Sex Determination During Inflorescence Bud Differentiation in Monoecious Pistacia chinensis Bunge
Round 1
Reviewer 1 Report
This is a complete descriptive study of the sex determination in Pistacia chinensis. The most important finding is that it occurs during floral differentiation.
The most interesting question undetermined is why the sex expression of grafted trees was inconsistent with that of the scion, suggesting that the monoecious character did not result from a bud mutation.
Author Response
Thank you for your positive comments. We have modified the language and style of the manuscript with the English editing service of MDPI. We hope that the revisions in the manuscript will now be suitable for publication in “Forests”.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript, the authors dealed with a very relevant subject: the reproductive biology of an important species for bioenergy purposes in China: Pistacia chinensis.
The results seem relevant and the combination of molecular and anatomical/morphological methods is attractive and help a wider view of the issue.
However, I found the current manuscript difficult to follow and probably needing some rethinking, in the following items:
Firstly, perhaps is my fault, but I frequently got lost among the variety of terms used for referring to the sex/gender expression for individual buds, branches and trees. I think the terms sex and gender are used sparsely as synonyms, but often in a confusing manner (see specific comments).
Second, it strikes me that no attempt of quantification was done in the morphological/anatomical study, which turns it 100% descriptive. I mean, just basic statistics, supported by plots of the developmental phases and changes observed could really help a more thorough discussion. I also wonder if all the morphological terms used are clear for a wide scope of readers (see specific comments).
Third, It seems that the fact that the ancestral character of Pistacia was monoecious is not properly commented at the Introduction and used as the basis for the hypothesis/expectations, but rather commented only at the Discussion (l. 364-366). I think the Introduction should be more complete in this aspect, and also commenting more extensively the findings in other genera with variable gender expression.
Fourth, the manuscript indeed need a thorough English editing. There are too many odd phrases and grammatical mistakes that make the text difficult to follow.
Specific comments
L 25: please use ‘anatomical analysis’, or ‘microscopy’ instead of ‘paraffin section’
L 33-35: I think these are no proper conclusions
L 47: I think ‘fortunately’ is out of the point
L 54: please change ‘discovery’ into ‘finding’ or ‘identification’
L 61: change ‘can’ into ‘could’
L 63: change ‘always’ into ‘accurately’
L 69: While determining the sex molecular mechanism is hard to achieve...
L 87-88: The phrase starting ‘For different…’ doesn’t read well. Please rewrite.
L 90: change ‘solve these issues’ into ‘answer these questions’
L 95: ‘sex types of monoecism as scions’ ???
L 95-98: This sentences are out of place here. Only Objectives/Hypothesis should be end the Introduction
L 102-104: Please, improve the description: is this a natural population, or a plantation? What is the exact meaning of 'wild'?
L 104, 108, 109, 143, 150, 174, 175, 177, 178, 184, 190, 195-197, 203 (Table 3), 258: please, check the consistency of the usage of terms between sex, gender, monoecious, bisexual, etc.
L 248, 358: I think ‘alternative is more adequate than ‘opposing’
L 208: Is ‘shoulder’ the right morphological term for this?
L. 212: I think ‘anatomical’ is more accurate than ‘internal morphological’
L 222: I wonder if ‘apical point’ is a right morphological term
L 225: medial bract
L 233: delete ‘to’
L 252: change ‘round’ into ‘phase’
L274: please, change ‘germinated’ into ‘burst’!!! Buds DO not germinate, only seeds or pollen
L 324: ‘differences IN…’
L 337: please change ‘can directly affect’ into ‘is inherently linked to’
L 343-345: Please do not repeat methodology description here
L 352: ‘nutritions’ is not a sound term
L 364-367: This should be commented more deeply at the introduction, as a starting point and as the basis for the hypotheses to be checked
L 381-382: This is not a conclusion
Author Response
(Notes: If the images cannot be displayed here, please see the images in Word version. All the line numbers correspond to the track changes version.)
Comments: In this manuscript, the authors dealed with a very relevant subject: the reproductive biology of an important species for bioenergy purposes in China: Pistacia chinensis.
The results seem relevant and the combination of molecular and anatomical/morphological methods is attractive and help a wider view of the issue.
