Improving High Light Tolerance of Tobacco Plants: Adequate Magnesium Supply Enhances Photosynthetic Performance
, Jingjuan Gao 1, Zongfeng Yang 2, Yuemin Wang 2, Liangyuan Tong 1, Yuqin Ke 3, Chunjian Li 1,4, Chaoyuan Zheng 1,* and Wenqing Li 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear responsible author,
Thank you for your invitation to review this article.
Due to global climate change, abiotic stresses are increasingly affecting plant production. In addition to abiotic stresses such as drought and salinity which are already on the agenda, other stresses such as high light, radiation and waterlogging have been added to the list. Tobacco is a plant of high economic value on a global scale. The large leaves of this plant are both the main organ for its industrial consumption and the most vulnerable area to external stresses. High light is known to be a factor that disrupts chlorophyll balance in all plants. The fact that the central atom of chlorophyll is magnesium has from time to time encouraged different research groups to work on the topics of "magnesium supplementation & chlorophyll balancing & photosynthetic efficiency". In this study, the researchers examined the effect of different magnesium doses on various parameters, especially chlorophyll content, of tobacco plants under high light intensity. In this context, it is a well-chosen subject. The title is successful in reflecting the content of the study. However, some recommendations for the study are as follows.
In the abstract, it should be written what the control treatment was that influenced the overall study. In its current form, two light intensities x 4 Mg concentrations are understood as the experimental factor. However, combinations of all these factors were not used in the study.
The introduction is generally simple and adequate. However, (as the authors point out) there are many studies on magnesium and chlorophyll contents. The difference between this study from the others should be made clearer. Brief references should be made to the studies shared below and similar ones and the results obtained should be compared, especially in the discussion section.
In the Material and Method section, the reason (if any) for the choice of tobacco variety used should be written. In accordance with the general academic writing tradition, the year, and the institution where the study was conducted should be indicated.
According to the information shared in the abstract, the experimental factors were two light intensities and four magnesium doses. In this case, it is hoped that all measurements and observations will be taken from eight different groups and that the magnesium x light interaction will be evaluated at the end of the study. However, in the methods section, it was seen that 750 μmol m−2 s−1 light dose was included in only 1 treatment (combined with 1 mol magnesium). Although the authors stated that this selection was based on previous studies, it is a great deficiency that the effects of other magnesium doses were not examined at 750 μmol m−2 s−1 light dose. However, the data shared in "Supplemental Figure S1." in the Supplemental documents are very valuable. If available, other parameter results obtained for different magnesium doses at 750 μmol m−2 s −1 light intensity should also be shared, so that readers can determine whether any changes are primarily related to light intensity or magnesium. Moreover, the interaction effect is thus considered statistically.
The Reference list is generally sufficient and up-to-date. However, I would recommend adding resources directly related to the topic I mentioned below to the introduction and discussion section.
Yang, Y., Fu, Q., Yang, C., Rao, X., Wu, Z., Wu, Z., ... & Yu, Z. (2023). Effects of Mg on chlorophyll degradation and leaf chroma during the airing of cigar tobacco leaves. Acta Societatis Botanicorum Poloniae, 92, 1g-1g.
GUAN, G. S., TU, N. M., XIAO, H. Q., ZHU, L. S., ZHU, Y. H., & WANG, Z. M. (2008). Effects of magnesium on tobacco growth and chlorophyll fluorescence parameters of tobacco leaves. Journal of Plant Nutrition and Fertilizers, 14(1), 151-155.
Li, J., Muneer, M. A., Sun, A., Guo, Q., Wang, Y., Huang, Z., ... & Zheng, C. (2023). Magnesium application improves the morphology, nutrients uptake, photosynthetic traits, and quality of tobacco (Nicotiana tabacum L.) under cold stress. Frontiers in Plant Science, 14, 1078128.
Biswal, A. K., Pattanayak, G. K., Pandey, S. S., Leelavathi, S., Reddy, V. S., Govindjee, & Tripathy, B. C. (2012). Light intensity-dependent modulation of chlorophyll b biosynthesis and photosynthesis by overexpression of chlorophyllide a oxygenase in tobacco. Plant physiology, 159(1), 433-449.
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Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
It is obvious that you put a lot of effort into the research part and you got large quantity of interesting data. Sometimes it is extremely difficult to summarize a large amount of data on a small number of pages.
The title of the paper is appropriate and concise.
The objective and the hypothesis should be stated more clearly.
Tables and figures are scattered in the text of the paper and supplements, which makes it difficult to read the paper fluently.
Figures titles can be more concise.
The discussion is well aligned with the research results and the previous text.
I suggest that you review the manuscript in detail and minor typos corrected, with particular attention to uniform writing of SI units and abbreviations and more readable format of tables.
1. In “Material and methods, plant cultivation and sampling”; please describe the conditions in the greenhouse.
2. In “Material and methods, plant cultivation and sampling” - Have you used seed pretreatment to prevent fungus and mold growth? If so, state it in the text. How many seedlings were in the pot?
