Cold Treatment Modulates Changes in Primary Metabolites and Flowering of Cut Flower Tulip Hybrids

Round 1

Reviewer 1 Report

Vernalization is very important for tulips planting. In this paper, the authors used two tulips cultivars and temperature treatments to investigate the Physiological substances change and flowering. It is very useful for tulips planting. I think it could be accepted after minor revisions.

 

1. About figure 1, move the cultivar and temperature to the up of the figure, and analyze the relationship between different cultivars and temperatures. Which substance is very important for flowering, the authors should be discussed in the manuscript. The abbreviation in the figure should be introduced in the figure legend
2. the title should be revised, such as Physiological substances change and flowering of …., and the main text should also be revised.
3. Flower color should be measured, it is vey important characters for flowering quality.

Author Response

Vernalization is very important for tulips planting. In this paper, the authors used two tulips cultivars and temperature treatments to investigate the Physiological substances change and flowering. It is very useful for tulips planting. I think it could be accepted after minor revisions.

1. About figure 1, move the cultivar and temperature to the up of the figure, and analyze the relationship between different cultivars and temperatures. Which substance is very important for flowering, the authors should be discussed in the manuscript. The abbreviation in the figure should be introduced in the figure legend

The figure has been changed according to the suggestion of the reviewer.

The relationship between different cultivars and temperatures, particularly in bulbs before flowering, were discussed and the delay in flowering of Ad rem pre-treated at 5 °C correlated to the incapacity to accumulate sucrose, as proved by the significant increase of metabolites previously found involved in cold stress and /or acclimation, such as threonine, sucrose, proline or GABA [25,26,27] (Figure 1). Proline and GABA may be rapidly accumulated for cell protection against stress, mainly as ROS scavengers for stabilizing membranes and macromolecules against oxidative stress, and then broken down upon relief of stress to supply carbon, nitrogen and energy to recover and repair stress induced damages [25]. Moreover the synthesis of GABA is a proton consuming reaction able to cope with the cytosolic acidosis that can happen in response to an abrupt transfer to lower temperature [28]. Sucrose, that highly increased, together with threonine, in particular in H1 bulbs pre-treated at 5 °C, in addition to having a protective role against cold stress being also indirectly helpful as a substrate for metabolism, can directly regulate cold-acclimation by mediating an in-crease in GUS activity, as a reporter for the activity of the cold-responsive COR78 promoter in Arabidopsis [29]. Regarding threonine, Klein et al. [30] and Yuan et al. [31] have both explained that its increase under cold acclimation could depend on its function as precursor of the aspartate-derived amino acid pathway, in particular for the synthesis of branched chain amino acids (BCAAs). BCAAs may serve as substrate for the synthesis of stress-induced proteins, or act as signaling molecules to regulate stress related genes’ expression [32]. BCAAs may also play a role as ROS scavengers through a not fully elucidated mechanism and/or function as alternative electron donors for the mitochondrial electron transport chain to allow plant growth after stress relief [33]. In H2 bulbs pre-treated at 9 °C, the symptoms of cold stress were perceived as milder, as shown by the lower proline and threonine levels than in other bulbs before planting (Figure 1).

2. the title should be revised, such as Physiological substances change and flowering of …., and the main text should also be revised.

The title has been revised taking into account the suggestion.

3. Flower color should be measured, it is very important characters for flowering quality.

We thank the reviewer for this comment. We agree that flower colour is a relevant feature in cut flowers and ornamental crops. Since this measure was not performed in the present experiment which focused mainly on metabolic aspects, this is a useful suggestion for ongoing research on flower bulbs under similar treatments.

Author Response File: Author Response.docx

Reviewer 2 Report

At first, there is a fundamental mistake in authors’ understanding of plant physiology terms. “Vernalization” is a phenomenon in which flower bud differentiation is induced by encountering a predetermined amount of low temperature. However, after the tulips dig up the bulbs in the late spring, the bulbs themselves are dormant, but the flower bud differentiation progress and the flower bud formation completed in about August. Therefore, the vernalization phenomenon does not occur in tulips. Low temperatures after autumn are necessary conditions for flower stem elongation and normal flowering.

However, the authors explain the low temperature effects as vernalization and even put it in the title. Although the points are clearly stated in the references 2 and 9 cited by the authors, this paper needs to be completely rewritten in that the argument is being advanced with a misunderstanding.

In addition, the International Code of Nomenclature for Cultivated Plants stipulates that cultivar names of cultivated plants should be enclosed in single quotation marks. In this paper, the cultivar names are shown in italics, and there is a risk of causing a great misunderstanding as the genus name and species name.

Furthermore, the unit of EC value is indicated by “dS / m” in the International System of Units regulations. “μS / cm” is inappropriate.