However, I found the current manuscript difficult to follow and probably needing some rethinking, in the following items:
Firstly, perhaps is my fault, but I frequently got lost among the variety of terms used for referring to the sex/gender expression for individual buds, branches and trees. I think the terms sex and gender are used sparsely as synonyms, but often in a confusing manner (see specific comments).
Second, it strikes me that no attempt of quantification was done in the morphological/anatomical study, which turns it 100% descriptive. I mean, just basic statistics, supported by plots of the developmental phases and changes observed could really help a more thorough discussion. I also wonder if all the morphological terms used are clear for a wide scope of readers (see specific comments).
Third, It seems that the fact that the ancestral character of Pistacia was monoecious is not properly commented at the Introduction and used as the basis for the hypothesis/expectations, but rather commented only at the Discussion (l. 364-366). I think the Introduction should be more complete in this aspect, and also commenting more extensively the findings in other genera with variable gender expression.
Fourth, the manuscript indeed need a thorough English editing. There are too many odd phrases and grammatical mistakes that make the text difficult to follow.
Responses: Thank you for your patience! Your comments were highly insightful and enabled us to greatly improve the quality of our manuscript. Our responses to your main comments have been listed as followed:
Firstly, we are sorry to make you confused. We have changed all ‘gender’ into ‘sex’ and ‘genders’ into ‘sexes’ throughout the manuscript. There are only three sex types for a floret, including female, male and bisexual (with pistil and stamens). But the inflorescences could bear several hundred individual florets, involving female, male, monoecious (when the florets have mixed sex types), and bisexual type (when the florets are bisexual). Inflorescence buds occurred on one-year-old shoots, so the sex types for single shoots were identified by flowering observations, involves female, male, monoecious (bearing mixed inflorescences or florets), bisexual (bearing bisexual florets), or non-flower. In the same way, the sex expressions of branches and trees include female, male and monoecious in this study, because no individual tree bears fully bisexual florets has been found. In addition, in the early stages of floral development, florets of all sex types have both pistil and stamen primordia, and this stage is also called bisexual stage.
Second, thank you for your valuable advice on quantification. Below are our explanations for the lack of statistical data:
1) The study purpose is to find the sex differentiation stage and the method for judgment the internal processes without anatomy. Initially, we tried to correlate sexes with certain indicators such as the length of shoot, transverse diameter and longitudinal diameter of the bud, while the differences among different plants were greater than that between sexes, and the bud size of the same sex on the same tree was also uneven, so the data were abandoned. Then, we found that internal processes correspond to phenological phase and external morphology, so we list the ontogenetic timeline of floral development (Table 4), and showed the corresponding external morphology link to the internal process in the figures.
2) Also, we have tried to show sex differences in leaf nutrients between sexes during floral development (Figure 1, 2).
Fig. 1. The dynamic changes of sugar, starch and soluble sugar content during flower bud differentiation periods in monoecious P. chinensis
Fig. 2. The dynamic changes of N, P and K content during flower bud differentiation periods phase in monoecious P. chinensis
However, the results were not promising. An expert gave us the following advice:
“Your descriptions and discussion of the male and female inflorescence differentiation is new information and very well done.
However, I think you weaken an excellent paper by presenting the nutrient and carbohydrate data.
First, there are no statistics on the data to indicate significant differences.
Second, a female shoot would have fruit on the one year old wood that in the edible bearing Pistacia species strongly affect nutrient and carbohydrate status; you did not account for this.
Third, the increase in carbohydrates could related to leaf maturity, when the leave on current year's shoot growth became net exporter of photosynthates instead of supporting shoot growth.
I suggest you rewrite this manuscript without the nutrient and carbohydrate discussion and focus on the bud differentiation which is new, and excellent, information.”
Therefore, we removed this section and integrated the molecular and grafting data to focus on the topic of sex determination occurring in inflorescence bud differentiation in monoecious Pistacia chinensis Bunge.
3) Your suggestion provides us with a good research direction. We will add more quantitative data in the follow-up work, including internal physiological changes and external meteorological factors. Also, we have added it at the Disccusion in lines 623-629.
Third, many studies have hypothesized that dioecism evolved from hermaphroditic ancestors. Based on this hypothesis and in combination with the sex expressions of the Pistacia species, we hypothesized that their ancestors were also monoecious. We have added this aspect at the Introduction accordingly (see specific comments).
