Hyperhydricity-Induced Physiological Changes and Catechin Accumulation in Blueberry Hybrids (Vaccinium corymbosum × V. angustifolium)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsRajesh Barua and colleagues present an analysis of hyperhydricity (HH) in two blueberry hybrids. The study integrates both historical and metabolic data, revealing that HH may not only be a disadvantageous phenotype in propagation but also a potential means to enhance catechin accumulation in tissue cultures.
Overall, the manuscript is well-written, and the data are properly interpreted. However, the figures, particularly the SEM images, could be presented more professionally. Therefore, I recommend a minor revision to improve clarity and presentation.
Specific Comments:
- Title (Line 1): It is preferable to avoid abbreviations in the title for better clarity.
- Line 26: Please specify the full form of “HH” at its first mention.
- Line 30: Consider adding a concluding sentence to highlight the significance of the enriched compounds.
- Justification for Hybrid Selection: Please clarify the rationale for selecting these two hybrids. Are they diploid or tetraploid? Additionally, is HH specific to these hybrids, or do their parental lines also exhibit HH symptoms?
- SEM Figures: The SEM images could be presented more professionally. Consider adding scale bars for accuracy and indicating which images correspond to HH samples and which are controls to enhance readability.
These minor adjustments will improve the clarity and presentation of the study. I look forward to seeing the revised version.
Author Response
- Title (Line 1): It is preferable to avoid abbreviations in the title for better clarity.
Ans: Deleted all abbreviations in line number 1.
- Line 26: Please specify the full form of “HH” at its first mention.
Ans: Changed into full form “hyperhydricity” in line number 26.
- Line 30: Consider adding a concluding sentence to highlight the significance of the enriched compounds.
Ans: Updated in line between 30 – 32.
- Justification for Hybrid Selection: Please clarify the rationale for selecting these two hybrids. Are they diploid or tetraploid? Additionally, is HH specific to these hybrids, or do their parental lines also exhibit HH symptoms?
Ans: These two hybrids were available in our lab in the Center. Therefore we used them that provided a unique opportunity to conduct research on hyperhydricity. Half-highbush blueberries (Vaccinium corymbosum × V. angustifolium) are typically tetraploid (4x = 48). They inherit the tetraploid genome from the highbush blueberry (V. corymbosum, 4x) and the tetraploid genome from the lowbush blueberry (Vaccinium angustifolium, 4x), resulting in a tetraploid hybrid. Since only these two hybrids were available in our lab, our study focused on their response to different culture systems. We observed that hyperhydricity was more prevalent in liquid cultures than in semisolid media (authors mentioned in the line of 319-320).
- SEM Figures: The SEM images could be presented more professionally. Consider adding scale bars for accuracy and indicating which images correspond to HH samples and which are controls to enhance readability.
Ans: Scale bars are added in each image. Although for the scale bar in SEM figures, we provided detailed specifications for each image, including microscope settings, date, time, magnification, and other relevant parameters (one example shown below large version of Figure 3F).
All HH plants mentioned in line number 211, 212, 213, 214, 215, 216, 217 (D, M, E, N, F, O).
In line number 213, 214, 215, 216, 217 (G, P, H, Q, I, R) are naturally grown greenhouse plants’ figures which are control to enhance readability for hyperhydricity experiment.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article “NMR and SEM analysis of hyperhydricity-induced physiological changes and catechin accumulation in blueberry hybrids” by Rajesh Barua, Sayani Kundu, Abir U. Igamberdiev, Samir C. C Debnath presents a study on hyperhydricity in blueberry hybrids grown in vitro, using Scanning Electron Microscopy (SEM) and Nuclear Magnetic Resonance (NMR) analysis to shed light on physiological changes and the accumulation of bioactive compounds. My comments below are intended to help the authors.
In the introduction, some phrases are a little long or repetitive (e.g. “blueberry” appears several times in a row). I also note that it would be interesting to briefly mention why the two hybrids (HB1 and HB2) were chosen, although the details are left for the methods section.
In the material and methods section, I see the need to clarify the identity of the hybrids, which has been concealed in the text, which will limit reproducibility in other research and future comparisons with other studies. If the authors don't want to mention it, they should justify why. Is there a conflict of interest?
The results are well organized, clear, confirming the objectives and hypotheses formulated, with a comprehensive analysis of the effects of hyperhydricity.
