questions but didn’t make any changes for some of them. See my comments below. Did the authors upload a different version (probably the previous version)?

Major point:

1. Figure 1: I think the authors didn’t read my comments carefully or had some misunderstanding. My point was that the data shown by the authors and other groups suggested that the effect of cold temperatures on the lignin contents is very small. However, other studies showed that there was an increase in related enzyme activities and in gene expression. I suggested that the authors should discuss this in the manuscript. I didn’t see any changes in the text.

2. Figure 2: Again, please add statistical analyses in Figure 2B.

3. Figure 3: I’m fine with the modifications.

4. Results 3.5, Paragraph 1: please indicate Supplemental Table 7 in the text.

5. Figure 4B: I don’t see any changes in the figure.

Minor point:

1. I meant an illustration, but I’m fine if the authors decide not to include one.

2. Page 2, Paragraph 1: I meant the ones in lines 44-45 and I didn’t see any changes in the text.

3. Page 2, Paragraph 2: there must be something wrong with the manuscript I received. ERF8 is in line 67 and I don’t see the full named added here.

4. Page 2, Paragraph 2: Please add the information in the text. I don’t see any changes in the text.

5. Methods part (Page 4, Paragraphs 1 & 2; Page 5, Paragraph 1): do you mean that you did three biological replicates and three technical replicates for each biological replicate? Please add this information in the methods part.

6. Page 5, Paragraph 2: I don’t see the modification.

7. Page 5, Paragraph 3: I don’t see the modification.

8. The Figure 4 and Figure 5 legends should be exchanged: I don’t see any changes.

9. Page 6, Paragraph 6: Fig. 2B should be Fig. 3B. It’s in line 289. I don’t see the modification.

10. Page 6, Paragraph 6: “subclasses 3 and 6 whose expression continually decreased” should be “subclasses 3 and 6 whose expression continually increased”. I don’t see the modification in line 293.

11. Page 6, Paragraph 6: What do you mean by “generally increased and decreased”? Please change the expression.

12. Page 7, Paragraphs 1 & 2: Fig. 3B should be Fig. 4B. I don’t see the changes in lines 304 and 322.

Author Response

There is no option to upload modified pictures and revised manuscripts in the system, so I uploaded my revised manuscript with pictures in the following attachment.

Major point:

 

1. Figure 1: I think the authors didn’t read my comments carefully or had some misunderstanding. My point was that the data shown by the authors and other groups suggested that the effect of cold temperatures on the lignin contents is very small. However, other studies showed that there was an increase in related enzyme activities and in gene expression. I suggested that the authors should discuss this in the manuscript. I didn’t see any changes in the text.

A: Thank you for your suggestion. In line 366-376, we discussed the impact of low temperature on lignin content “Although the effects of low temperatures on lignin vary with plant species, developmental period and organs, short-term treatment with low temperature reduced the content of lignin in the stems of poplar plantlets [6,34,37,38].” Different hypothermic conditioned stimuli, brassica napus plants certain enzymes such as ρ-coumaric acid, ferulic acid and synapic acid in the cells of the leaf mesophyll levels were elevated. However, we found the contents of caffeate, ferulic acid, coniferaldehyde, and p-coumaraldehyde decreased after cold stress. The reason for this difference may be the low temperature conditions and handling time and the plant samples (we used tissue culture plantlets). When treatment with 10℃, PAL activity of glycine max roots increased. In our results, L-phenylalanine activity was also increased at 72 hours after low temperature treatment. Line 366-383.

2. Figure 2: Again, please add statistical analyses in Figure 2B.

A: We added it.

 

3. Figure 3: I’m fine with the modifications.

A: Thank you for your recognition.

4. Results 3.5, Paragraph 1: please indicate Supplemental Table 7 in the text.

A: We added it in line 322, 331, 337.

5. Figure 4B: I don’t see any changes in the figure.

A: Thank you for your suggestion. Our heat maps have been able to fully show the trend of rising and falling genes, and much of the literature has done the same.

Minor point:

 

1. I meant an illustration, but I’m fine if the authors decide not to include one.

A: Thank you for your recognition.

2. Page 2, Paragraph 1: I meant the ones in lines 44-45 and I didn’t see any changes in the text.

A: We rewrote the sentence with “Lignin is generally recognized to be composed of the three monomers syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H), which are polymerized by sinapyl alcohol, p-coumaryl, and hydroxycinnamyl alcohols, respectively”.

3. Page 2, Paragraph 2: there must be something wrong with the manuscript I received. ERF8 is in line 67 and I don’t see the full named added here.

A: We modified it with “REDUCED EPIDERMAL FLUORESCENCE 8, REF8” in line 72-73.

4. Page 2, Paragraph 2: Please add the information in the text. I don’t see any changes in the text.

A: Transgenic plants means C3’H gene was knocked down and its expression was inhibited.

5. Methods part (Page 4, Paragraphs 1 & 2; Page 5, Paragraph 1): do you mean that you did three biological replicates and three technical replicates for each biological replicate? Please add this information in the methods part.

A: Biological replications were conducted.

6. Page 5, Paragraph 2: I don’t see the modification.

A: We modified it in line 230.

7. Page 5, Paragraph 3: I don’t see the modification.

A: We modified it in line 253.

8. The Figure 4 and Figure 5 legends should be exchanged: I don’t see any changes.

A: We exchanged it.  

9. Page 6, Paragraph 6: Fig. 2B should be Fig. 3B. It’s in line 289. I don’t see the modification.

A: We modified it in line 310.

10. Page 6, Paragraph 6: “subclasses 3 and 6 whose expression continually decreased” should be “subclasses 3 and 6 whose expression continually increased”. I don’t see the modification in line 293.

A: We modified it in line 316.

11. Page 6, Paragraph 6: What do you mean by “generally increased and decreased”? Please change the expression.

A: We mean the level of expression of the genes in subclasses 4 and 5 goes up and down.

12. Page 7, Paragraphs 1 & 2: Fig. 3B should be Fig. 4B. I don’t see the changes in lines 304 and 322.

A: We modified it.

Author Response File: Author Response.doc