Reassimilation of Leaf Internal CO2 Contributes to Isoprene Emission in the Neotropical Species Inga edulis Mart.
, Kolby Jardine 2, Vinicius F. de Souza 3, Rodrigo A. F. de Souza 4, Sergio Duvoisin Junior 3 and José Francisco de C. Gonçalves 1,*Round 1
Reviewer 1 Report
This manuscript is clearly a follow-up paper of “Jardine et al. 2014”, cited several times throughout the text and in part it presents similar conclusions. Nevertheless, the manuscript “Re-assimilation of leaf internal CO2 contributes to isoprene emission in the neotropical species Inga edulis Mart” also presents some novelties as it widens the alternative carbon sources for isoprene synthesis to all the decarboxylation processes within the leaf and not only to photorespiration.
However, the manuscript presents several weaknesses:
-The main weak point is the fact that some measurements (inhibitor experiment with DCMU and dark experiment) have been performed only in one leaf as stated in figure legends (n=1) (Figure 3 and Figure 6). Only one sample is too low to be able to consider the result as significant and /or publishable. Authors should present the result of, as least, three different leaflets.
- The discussion section is quite long. It should be shortened, considering, overall, that it is extremely repetitive.
For instance, the concept explained in page 10, lines 303-309, is repeated in page 11, lines 354-360, in page 12, line 412-413 and in page 12 lines 427-430.
Other slight comments are:
-Some English mistakes:
Page 2, line 54. “may not possible” is not correct. You should add another verb.
Page 4, line 178: “are stimulated” instead of “are stimulate”
Page 10, line 352: Better “authors suggested”
-“Is” and “isoprene emissions” are used throughout the text. I proposed to used always “Is”. Moreover, it is not clear whether “Is” substitutes “Isoprene” or “Isoprene emission”.
Material and methods:
Page 3, line 119. Though this methodology is explained in “Jardine et al. 2014”, authors should briefly include some information. Were gas exchange and isoprene emission rate determinations performed simultaneously? Which fraction of air exiting the leaf chamber was used for GC-MS and for PTR-MS measurements?
Moreover, the air flow rate entering the leaf chamber should be measured in ml/min (line 121).
Results
Page 7, lines 254-256:
The result of this measurement is the same that the one described in page 6, lines 226-229. Do not refer to it as a new result but as a confirmation of a previous result.
Discussion
Page 10, lines 346-349. It is discussed that in the absence of O2 and under 0 mmol mol-1 CO2, no isoprene emission was observed. I wonder why authors have not done this same experiment to check that, indeed, isoprene emission rates were high due to the stimulation of photorespiration in a CO2 free atmosphere, and not due to other alternative carbon sources. I would consider seriously to perform this measurement.
Figures:
-I propose to change the colors in figure 3 and figure 6. The difference between dark and light green, dark and light orange, and dark and light blue is not very clear.
-Figure 4: in the figure legend it is specified that” n=7”, whereas in the “material and methods” section (Page 3, line 136) it is indicated that “n=4”.
-Figure 4b has only four points with high standard deviations. It would be clearer to show all single measurements (4 for each temperature) and then show the regression line. This could be done also in figure 1b.
Author Response
Reviewer 1:
Comments and Suggestions for Authors
This manuscript is clearly a follow-up paper of “Jardine et al. 2014”, cited several times throughout the text and in part it presents similar conclusions. Nevertheless, the manuscript “Re-assimilation of leaf internal CO2 contributes to isoprene emission in the neotropical species Inga edulis Mart” also presents some novelties as it widens the alternative carbon sources for isoprene synthesis to all the decarboxylation processes within the leaf and not only to photorespiration.
However, the manuscript presents several weaknesses:
-The main weak point is the fact that some measurements (inhibitor experiment with DCMU and dark experiment) have been performed only in one leaf as stated in figure legends (n=1) (Figure 3 and Figure 6). Only one sample is too low to be able to consider the result as significant and /or publishable. Authors should present the result of, as least, three different leaflets.
The authors thank you and understand your concern. In the first moment, the authors made these measurements (with DCMU and dark) as pre-tests, to verify what was observed in the literature, that DCMU and the dark blocks isoprene synthesis. We tested this in the Inga edulis species and we saw the same pattern. We decide to include these data after the other measurements to clarify as much as possible our message and to guide the main results of this paper.
The discussion section is quite long. It should be shortened, considering, overall, that it is extremely repetitive.
For instance, the concept explained in page 10, lines 303-309, is repeated in page 11, lines 354-360, in page 12, line 412-413 and in page 12 lines 427-430.
We agree with your comment. The discussion section was revised and the parts that were repetitive were taken off.
Other slight comments are:
-Some English mistakes:
Page 2, line 54. “may not possible” is not correct. You should add another verb.
Page 4, line 178: “are stimulated” instead of “are stimulate”
Page 10, line 352: Better “authors suggested”
The text was changed.
-“Is” and “isoprene emissions” are used throughout the text. I proposed to used always “Is”. Moreover, it is not clear whether “Is” substitutes “Isoprene” or “Isoprene emission”.
We agree to use “Is” instead “isoprene emissions”.
