Chrysanthemum CmHSP90.5 as a Tool to Regulate Heat and Salt Stress Tolerance

Round 1

Reviewer 1 Report

The manuscript submitted by Wang et al, titled " Chrysanthemum CmHSP90.5 negatively regulates heat and salt stress tolerance in Arabidopsis” evaluated the roles of CmHSP90.5 in response to abiotic stresses in chrysanthemum.  Overall, the manuscript is well written. The method section needs to be elaborated. Further, the author has nicely correlated the tolerance phenotype to the Na⁺ accumulation, gene expression of known transporter to extrude salt, quantified proline, and expression of the key rate-limiting enzyme. This result needs to be discussed a bit more. I have a few comments that will improve the readability of this paper.

 

Some comments

• Consistently italicize or not italicize for Arabidopsis.
• Fig 1A- what are other plant species used for the alignment? The alignment figure is too small to read, enlarge the figure. Also, fig 1 B and C need to be enlarged, the font is too small to comment. I realized after reading the entire manuscript that figures have a small font. So, if you want readers to read it then need to increase the font size.
• Fig 2- what is panel B for?
• Fig 4A- mark the size of a ladder in bp.
• I m skeptical about most of the comparisons for heat and salt stress when it is compared with Arabidopsis wild-type plants for sensitivity tests, but we can compare for induced tolerance to heat or salt. Arabidopsis is a salt-sensitive plant species and cannot recover from heat stress if it is given for more than 24 hours.
• Create a summary figure that will help the readers to understand the findings from this study clearly.
• Accumulation of ROS increased the abundance of proline and other amino acids, and sugar with the known function of antioxidants is known to increase upon salt stress. When the overexpressed lines are sensitive to salt then the decreased gene expression and respective metabolite in the current study is nicely matching. In the discussion, the section cites the following paper when discussing increased expression of rate-limiting enzymes in proline biosynthesis and proline accumulation leading to the salt tolerance phenotype.

1. Kant S, Kant P, Raveh E, Barak S (2006) Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant, Cell Environ 29: 1220–1234
2. Pantha P, Oh D-H, Longstreth DJ, Dassanayake M (2021) Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress. bioRxiv 2021.07.01.450778

Author Response

The manuscript submitted by Wang et al, titled " Chrysanthemum CmHSP90.5 negatively regulates heat and salt stress tolerance in Arabidopsis” evaluated the roles of CmHSP90.5 in response to abiotic stresses in chrysanthemum.  Overall, the manuscript is well written. The method section needs to be elaborated. Further, the author has nicely correlated the tolerance phenotype to the Na⁺ accumulation, gene expression of known transporter to extrude salt, quantified proline, and expression of the key rate-limiting enzyme. This result needs to be discussed a bit more. I have a few comments that will improve the readability of this paper.

Some comments

• Consistently italicize or not italicize for Arabidopsis.

Thank you for your suggestion, Arabidopsis in manuscript is in Italicize.

• Fig 1A- what are other plant species used for the alignment? The alignment figure is too small to read, enlarge the figure. Also, fig 1 B and C need to be enlarged, the font is too small to comment. I realized after reading the entire manuscript that figures have a small font. So, if you want readers to read it then need to increase the font size.

The font size in the figures was enlarged. The species in Fig.1A are listed in the manuscript. Please refer to Page6, line220.

• Fig 2- what is panel B for?

The legend of panel B, reproductive stages, was added to present Fig.3, please refer to Page7, line255.

• Fig 4A- mark the size of a ladder in bp.

The size of a ladder in bp was added to the present Fig 5A, please refer to Page10, line304.

• I m skeptical about most of the comparisons for heat and salt stress when it is compared with Arabidopsis wild-type plants for sensitivity tests, but we can compare for induced tolerance to heat or salt. Arabidopsis is a salt-sensitive plant species and cannot recover from heat stress if it is given for more than 24 hours.

Thank you for your nice suggestion. it should be very interesting to explore induced resistance for better understanding the mechanisms by which Hsp90.5 confers to stress sensitivity, in present study, we mainly focused on the identification of the function of CmHSP90.5. Your suggestion will trigger us to study the induced resistance in chrysanthemum in the future. For the heat tolerance treatment, we followed the methods described by Huang et al.

Huang LJ, Cheng GX, Khan A, et al. CaHSP16.4, a small heat shock protein gene in pepper, is involved in heat and drought tolerance[J]. Protoplasma, 2018, 256(1) 39-51.

• Create a summary figure that will help the readers to understand the findings from this study clearly.

Thank you for your suggestion. A summary figure, Fig 11, was added, please refer to page14, line364.

• Accumulation of ROS increased the abundance of proline and other amino acids, and sugar with the known function of antioxidants is known to increase upon salt stress. When the overexpressed lines are sensitive to salt then the decreased gene expression and respective metabolite in the current study is nicely matching. In the discussion, the section cites the following paper when discussing increased expression of rate-limiting enzymes in proline biosynthesis and proline accumulation leading to the salt tolerance phenotype. We have cited the following references as suggested, please refer to page21, line661.

