Stevia rebaudiana Bertoni: The Interaction of Night Interruption on Gas Exchange, Flowering Delay, and Steviol Glycosides Synthesis

Round 1

Reviewer 1 Report

The manuscript titled “Stevia rebaudiana Bertoni: The interaction of steviol glycosides and leaf biomass production under night interruption” shows a valuable investigation for artificial sweetner industry. However, it is not well-organized and many necessary information are missing. 1. What is the main target component in S. rebaudiana in this study? According to 1st paragraph in the introduction, SvGly is mentioned as a major one, while Reb-A is mentioned in the 2nd paragraph. Also, stevioside is mentioned in the 5th paragraph. Please clarify which one is a major target and use uniform abbreviations for one word (e.g. steviol glycoside> SvGly or SG?). Did ‘rebaudioside A/stevioside’ in the line 45 mean rebaudioside A / steviol glycosides? In addition, I wonder what the relationship between 3 mentioned molecules is. Please explain sweetening components in S. rebaudiana in the introduction. 2. Please explain how NI treatment was applied to the plants in detail. For example, lights turn on 9 PM, 12 AM, 3 AM three time during night, every time lights on for 20 min. Also, how were long nights during experiments? How many time were leaves harvested during experiments? When explaining makers measured, please show whole scheme of experiments: when and how many times markers were collected. 3. How were RGR calculated? 4. Why were time points different in each parameters? For example, internode length was measured in 64 days plantlets while leaf area was measured in 15-71 days after bud flowering? 5. Labeling is confusing in the Tables. Why did authors mark lower case for comparison within each treatment? Generally, leaf area is increasing with plant growth. 6. Why was leaf area of genotype 2 double up from 36 to 43 DABF under 10 min and 20 min NI, not 0 min NI? 7. What is specific leaf weight and how was it measured? What is LAR and how was it measured? 8. I cannot access any supplementary data in this manuscript. 9. According to the line 212-213, Y axis of Figure 4 is not flowering delay but flowering day after harvesting. Also, information in the paragraph in the line 206-223 need to mention in the materials and methods because it contains harvesting timing which is critical for whole experimental design. 10. Chapter 3.4 is repeated information of whole results, which were already explained in the previous chapters. 11. Correlation values in the discussion were not mentioned in the results. How were they calculated? 12. Did both SvGly and GA synthesis pathway share any part of pathways? Is positive correlation of NI and internode length linked to sweetner production? What is the relationship of internode length with GA production? 13. How were authors sure there is no photodamage during the experiments? 14. The paragraph in the line 387-414 is meaningless. If it is necessary to compare the productivity of the experiments with other cultivation, please compare them. 15. Why did authors choose R/FR ratio as 11.4 not 0.16? 16. How were sweetner production increased by NI? Is NI itself an inducer of sweetner pathways or did more lights due to NI activate sweetner pathways? 17. The paragraph in the line 532-546 is same as in the line 343-357. 18. Unify the name of Stevia, Stevia rebaudiana and etc. If it means the scientific name of this plant, utilize italic letters. 19. Unify a reference style fit to the guidance of the journal (e.g. [35] or (36) in the line 319 and 324). 20. I recommend professional English editing for the manuscript.

Author Response

See letter in attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript  deals with to understand the effect of night interruption  for modification of biomass yield and accumulation of steviol glycosides in the selected accession of stevia. The authors have beautifully filled a knowledge gap.

After careful reading of the manuscript, I have found that the manuscript is interesting.  The hypothesis and aims are clear with proper design of experiment. The manuscript  can fit into the scope of the journal.

However, there are some flaws in the present form which are needed to be addressed properly. 

The title should be changed: the interaction should be accession and night interruption. 

The methodology sections is confusing.  Need to be expanded properly. 

The figure and tables presentations are confusing. 

The discussion section should be reduced. 

Abstract is very poor. The key findings should be mentioned. 

Line 33-34: statement is not correct.

L35: check the data for authentication. 

L51: is it raw leaf or finished product?

L57: 50% DW, not clear.

Check the symbol of net photosynthesis. 

Author Response

See letter in attachment

Author Response File: Author Response.docx

Reviewer 3 Report

Title: The interaction of steviol glycosides and leaf biomass production under night interruption. But there was no result and discussion about the interaction.

Line 39-40  ‘The global rebaudioside A (Reb-A) market includes forecast data, demand, application details, price trends and Reb-A actions by geographic region ’. what do this mean and is related to the object of this manuscript? 
Line 93 ‘Cuttings of genotype 2 (a long night plant; LN), and genotype 4 (a short night plant; SN)’  There should be illustrated clearly about the light-response character of this two genotypes.
Line 102-103 ‘ three times of night interruption (0, 10, and 20 min)’   what is the time of night interruption? such as from 20:00-20:10.
Line 114-115 ‘specific leaf weight (SLW), leaf area ratio (LAR), RGR,’ How to calculate these? 
Line 156-158 what do this sentence mean ?
Figure 3 A and Figure 4  There are mistakes about the significance.
Line 326 how ‘seems to automatically move other materials from the organs ’?
Line 341 and 356 There are repeated sentences.
Line 380 ‘ the times used in this study were not sufficient to achieve a stimulus in the hormones involved in cell elongation’ and line431-432 ‘ in this study probably were not enough to induce a response in the phytochrome that regulates important variations in the LA’ were contradictory to line 479 ‘small flashes of light are sufficient to activate phytochromes’
Line 499-515  how do these related to this study?
Line 532-545  why do these repeat at this section?