Fourth, we have modified the manuscript accordingly and made paper more readable with the help of English editing service of MDPI. We hope that the revisions in the manuscript will now be suitable for publication in “Forests”.
Below we explain in detail how they were addressed in the revised manuscript.
Specific comments
Point 1: L 25: please use ‘anatomical analysis’, or ‘microscopy’ instead of ‘paraffin section’
Response 1: Thank you and we have revised ‘paraffin section’ into ‘anatomical analysis’ in line 24.
Point 2: L 33-35: I think these are no proper conclusions
Response 2: Thank you and we have revised the conclusions into ‘Taken together, no sex-associated DNA marker was found, sex expressions were unstable after grafting, and the alternative sex organs appeared in the early stage of sex differentiation, suggesting that sex determination occurred during floral development instead of the early vegetative period. These results indicated that the sex expressions may be affected by environmental factors, increasing the understanding of sex determination mechanisms in P. chinensis and other species’ in lines 32-37.
Point 3: L 47: I think ‘fortunately’ is out of the point
Response 3: Thank you and we have changed it into ‘However’ in line 72.
Point 4: L 54: please change ‘discovery’ into ‘finding’ or ‘identification’
Response 4: Thank you and we have changed ‘discovery’ into ‘finding’ in line 79.
Point 5: L 61: change ‘can’ into ‘could’
Response 5: Thank you and we have changed ‘can’ into ‘could’ in line 86.
Point 6: L 63: change ‘always’ into ‘accurately’
Response 6: Thank you and we have changed ‘always’ into ‘accurately’ in line 88.
Point 7: L 69: While determining the sex molecular mechanism is hard to achieve...
Response 7: Thank you and we have revised ‘It is hard to determine the sex determination mechanism at the molecular level, but’ into ‘While determining the sex molecular mechanism is hard to achieve,’ in line 94.
Point 8: L 87-88: The phrase starting ‘For different…’ doesn’t read well. Please rewrite.
Response 8: We are sorry for that and have revised ‘For different parts that express different genders on a monoecious tree, are their amplification patterns the same’ into ‘Do different parts that express different sexes on the same monoecious trees show different amplification patterns’ in lines 140-142.
Point 9: L 90: change ‘solve these issues’ into ‘answer these questions’
Response 9: Thank you and we have changed ‘solve these issues’ into ‘answer these questions’ in line 144.
Point 10: L 95: ‘sex types of monoecism as scions’ ???
Response 10: We are sorry for that and have revised ‘gender stability was observed by grafting using various sex types of monoecisms as scions’ into ‘sex stability was observed by grafting the shoots that expressed different sex types on monoecious plants’ in lines 147-149.
Point 11: L 95-98: This sentences are out of place here. Only Objectives/Hypothesis should be end the Introduction
Response 11: Thank you and we have revised ‘More importantly, the sex differentiation of male and female flower buds on monoecious P. chinensis is reported for the first time in this study. These studies can provide new information regarding sex expression in Pistacia and promote the application of monoecious P. chinensis’ into ‘Moreover, the sexual development of male and female flower buds on monoecious P. chinensis was observed to provide new information regarding sex expression in monoecious P. chinensis’ in lines 149-151.
Point 12: L 102-104: Please, improve the description: is this a natural population, or a plantation? What is the exact meaning of 'wild'?
Response 12: Thank you and we have changed ‘wild and discretely distributed’ into ‘discretely distributed in a natural population’ in line 156.
Point 13: L 104, 108, 109, 143, 150, 174, 175, 177, 178, 184, 190, 195-197, 203 (Table 3), 258: please, check the consistency of the usage of terms between sex, gender, monoecious, bisexual, etc.
Response 13: We are sorry to make you confused. We have changed all ‘gender’ into ‘sex’ and ‘genders’ into ‘sexes’ throughout the manuscript. There are only three sex types for a floret, including female, male and bisexual (with pistil and stamens). But the inflorescences bear up to several hundred individual florets, involving female, male, monoecious (when the florets have mixed sex types), and bisexual type (when the florets are bisexual). Inflorescence buds occurred laterally on one-year-old shoots, so the sex types for single shoots were identified by flowering observations, involves female, male, monoecious (bearing mixed inflorescences or florets), bisexual (bearing bisexual florets), or non-flower. In the same way, the sex expressions of branches and trees include female, male and monoecious in this study, because no individual tree bears fully bisexual florets has been found. In addition, in the early stages of floral development, florets of all sex types have both pistil and stamen primordia, and this stage is also called bisexual stage.