3. Row 88. Please indicate the references of the method with which you determined the elemental composition of the sand and water. Do not forget to put them in the References section. I suggest you do an analysis of nitrogen in the sand and water.
4. I suggest that you delete Table S3 because it is well described in text.
5. Row 106. “The NL-Mg1 treatment was determined to have the best effect on tobacco growth physiology based on the results of preliminary experiments (Figure. S1)” but when you read in supplement it says Figure S1. Impact of magnesium on plant hydrogen peroxide content and relative electrolytic leakage under normal light. Figure S1 does not show the results of preliminary experiments.
6. Why did you not take NL Mg1 as a control group because it is obvious that the Mg 1 group is in deficit with Mg and therefore in stressful conditions?
7. Row 109. “Carbon (N) Concentration” it should say “(C)”.
8. Row 116. “weight of 70°C” it should say “weight at 70°C”.
9. Row 122. “in vivo” should be italic.
10. Please provide references for all the methods you have done and do not forget to write them in the References section.
11. In the statistical analysis chapter, please describe in detail how many repetitions there were and how many plants there were per repetition. Also note which treatment was the control.
12. When you write “(Figure. XY)” please write without a point. (106; 176; etc.)
13. Figure 1 (C); should say “Mg percentage”
14. Row 185. “(C) Magnesium (Mg) concentration. (D) Hydrogen peroxide” Should say “(C) Magnesium (Mg) percentage. (D) Hydrogen peroxide concentration “
15. Row 205. Should say “ N percentage “.
16. Row 208. - 216. – “HMg0-, HMg1-, HMg2-, and HMg5-“ - Please write abbreviations uniform trough whole text.
17. It would be interesting to calculate and display the correlation.
18. In which leaf was the enzyme activity analyzed (upper or lower; old or young). Please state it.
19. Figure 2. A and B – should say “C percentage” and “N percentage”.
20. Figure 3. c – should say “mmol H2O” not H2O2 .
21. Row 250. “Net photosynthetic rate. (B) Intercellular” should say “Net photosynthetic rate (Pn). (B) Intercellular”.
22. Chart in Figure 3 and Figure 4B is not accessibility compliant. I suggest you find different way to show your data.
23. Row 270. “(Table 4) with R2 values near 1” it is Table 1.
24. Row 314. – MgD please write full name not abbreviation at least for the first mentioning in text
25. Figure 5 instead of “C” in last part should say “D” because C already exist
26. Table 1 - you do not have to indicate insignificant differences. Whenever applicable, write numbers to two decimal places. Describe the statistical significance marks in the title. Write the marks of statistical significance in the second row of the cell for better visibility.
27. Row 468. “Vicia faba” italic; Row 451. “Citrus reticulata” italic. Row 523. – species - italic
28. Row 512. – doi - without the underlined line.
29. References listed in Table S4 are not listed in the Reference section.
30. In text, you often write “tobacco seedings” but you probably mean seedlings.
Please try to explain in discussion why is C percentage in upper leaf lower than in lower leafs (lower leafs were deteriorated), and for nitrogen it was the opposite.
The conclusion could be better and longer written due to the large amount of data.
I hope my suggestions will help you. I wish you all the best and thank you.
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsComments for author File: Comments.pdf
Author Response
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Reviewer 4 Report
Comments and Suggestions for AuthorsThis manuscript examines the hypothesis that elevated Mg2+ levels can help to alleviate stress caused by high light levels. The negative effects of high light are related to production of ROS by the photosynthetic apparatus. This might be a reasonable hypothesis, however I am not convinced that the data in the manuscript provide solid evidence in support of the hypothesis.
My detailed comments are listed below, in no particular order (they are not ranked in order to importance):
1) Line 44 – “Furthermore, Mg plays an essential role in the electron transport chain of chloroplasts [4,14,15], transferring energy from photosystem II (PSII) to nicotinamide adenine dinucleotide phosphate (NADP+), while also mitigating thylakoid membrane damage caused by reactive oxygen species (ROSs) [16,17].”
This is vague. The authors mention that Mg2+ is a component of chlorophyll; that is a specific role of Mg2+ and is informative to the reader. But the other listed roles of Mg2+ are vague and not explained (for example, Mg limitation could lead to problems with phloem loading of sucrose because of depressed Mg2+-ATP levels during Mg limited growth).
2) Line 112 – “The upper and lower leaves were halved based on the total number of flue-cured tobacco leaves.”
I do not understand. What does flue-curing have to do with this experiment?
3) Line 116 – “The different plant parts were then ground into a fine powder for various treatments, and the powdered samples were tested for N, C, and Mg concentrations”
How were these analyses performed?
4) Line 132 – “The concentration of H2O2 was measured using the Sino- bestbio hydrogen peroxide content assay kit (YX-C-A400, Sino Best Biological Technology Co., Ltd., Shanghai, China) and a UV spectrophotometer (Mapada UV-P5; Shanghai Mapada Instruments Co., Ltd., Shanghai, China).”