The discussion has a good approach to the literature and is very well contextualized, in addition to being well aligned with the results observed. However, the study does not perform any genetic or molecular analysis to explain why HB2 was more susceptible to HH, missing the opportunity to explore genotypic variations. Furthermore, the discussion could explore other important metabolites such as quercetin, chlorogenic acid. I would also suggest exploring physiological tests such as photosynthesis, stomatal conductance and cellular respiration, which impact the actual functional assessment of the changes.
Author Response
In the introduction, some phrases are a little long or repetitive (e.g. “blueberry” appears several times in a row). I also note that it would be interesting to briefly mention why the two hybrids (HB1 and HB2) were chosen, although the details are left for the methods section.
Ans: Updated introduction and remove repetitive words/phrases in the line number 37, 38, 43, 47, 49, 52, 57, 59, 60. And, these two hybrids were available in our lab center therefore these we used it which providing a unique opportunity to conduct research based on hyperhydricity.
In the material and methods section, I see the need to clarify the identity of the hybrids, which has been concealed in the text, which will limit reproducibility in other research and future comparisons with other studies. If the authors don't want to mention it, they should justify why. Is there a conflict of interest?
Ans: According to Agriculture and Agri-Food Canada’s intellectual property rights, confidentiality agreements, regulatory policies as well as The Canadian Food Inspection Agency administers the Plant Breeders' Rights Act (1990) and regulations which provide legal protection to plant breeders for new plant varieties for up to 25 years for a variety of tree and vine (including their rootstocks), and 20 years for all other varieties of plants.
In general, these two half-highbush blueberries (Vaccinium corymbosum × V. angustifolium) typically inherit the tetraploid genome from the highbush blueberry (Vaccinium corymbosum, 4x) and the tetraploid genome from the lowbush blueberry (V. angustifolium, 4x), resulting in a tetraploid hybrid. These two hybrids were developed at the St. John’s Research and Development Centre, Agriculture and Agri-Food Canada, Newfoundland and Labrador, Canada. The plant materials used in this study comply with relevant institutional, national, and international guidelines and legislation.
The discussion has a good approach to the literature and is very well contextualized, in addition to being well aligned with the results observed. However, the study does not perform any genetic or molecular analysis to explain why HB2 was more susceptible to HH, missing the opportunity to explore genotypic variations. Furthermore, the discussion could explore other important metabolites such as quercetin, chlorogenic acid. I would also suggest exploring physiological tests such as photosynthesis, stomatal conductance and cellular respiration, which impact the actual functional assessment of the changes.
Ans: This study evaluates hyperhydricity (HH) conditions by analyzing morphological traits and water content in two half-highbush blueberry hybrids. Besides, scanning electron microscopy (SEM) and histological analysis were utilized to investigate physiological and cellular changes, including stomatal density, structural modifications, vascular bundle (xylem and phloem) orientation, and trichome development. Additionally, nuclear magnetic resonance (NMR) was employed for the first time to explore HH-induced metabolic alterations. Further study will be conducted on global transcriptome analysis and whole-genome sequencing to clarify different genes involvement and the genetic variations associated with hyperhydricity. And in the discussion section, authors mentioned various untargeted metabolic compounds: β-P-arbutin, 2-O-caffeoyl arbutin, Quercetin-3-O-glucuronide, Quercetin-3-O-glucoside, Chlorogenic acid, -CH=CH- of fatty acids, β-glucose, fatty acid chains (-CH2-), the acetyl group, CH3- of alpha-linolenic acid and β-coumaroyl.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe difference in tissue anatomy is not due to who manufactured the culture vessel (Growtek vs Sigma) but instead is liquid medium vs. semis-solid agar. This is necessary to compare these results with other prior works. Also the Sigma vessel is more commonly referred to as a baby food jar with Magenta-type polypropylene closure. Sigma does not make the vessel, it is merely a distributor of a common food service item.
Anatomical work was done on plants from liquid and agar, as I understand, but selections were not done for hyprehydric vs. non-hyperhydric tissues within those vessels. However, the results and discussion of NMR data refer to metabolites produced in hyperhydric vs. non-hyperhydric plants. I am especially interested to see the RWC comparisons of hypehydric vs non-hypehyric plants within the same vessel, since this difference is due to plant developmental anomalies, not the microenvironment. The two types of comparisons are not equivalent, although the authors handle them as equivalent comparisons.