Point 14: L 248, 358: I think ‘alternative is more adequate than ‘opposing’
Response 14: Thank you and we have changed ‘opposing’ into ‘alternative’ in line 395, and 533.
Point 15: L 208: Is ‘shoulder’ the right morphological term for this?
Response 15: Thank you for your question. The inflorescence of P. chinensis is panicle which is similar to that of grape, and we tried to express the meaning of secondary spike which probably means the “shoulder” according to the figure below (Christensen 2000).
Reference:
Christensen, L. P. (Ed.). (2000). Raisin production manual (Vol. 3393). UCANR Publications.
Point 16: L. 212: I think ‘anatomical’ is more accurate than ‘internal morphological’
Response 16: Thank you and we have changed ‘internal morphological’ into ‘anatomical’ in line 352.
Point 17: L 222: I wonder if ‘apical point’ is a right morphological term
Response 17: Thank you. We used ‘apical point’ to show the growing point according to Zhang’s study which described the process of female bud differentiation in P. chinensis (see the figure below). Also, it was used in some other references (Du et al. 1996; Graman 1972).
References:
Zhang, Y.Q.; Qi, G.H.; Li, B.G.; Guo, S.P.; Zhang, X.M.; Qi, K. Female Flower Bud Differentiation in Pistacia chinensis. Acta Bot. Boreal-Occident Sin. 2011, 31, 972–976.
Du, L.; Li, Y.; Ma, M.; Zhang, Z.; Zhu, Z.; Gong, Z. Preliminary study on transformation of wheat apical point. Acta Bot. Sin. 1996, 38, 921–924.
Graman, J. Development of apical point of broadbean from emergence to beginning of period of flowering. Rost. Vyroba 1972, 18, 431–41.
Point 18: L 225: medial bract
Response 18: We are sorry for the mistake. We have changed ‘each bract medial formed a shoulder’ into ‘a shoulder formed inside each bract’ in ine 365.
Point 19: L 233: delete ‘to’
Response 19: Thank you and we have deleted ‘to’ in line 373.
Point 20: L 252: change ‘round’ into ‘phase’
Response 20: Sorry, it seems mistaken expression in the original manuscript. We have revised ‘female buds entered the second round of the petal differentiation stage while the first round petals developed’ into ‘female buds entered the differentiation stage of the second round petals while the first round petals continued to develop’ in lines 403-404.
Point 21: L274: please, change ‘germinated’ into ‘burst’!!! Buds DO not germinate, only seeds or pollen
Response 21: We are really sorry for this error and have changed ‘germinated’ into ‘burst’ throughout the manuscript.
Point 22: L 324: ‘differences IN…’
Response 22: We are sorry for that and have added ‘in’ after ‘differences’ in line 511.
Point 23: L 337: please change ‘can directly affect’ into ‘is inherently linked to’
Response 23: Thank you and we have changed ‘can directly affect’ into ‘is inherently linked to’ in line 505.
Point 24: L 343-345: Please do not repeat methodology description here
Response 24: Thank you and we have deleted ‘For the different sexes of monoecious P. chinensis, the banding patterns of leaf DNA were compared and the shoots were grafted and observed, showing that no difference among sexes at the DNA level has been found and the gender expressions were unstable’ in line 520.
Point 25: L 352: ‘nutritions’ is not a sound term
Response 25: Thank you and we have changed ‘nutritions’ into ‘nutrients’ in line 526.
Point 26: L 364-367: This should be commented more deeply at the introduction, as a starting point and as the basis for the hypotheses to be checked
Response 26: Thank you and we have added relevant contents at the introduction in lines 96-137.