I think that more details are needed about the protocol for H2O2 measurement. H2O2 is very reactive in biological systems (the half-life is much less than one second because it rapidly encounters oxidizable molecules). Under what conditions was H2O2 measured? In detached leaves? What was the light environment during measurement?
5) Fig. 3 – Why does stomatal conductance decrease as light increases? Why is stomatal conductance a maximum in the dark? I cannot think of a physiological explanation for this observation, and the authors do not address this. I strongly suspect that there is an error associated with the stomatal conductance data, and also with the associated transpiration data (both stomatal conductance and transpiration should be at a minimum in darkness, and increase as light increases).
6) Line 323 – “Under MgD, the exacerbation of HL stress on leaves occurs, with an increase in total carbon content”
If total C increases, that does not distinguish between structural and non-structural C. I assume that the authors are most interested in non-structural C? The hypothesis being that Mg limitation affects sucrose and starch metabolism.
7) Line 351 – “through the Meler reaction”
Mehler reaction.
8) Figs. 5A to 5C – I do not understand what is being shown in these figures (other than black ellipses represent inactive PSIIs). As well, the figure legend suggests that Fig. 5B has black ellipses, but I do not see any. Lastly, the second Fig. 5C is probably Fig. 5D?
9) There are no signs that plants were stressed under any treatments. There are no signs of photoinhibition (measured as either Fv/Fm) or as the initial slope of a P vs I curve. Pmax values are not that different between treatments, and enzyme activities are similar.
10) Is there clear evidence for Mg limitation in this work? There is perhaps some evidence that the HMg0 is mildly deficient in Mg (based on Fig. 1B), but other than that there seems to be excess Mg. How was the Mg added? For example, in the HMg1 treatment, how was the 1 mol of Mg added to the plant? Was it dissolved in the modified Hoagland’s solution? What volume of Hoagland's was added every 3 to 5 days? Did solution run out of the bottom of the pot?
If we assume that 1L of Hoagland’s solution was applied every 3-5 days, and the Mg was added as part of the Hoagland’s solution, the nutrient ratios on the Hoagland’s would very skewed to high Mg. The N concentration was 4.98 mM, while the Mg concentration would be 1M (1000 mM). Plants simply have no use for Mg at that level (plants need FAR more N than Mg). It is quite possible that all treatments provided sufficient Mg, with perhaps a minor Mg deficiency in the HMg0 treatment.
Then again, the biomass results may be based on only three plants (n = 3) if I understand the experimental design. Three plants is not a large sample size for comparing biomass values.
11) in terms of experimental design I have suggest:
a) acid-washing the sand prior to planting tobacco, in order to remove contaminating nutrients (especially Mg)
b) adding lower levels of Mg, including levels that would lead to both Mg deficiency and sufficiency, would have been useful for comparisons
In summary, I do not think that there is much of an effect of the various Mg2+ levels in this experiment (there was a LOT of Mg2+).
Comments on the Quality of English Language
Could use some minor editing.
Author Response
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Round 2
Reviewer 4 Report
Comments and Suggestions for AuthorsIn my original review I pointed out that the data for stomatal conductance and transpiration rates were unusual (with the highest stomatal conductance measured in darkness). The authors state that they checked their calculations, and now everything seems to be correct (Fig. 3C in v2 seems to have the data reversed from Fig. 3C in v1, which I assume to be the correction). While the data in v2 Fig. 3C are more reasonable than in v1 Fig. 3C, there is a huge variation in stomatal conductance values in darkness (and some of the dark values are extremely high). I am guessing that the large error bars might explain the results. In terms of stomatal conductance in tobacco, as affected by PFD, more typical results are found in:
Chinese Journal of Eco-Agriculture, Aug. 2013, 21(8): 966−972 (see Fig. 3)
In the end, I am still having difficulties accepting the stomatal conductance data.
Line 380 – “Mg affects many physiological processes, and previous studies have confirmed that MgD can lead to phloem loading”
Is there is a missing word? MgD can lead to decreased phloem loading? Or MgD can affect phloem loading?
Line 413 – “Absorption of excess light energy by Chl may lead to the production of triplet chlorophyll (3Chl), which subsequently transfers energy to oxygen molecules (O2) through the Mehler reaction, resulting in singlet oxygen (1O2) formation, which are then converted to H2O2 through enzyme action.”
Triplet chlorophyll transfers energy to oxygen (O2), generating singlet O2 (1O2). In contrast, the Mehler reaction produces superoxide (O2-) by accepting an electron from reduced ferredoxin (superoxide is O2 with an extra electron, while singlet O2 is O2 with an electron in a higher energy orbital). Superoxide is converted to hydrogen peroxide however singlet oxygen is not converted to hydrogen peroxide. Singlet oxygen is typically detoxified via carotenoids.
I apologize if I missed this point in the first review.
Comments on the Quality of English Language
Mostly good.
Author Response
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