Author Response

See letter in attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

1. Most information related to how to do those experiments I asked in the 1^st review was provided by authors. Still, I believe those information should be provided all researchers who will read this manuscript. So, all information should be included in materials and methods parts.

Reviewer 1: Please explain how NI treatment was applied to the plants in detail. For example, lights turn on 9 PM, 12 AM, 3 AM three time during night, every time lights on for 20 min. 

Authors: We apologize for the lack of information and inform you that a better description has been added on lines 107-110.

 

Reviewer 1: Also, how were long nights during experiments?

Authors: The city of Montería, where the experiments were carried out, is located at 8º47’ N and at this latitude, during the experiment, the nights length are close like day length, with a slight tendency to a shorter night, approximately 11.5 hours night. So, during the experiment the nights were always interrupted by the pulse of light. Therefore, there were never long nights, even in the control experiment that did not receive additional light.

Reviewer 1: How many time were leaves harvested during experiments?

Authors: The S. rebaudiana plants were leaf harvested at 14th, 43rd, and 76th day, so there were three leaf harvests. This item is described in item 3.2, lines 227-240.

 

Reviewer 1: When explaining makers measured, please show whole scheme of experiments: when and how many times markers were collected.

Authors: This question is answered above.

 

2. Still, labeling is confusing. I know all units could be different depending on the analysis methods. However, I do not know when those samples were collected at all. For example, what does 15 DABF mean as plants’ age? 60 days of plantlets? 70 days of plantlets? Please uniform the time point when authors explained the data.
3. In addition, according to authors’ answer in my review, leaves were harvested at 14^th, 43^rd, 76^th But internode length was measured in 64 days plantlets due to destructive analysis. I wonder total time period of this experiment. 76 days or 64 days?
4. According to Table 1, leaf area and weight were decreased from 22 to 29 DABF. Why?
5. In the discussion, authors tried to insist better productivity of this experiment in the line 395-411 with literatures. However, I did not find the direct comparison between literatures and this experiment. Please compare them in the discussion.
6. This parts should be included in the discussion. This is a main point of this manuscript.

Reviewer 1: How were sweetner production increased by NI? Is NI itself an inducer of sweetner pathways or did more lights due to NI activate sweetner pathways?

Authors: So, the process of activating the synthesis of biomolecules by light is a whole and intricate process. In accordance with García-Martinez & Gil, 2002 (J Plant Growth Regul. 20:354-368, 2002. doi: https://doi.org/10.1007/s003440010033) many phenotypic and biochemical effects produced in plants by light are similar to the same processes activated by chemical mechanisms via light perception by phytochromes (Curr Opin Plant Biol. 2(5): 398-403, 1999. doi: https://doi.org/ 10.1016/S1369-5266(99)00012-6). It is known that a terpenoid is a compound that results from the junction of 5 carbon units, isoprene or isopentenyl pyrophosphate (IPP), which is the initial precursor molecule in the synthesis of geranylgeranyl diphosphate (GGPP) and remains the same until the ent-kaurenoic acid and then can lead to gibberellin or SvGly synthesis (Biochim Biophys Acta, Mol Cell. 920(2): 140-148, 1987. doi: https://doi.org/10.1016/0005-2760(87)90253-0. As shown in line 63, S. rebaudiana is a long night (LN) plant with a critical day length of about 12-13 h. Under these conditions the plant grows, blooms and completes its life cycle. However, it was found that a light pulses at night was enough to break the night and modify the synthesis of various molecules, and/or biological processes. In the specific case of S. rebaudiana, night interruption (NI) delays flowering (Phytochemistry 137: 57-65, 2017. doi: http://dx.doi.org/10.1016/j.phytochem.2017.02.002; J Plant Physiol 169: 749-752, 2012. doi: https://doi.org/10.1016/j.jplph.2012.01.006; J Appl Res Med Aromat Plants. 7: 64-73, 2017. doi: http://dx.doi.org/10.1016/j.jarmap.2017.06.001) and thus more leaves are produced before practically all the carbon generated by photosynthesis is drained to form flowers, fruits and seeds. Furthermore, as light activates the formation of GGPP the synthesis of SvGly can also be increased and there are hundreds of articles describing how the night interruption increases the production of SvGly and Reb-A. So, it is not enough to increase the number of hours of light, but to reduce the plant's dark time so that its metabolism is modified and the plant's biological cycle is altered to suppress flowering and the induction of SvGly and Reb-A synthesis). In accordance of Serfaty et al., 2013 (Ind Crops Prod 50: 731-736, 2013. doi: http://dx.doi.org/10.1016/j.indcrop. 2013.08.063), the main phenological factor affecting the steviol-glycosides content in Stevia plants is flowering, which is induced by day length.

Author Response

see the letter in Response to referee 1.docx

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have modified the manuscript in light of the suggestions provided. Thus, I recommend acceptance of the present version of the manuscript for publication in the Journal of “Horticulture”.

 

Author Response

Thanks for your attention, review, and comments, they were very important to improving the manuscript.

Reviewer 3 Report

1   But there was no results or discussion about the  interaction of steviol glycosides and leaf biomass production in  the manuscript.

2  Line 62  ‘S. rebaudiana is a long night (LN) plant with a critical day length of about 12-13 h,’ 

Why in  Line 96 ‘Cuttings of genotype 2 (a long night plant; LN), and genotype 4 (a short night plant; SN)’ ?    How about the photoperiod-response character of this two genotypes?

3  I do not think there was ‘automatically move  other materials from the organs’. ‘automatically ’  do not used in plant physiology.

Author Response

see a letter with a response to referee 3.docx

Author Response File: Author Response.docx