As mentioned above, because it lacks reliability due to many mistakes in basic descriptions such as plant physiology, horticulture, and soil chemistry, this manuscript is rejected.

Author Response

At first, there is a fundamental mistake in authors’ understanding of plant physiology terms. “Vernalization” is a phenomenon in which flower bud differentiation is induced by encountering a predetermined amount of low temperature. However, after the tulips dig up the bulbs in the late spring, the bulbs themselves are dormant, but the flower bud differentiation progress and the flower bud formation completed in about August. Therefore, the vernalization phenomenon does not occur in tulips. Low temperatures after autumn are necessary conditions for flower stem elongation and normal flowering.

However, the authors explain the low temperature effects as vernalization and even put it in the title. Although the points are clearly stated in the references 2 and 9 cited by the authors, this paper needs to be completely rewritten in that the argument is being advanced with a misunderstanding.

We thank the Reviewer for highlighting this mistake. Indeed, the response to low temperature in tulips is different from the classical vernalization response, as the cold exposure promotes the stem elongation and the anthesis rather than the meristem flower induction, and these effects occur after warm temperatures have already induced the flower transition. The term “vernalization” is quite common for tulip in informal language among growers, and our collaboration with the farm was misleading in choosing the proper terminology. We have replaced the word “vernalization” with “preparation procedure” or “cold treatment” throughout the manuscript. In addition, the English language has been checked and improved.

 

In addition, the International Code of Nomenclature for Cultivated Plants stipulates that cultivar names of cultivated plants should be enclosed in single quotation marks. In this paper, the cultivar names are shown in italics, and there is a risk of causing a great misunderstanding as the genus name and species name.

We thank the Reviewer for highlighting this imprecision. To our best knowledge, the name of commercial hybrids does not necessarily follows the rules regulating nomenclature of cultivated varieties (cultivars), and this is what we understood also by reading the International Code of Nomenclature for Cultivated Plants, article 2 (Brickell et al., 2016, 9th edition, Published by the International Society for Horticultural Science - ISHS). However, since the absence of single quotation marks can actually cause a misunderstanding, we have corrected the name of the hybrids throughout the manuscript.

 

Furthermore, the unit of EC value is indicated by “dS / m” in the International System of Units regulations. “μS/cm” is inappropriate.

We thank the Reviewer for highlighting this error. The unit of measure has been corrected and the values of electrical conductively have been converted accordingly.

 

As mentioned above, because it lacks reliability due to many mistakes in basic descriptions such as plant physiology, horticulture, and soil chemistry, this manuscript is rejected.

After reading this final comment, we have read the manuscript carefully in order to identify the mistakes in physiology, horticulture, and soil chemistry mentioned by the reviewer. While we found the comment about the lack of precision in describing the effects of cold temperature (Vernalization) on plant development in tulip (physiological and horticultural issues) as useful to improve the manuscript, we could not recognize the weakness in soil chemistry. We would be grateful if the reviewer could provide a further explanation on this aspect.

Author Response File: Author Response.docx

Reviewer 3 Report

 

It is a good article and will be beneficial mainly to flower producers. However, there are minor English spells required.

The authors mentioned in the materials and methods section that chlorophyll and carotenoids were analyzed but were not clear in the data, so please clarify. In addition, some of the abbreviations in the data do not have full names in the text, so please check all the abbreviations. 

 

 

Author Response

It is a good article and will be beneficial mainly to flower producers. However, there are minor English spells required.

English language has been checked throughout the manuscript.

 

The authors mentioned in the materials and methods section that chlorophyll and carotenoids were analyzed but were not clear in the data, so please clarify.

The reason why chlorophylls and carotenoids data were not included in the PCA and heat map analysis has been explained “The data of chlorophylls and carotenoids, measured only in the leaves, were not in-cluded in these analyses. This choice was done because if these data had been included, it would have been impossible to compare the metabolism changes in bulbs and leaves at the same time. Anyway, their differences were not significant, demonstrating that these parameters were unaffected by different vernalization procedures.” (see lines).

In addition, some of the abbreviations in the data do not have full names in the text, so please check all the abbreviations.

Full names and related abbreviations of amino acids have been added in the Table 4 caption.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

To authors of horticulturae-1658855

Ask for trivial corrections.

The name of the variety on the 19th line is changed from the double quotation mark to the single quotation mark.

The cultivar name ‘Ad rem’ has been changed to ‘Ad Rem’ throughout.

The cultivar name in Table 4 is italic, but it should be corrected to roman.

Thank you.

Author Response

We thank the reviewer for his last suggestions.

All the requests have been addressed in the current version of the manuscript.

Best regards,

Roberta Paradiso (on behalf of all the authors)

Author Response File: Author Response.docx