Material and methods:
Page 3, line 119. Though this methodology is explained in “Jardine et al. 2014”, authors should briefly include some information. Were gas exchange and isoprene emission rate determinations performed simultaneously? Which fraction of air exiting the leaf chamber was used for GC-MS and for PTR-MS measurements?
Moreover, the air flow rate entering the leaf chamber should be measured in ml/min (line 121).
We included more information about the methods, describing the amount of air from the leaf chamber that was used for GC-MS and for PTR-MS, as well as changed the units to mL min-1.
Results
Page 7, lines 254-256:
The result of this measurement is the same that the one described in page 6, lines 226-229. Do not refer to it as a new result but as a confirmation of a previous result.
The authors thank you for this remark but we believe that these two sentences address different results. The result described in page 6, lines 226-229 shows isoprene temperature-response curves under constant light and CO2 conditions of 1000 mmol m-2 s -1 and 450 mmol mol-1, respectively, in order to observe the isoprene emission pattern with and without CO2, when compared to figure 5, that shows isoprene temperature-response curves under CO2-free atmosphere. The result described in page 7, lines 254-256 shows isoprene temperature-response curves under CO2-free atmosphere in the presence and absence of light in order to verify is there is some isoprene emission from Inga edulis individual in the dark.
Discussion
Page 10, lines 346-349. It is discussed that in the absence of O2 and under 0 mmol mol-1 CO2, no isoprene emission was observed. I wonder why authors have not done this same experiment to check that, indeed, isoprene emission rates were high due to the stimulation of photorespiration in a CO2 free atmosphere, and not due to other alternative carbon sources. I would consider seriously to perform this measurement.
We agree that an experiment with O2-free atmosphere would support the findings of this manuscript. However, the authors think that the issues raised by this work are answered with the current results, once that we show that carboxylation process can contribute to isoprene synthesis, as show in the figure 7 and describe in the lines [275-283].
Figures:
-I propose to change the colors in figure 3 and figure 6. The difference between dark and light green, dark and light orange, and dark and light blue is not very clear.
The authors thank you for the suggestion and changed the color in figures 3 and 6.
-Figure 4: in the figure legend it is specified that” n=7”, whereas in the “material and methods” section (Page 3, line 136) it is indicated that “n=4”.
We thank you for pointing out this mistake in the Figure 4 legend. We modified the legend to the right ”n”.
-Figure 4b has only four points with high standard deviations. It would be clearer to show all single measurements (4 for each temperature) and then show the regression line. This could be done also in figure 1b.
In fact, both, figure 1b and 4b, has high standard deviations bars. However, the authors agree that the average with standard deviations clearly show the relation between isoprene emission and photosynthesis in a carbon unit. We thank the reviewer for the suggestion to add a regression line and did it in both figures.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments by Reviewer
General comments:
The authors present “Re-assimilation of leaf internal CO2 contributions to isoprene emission in the neotropical species Inga edulis Mart.”. The results from these measurements are interesting. The manuscript is well written and clearly structured. I have several comments that should be addressed prior to publication (minor revision).
Specific comments:
1. Line 47: secondary aerosols → secondary organic aerosols
2. Lines 136-138 and Figure 1(a): Isoprene emission rate is caused by the effect of changes in environment. Did the authors check the time required for stabilizing the isoprene emission rate after changing leaf temperature and PPFD (15 minutes enough?)? I think equilibrium time is short.
3. Figure 2(b): What is axis label (PPFD) and values (0 → 2010) above the graph?
4. Figure 2(b): Isoprene emission depends on temperature and light intensity. However, isoprene emission rate (Is) started to increase under PPFD 0 μmol m-2 s-1 conditions. Please mention.
5. Figure 5: isopreno → isoprene
Author Response
Reviewer 2:
General comments:
The authors present “Re-assimilation of leaf internal CO2 contributions to isoprene emission in the neotropical species Inga edulis Mart.”. The results from these measurements are interesting. The manuscript is well written and clearly structured. I have several comments that should be addressed prior to publication (minor revision).
Specific comments:
1. Line 47: secondary aerosols → secondary organic aerosols
The authors thank you the comments and we changed the text.
2. Lines 136-138 and Figure 1(a): Isoprene emission rate is caused by the effect of changes in environment. Did the authors check the time required for stabilizing the isoprene emission rate after changing leaf temperature and PPFD (15 minutes enough?)? I think equilibrium time is short.
The authors thank you for pointed out this. Indeed, isoprene emission is strongly dependent of changes in environment and the authors were careful to test the time required for stabilizing the isoprene emission rate that was about 10 minutes after all environmental variables (temperature, water, [CO2]) were stable.
3. Figure 2(b): What is axis label (PPFD) and values (0 → 2010) above the graph?
4. Figure 2(b): Isoprene emission depends on temperature and light intensity. However, isoprene emission rate (Is) started to increase under PPFD 0 μmol m-2 s-1 conditions. Please mention.
The authors thank reviewer 2 very much for pointed out these errors in figure 2(b). In fact, the data was plotted with a delay between bottom and upper x axes, once that the isoprene emission was measured by PTR-MS (bottom x axis) under PPFD increases by IRGA-Li-COR (upper x axis). The error has already been fixed.
5. Figure 5: isopreno → isoprene
The Figure 5 was changed.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The paper can be published in its present form