Line 12 survival,
Line 121 where did greenhouse plants origninate, tc? What was GH environment conditions
Line 182 was hyperhydirc and non hyperhydric tissue separated for water content data
Line 255 why cant figure 4 be organized like figure 3
Line 281, 404 here you make statmetns about hyperhydric and non-hyperhydric plants, why is this not done in anatomical work
Line 342 why is liquid system high nutrient, wasn’t the same nutrient formulation used
Line 429 you are using plant growth regulators (PGR) not hormones
Author Response
The difference in tissue anatomy is not due to who manufactured the culture vessel (Growtek vs Sigma) but instead is liquid medium vs. semis-solid agar. This is necessary to compare these results with other prior works. Also the Sigma vessel is more commonly referred to as a baby food jar with Magenta-type polypropylene closure. Sigma does not make the vessel, it is merely a distributor of a common food service item.
Ans: This experimental study mentioned the difference between liquid medium vs. semi-solid medium. “Sigma glass baby food jar” updated in line number 15, 88-89, 94, 96, 99.
Anatomical work was done on plants from liquid and agar, as I understand, but selections were not done for hyprehydric vs. non-hyperhydric tissues within those vessels. However, the results and discussion of NMR data refer to metabolites produced in hyperhydric vs. non-hyperhydric plants. I am especially interested to see the RWC comparisons of hypehydric vs non-hypehyric plants within the same vessel, since this difference is due to plant developmental anomalies, not the microenvironment. The two types of comparisons are not equivalent, although the authors handle them as equivalent comparisons.
Ans: This study evaluates hyperhydricity (HH) conditions by analyzing morphological traits and water content in two half-highbush blueberry hybrids using sigma glass baby food jar and Growtek vessel. Besides, scanning electron microscopy (SEM) and histological analysis were utilized to investigate physiological and cellular changes, including stomatal density, structural modifications, vascular bundle (xylem and phloem) orientation, and trichome development. Additionally, nuclear magnetic resonance (NMR) was employed for the first time to explore HH-induced metabolic alterations. The hyperhydricity experiment was done for the first time at St. John’s Agriculture and Agri-Food Canada lab. As hyperhydricity was not observed in semi-solid media, instead of high susceptible in liquid media (authors mentioned through reference in the line number 318-319, 341-342, 360-362, 399-400), therefore authors experimented liquid cultured Growtek vessel for hyperhydric plantlet observation.
Line 12 survival,
Ans: Updated.
Line 121 where did greenhouse plants origninate, tc? What was GH environment conditions?
Ans: Greenhouse plants originated from tissue culture explants; mentioned in the line number 90-91. Greenhouse environment conditions mentioned in the line number 90, 91, 93.
Line 182 was hyperhydirc and non hyperhydric tissue separated for water content data
Ans: For water content, separated tissue samples were collected from sigma glass baby food jar, Growtek vessel and greenhouse pot; individually. Authors mentioned in the line number of 187, 188.
Line 255 why can’t figure 4 be organized like figure 3
Ans: In figure 3, add scale bars in each image. Although for the scale bar in SEM figures, we provided detailed specifications for each image, including microscope settings, date, time, magnification, and other relevant parameters (one example shown below large version of Figure 3F).
All HH plants mentioned in line number 211, 212, 213, 214, 215, 216, 217 (D, M, E, N, F, O).
In line number 213, 214, 215, 216, 217 (G, P, H, Q, I, R) are naturally grown greenhouse plants’ figures which are control to enhance readability for hyperhydricity experiment.
In figure 4, mentioned which images are hyperhydric (C, I, D, J) in line number 257, 258, 259, 260. First 3 row belongs to HB1 hybrid and second 3 row belongs to HB2 hubrids of sigma, growtek and greenhouse plants respectively to enhance the readability.
Line 281, 404 here you make statements about hyperhydric and non-hyperhydric plants, why is this not done in anatomical work
Ans: In this experiment, authors described both hyperhydric and non-hyperhydric stomatal orientation, mesophyll tissue through scanning electron microscopy. Besides, authors elaborated both hyperhydric and non-hyperhydric stem surface and vascular bundle (xylem and phloem) distribution through histological analysis.
Line 342 why is liquid system high nutrient, wasn’t the same nutrient formulation used
Ans: In a liquid culture, plant tissues are fully immersed, ensuring direct contact with nutrients, leading to faster absorption compared to semi-solid media, where nutrients diffuse more slowly. Authors mentioned with references in line number 318-319, 324-325. The media composition for both semi-solid media and liquid media are same except semi solid media add extra gerlite and agar for solidification.
Line 429 you are using plant growth regulators (PGR) not hormones
Ans: Updated in line number 430 and 331.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have improved the MS accordingly. I agree to accept it.
Reviewer 2 Report
Comments and Suggestions for Authors Sugestion:I believe that authors should use the justifications provided for my questions throughout
the text, avoiding questions from the reader.