Point 27: L 381-382: This is not a conclusion
Response 27: Thank you and we have deleted ‘this study explored the sex determination mechanism of P. chinensis at the DNA level, phenotypic level, and organogenesis level’ in line 631.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript by Bai and co-workers attempted to gain insights into the mechanisms underlying the sex determination of buds in P.chinensis. The authors recorded the phenology of budding tress and tried to establish molecular methods through DNA based markers to identify the sex in early stages. The histological sections of growing buds have been examined and nicely presented.
Though the attempts to establish the molecular markers to determine the sex had little success, the authors may need to discuss other tools that could potentially circumvent this issue.
If the occurrences of the different sex expression are responsive to external cues, I wonder if the authors could attempt to link any environmental parameters (temperature/precipitation etc.) during the period of phenological changes. Furnishing such ecological parameters during the period of the experiments would be helpful.
Author Response
(Notes: If the image cannot be displayed here, please see it in Word version. All the line numbers correspond to the track changes version.)
Comments: The manuscript by Bai and co-workers attempted to gain insights into the mechanisms underlying the sex determination of buds in P.chinensis. The authors recorded the phenology of budding tress and tried to establish molecular methods through DNA based markers to identify the sex in early stages. The histological sections of growing buds have been examined and nicely presented.
Though the attempts to establish the molecular markers to determine the sex had little success, the authors may need to discuss other tools that could potentially circumvent this issue.
If the occurrences of the different sex expression are responsive to external cues, I wonder if the authors could attempt to link any environmental parameters (temperature/precipitation etc.) during the period of phenological changes. Furnishing such ecological parameters during the period of the experiments would be helpful.
Response: Thank you for your positive comments and valuable suggestions to our manuscript. To provide other tools to screen the sex associated markers, we have added ‘Thus, it might be more fruitful to look at RNA expression during the different ontogenetic stages described in this paper. Sex-specific quantitative transcriptomic data (mRNA and small RNA), which have the time–space specific expression, can be compared to produce a set of candidate genes [12,40–43]. Furthermore, protein is inherently linked to phenotypic differentiation, so the proteomics can provide a fundamental understanding of the biological changes [44]. Hence, proteomic analysis among different sex types during sex differentiation phases could offer more clues on sex differentiation. To have a global view on sex determination, subsequent research could integrate multi-omics and multiple levels of morphology, cytology, physiology, and molecular biology’ in lines 497-510.
Your suggestion about environmental parameters is of great value to our study. However, we failed to present the corresponding environmental parameters during the floral development. Below are our explanations for it:
1) The monoecious trees were found in the wild mountain areas, and the developmental processes varied greatly among individuals. Our previous study has shown that the blooming phenophase changed a lot among different individuals (see the Figure 7 below), and sex types were unstable in successive years (Bai et al. 2016). The differentiation processes of different individuals in the same region were not consistent, so we failed to correlate sex differentiation with certain environmental factors base on meteorological data.
2) The purpose of this study is to find the sex differentiation stage and the method for judgment the internal processes without anatomy. As we have explained at the Materials and Methods, the sex expression of the selected monoecious tree was stable in successive years according to previous observations. We found that internal processes correspond to phenological phase and external morphology, so we list the ontogenetic timeline of floral development (Table 4), and showed the corresponding external morphology link to the internal process in the figures. Using the phenological characteristics of shoots and the external morphology of buds, we can judge the internal differentiation process of other individuals, providing a basis for the accurate sampling of subsequent studies.
Fig. 7. Blooming phenophase of local P. chinensis
The detailed blooming date of normal trees and different sex types on the same monoecious tree. Nf: normal female trees, M1–M23: monoecious trees.
3) Besides, your suggestion provides us with a good research direction. We will add more data in the follow-up work base on this study, including internal physiological changes and external meteorological factors. Also, we have added ‘The sex differentiation may link to certain environmental parameters, such as temperature and precipitation, but we failed to present the corresponding environmental parameters during the floral development due to the differences in developmental processes among individuals. To further understand the sex determination mechanism and develop plant sex control technology, differentially expressed genes, hormones, nutrients, and environmental parameters should be studied’ in lines 624-629.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
I have read the thorough answer to my previous review and I am quite satistied with the changes undertaken and/or the explanations given (for example, about the lack of quantitative data). Also I think the English has improved considerably, although I am not a native speaker. Therefore I consider it suitable for publication apart from further editor suggestions.