1. Kant S, Kant P, Raveh E, Barak S (2006) Evidence that differential gene expression between the halophyte, The llungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant, Cell Environ 29: 1220–1234
2. Pantha P, Oh D-H, Longstreth DJ, Dassanayake M (2021) Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress. bioRxiv 2021.07.01.450778

Reviewer 2 Report

The main problem of the MS is the fact that only a single, imprecise figure (out of a total of 9) concerns , gene expression in chrysanthemum. Therefore,  it is necessary to add some text indicating a direct link to horticulture and / or which are the outcoming of the work useful for horticulture.

Moreover, other minor points need a correction:

Title. Please change it in "Chrysanthemum CmHSP90.5 as a tool to regulates heat and salt stress tolerance" or something similar avoiding to include the word "Arabidopsis", and in the text giving more emphasis on “CmHSP90.5 as a target/tool for increasing the resistance of horticultural plants to stress".

Please explain why transformed N. benthamiana leaves were used instead of e.g. transient expression in Chrysanthemum protoplasts.

Please use italics for the specie name Chrysanthemum morifolium.

Please write uniformly “Arabidopsis” always in italics or always normal character being both specie name and common name.

Line 109. Please add the the city where Takara is based and add in M&M the other material suppliers.

Lines 169-171. It is necessary to explain much better the methods employed for The enzyme activity index SOD and catalase assays. In fact, catalase normally is assayed spectrophotometrically at 240 nm (absorbance H₂O₂).

Figure 1 should be divided in two by separating the current Figure 1A from Figure 1 B/C.

The reading of Figure 2 would be improved by colouring the histograms.

Figure 5. Please add colors to the histograms and please arrange the various frames in two columns so that each frame is enlarged.

Figure 6-9 as above.

In the conclusions please underline the possible use of the CmHSP90.5 gene in horticulture and the prospects that this work opens up for the horticultural sector

Author Response

Comments and Suggestions for Authors

The main problem of the MS is the fact that only a single, imprecise figure (out of a total of 9) concerns, gene expression in chrysanthemum. Therefore, it is necessary to add some text indicating a direct link to horticulture and / or which are the outcoming of the work useful for horticulture.

Thank you for your suggestion. We have added several texts to indicate the link of present study to horticulture. Please refer to page2, line92~93; page2, line99~101; page15, line392~396; page15, line411, page17, line490~501.

Moreover, other minor points need a correction:

Title. Please change it in "Chrysanthemum CmHSP90.5 as a tool to regulates heat and salt stress tolerance" or something similar avoiding to include the word "Arabidopsis", and in the text giving more emphasis on “CmHSP90.5 as a target/tool for increasing the resistance of horticultural plants to stress".

We have changed the title into "Chrysanthemum CmHSP90.5 as a tool to regulates heat and salt stress tolerance". And we have emphasized that CmHSP90.5 as a target/tool for increasing the resistance of horticultural plants to stress in the text. Please refer to page3, line99.

Please explain why transformed N. benthamiana leaves were used instead of e.g. transient expression in Chrysanthemum protoplasts.

Thank you for your suggestion, the efficiency of transient transformation of protoplast of chrysanthemum is pretty low, and unstable. At present study, we have to turn to N. benthamiana leaves.

Please use italics for the specie name Chrysanthemum morifolium.

The specie name Chrysanthemum morifolium is in italics. Please refer to page3, line113; page3, line116; page3, line136; page3, line139; page5, line204; page15, line366; page15, line366; page15, line388; page16, line453.

Please write uniformly “Arabidopsis” always in italics or always normal character being both specie name and common name.

“Arabidopsis” always in italics was used in the manuscript.

Line 109. Please add the city where Takara is based and add in M&M the other material suppliers.

The cities of the material supplies were added.

Lines 169-171. It is necessary to explain much better the methods employed for the enzyme activity index SOD and catalase assays. In fact, catalase normally is assayed spectrophotometrically at 240 nm (absorbance H₂O₂).

Spectrophotometrical assay have been widely used for the enzyme activity assay. Recently, a number of commercialized kit were developed for the enzyme activity assay.

Figure 1 should be divided in two by separating the current Figure 1A from Figure 1 B/C.

Figure 1 had been divided in two figures. Please refer to page13, line219; page14, line225.

The reading of Figure 2 would be improved by colouring the histograms.

The present Figure 3 had been improved by colouring the histograms

Figure 5. Please add colors to the histograms and please arrange the various frames in two columns so that each frame is enlarged.

Figure 6-9 as above.

The Figure 6~10 had been improved by colouring the histograms and arranged the various frames in two columns.

In the conclusions, please underline the possible use of the CmHSP90.5 gene in horticulture and the prospects that this work opens up for the horticultural sector

 

We have emphasized that CmHSP90.5 as a target/tool for increasing the resistance of horticultural plants to stress in the text. Please refer to page3, line99.

Reviewer 3 Report

The authors of this manuscript examined the potential role of CmHSP90.5, a stress-responsive gene, in heat and salt tolerance. They overexpressed CmHSP90.5 in Arabidopsis, and compared the phenotype of wild-type and transgenic lines under heat and salt stress, as well as the expression of genes and proteins involved in stress tolerance. Overall the experiments are well-designed, and the results are well elucidated and support the author's conclusion. I recommend this manuscript for publication with minor revisions / clarifications as detailed below:

1. The resolution of fig.1 is too low and it is very difficult for me to read the labels of every proteins compared, and the functional motifs. It is also impossible for me to further check the evolutionary relationships.
2. Usually the heat shock proteins are considered to improve plant stress tolerance. In the introduction, the authors cited another study that HSP90.2 improves the stress tolerance of Arabidopsis. However, the authors' conclusion in this study appear to contradict the common understanding. Though the authors provided the explanation from the evidence that multiple stress tolerance conferring genes / proteins are down-regulated in the CmHSP90.5 overexpressing line, they didn't provide a deeper rationale beyond the evidence. However, they did mention in the introduction that the cellular localization of HSP90s appear to make a difference. For example, Arabidopsis overexpressing chloroplastic HSP90.5 is more stress-sensitive than the one overexpressing cytoplasmic HSP90.2. As the authors did prove that CmHSP90.5 is chloroplast-localized, it would be interesting to see more discussion in this direction. For example, here are a few questions that I have: (1) is the Arabidopsis overexpressing AtHSP90.5 more stress-sensitive than the wild-types? (3) Are there similar observations made in other species? (2) Are all organelle-located HSP90s are negative regulators for stress tolerance, or are they only for the chloroplastic ones? 

Author Response

Comments and Suggestions for Authors

The authors of this manuscript examined the potential role of CmHSP90.5, a stress-responsive gene, in heat and salt tolerance. They overexpressed CmHSP90.5 in Arabidopsis, and compared the phenotype of wild-type and transgenic lines under heat and salt stress, as well as the expression of genes and proteins involved in stress tolerance. Overall, the experiments are well-designed, and the results are well elucidated and support the author's conclusion. I recommend this manuscript for publication with minor revisions / clarifications as detailed below:

1. The resolution of fig.1 is too low and it is very difficult for me to read the labels of every protein compared, and the functional motifs. It is also impossible for me to further check the evolutionary relationships.

The font size in the figures was enlarged.

1. Usually, the heat shock proteins are considered to improve plant stress tolerance. In the introduction, the authors cited another study that HSP90.2 improves the stress tolerance of Arabidopsis. However, the authors' conclusion in this study appear to contradict the common understanding. Though the authors provided the explanation from the evidence that multiple stress tolerance conferring genes / proteins are down-regulated in the CmHSP90.5 overexpressing line, they didn't provide a deeper rationale beyond the evidence. However, they did mention in the introduction that the cellular localization of HSP90s appear to make a difference. For example, Arabidopsis overexpressing chloroplastic HSP90.5 is more stress-sensitive than the one overexpressing cytoplasmic HSP90.2. As the authors did prove that CmHSP90.5 is chloroplast-localized, it would be interesting to see more discussion in this direction. For example, here are a few questions that I have: (1) is the Arabidopsis overexpressing AtHSP90.5 more stress-sensitive than the wild-types? (3) Are there similar observations made in other species? (2) Are all organelle-located HSP90s are negative regulators for stress tolerance, or are they only for the chloroplastic ones? 

 (1) Overexpression chloroplast-located AtHsp90.5 showed higher sensitivity to salt stress than wild-types. (2) So far, besides overexpression chloroplast-located AtHsp90.5 had the same Phenotype with CmHSP90.5, it has not been found in other species for this phenomenon. (3) We found that not all plastid-localized HSP90 genes lead to stress-sensitive phenotypes. Like, OsHSP50.2, an HSP90 family gene which has predicted chloroplast transit peptide, positively regulates drought stress tolerance in rice.

Xiang, J.; Chen, X.; Hu, W.; Xiang, Y.; Yan, M.; Wang, J., Overexpressing heat-shock protein OsHSP50.2 improves drought tolerance in rice. Plant Cell Rep 2018, 37, (11), 1585-1595.

Reviewer 4 Report

In their article, the authors focused on studying the effect of heat shock proteins on the resistance of the Arabidopsis model culture to drought. The schemes and design of the experiment are logical and standards. Modern approaches of molecular biology are used. The toolkit of analytical methods for verifying the results of an experiment is beyond doubt. An interesting fact has been established that the increased expression of heat shock proteins in Arabidopsis culture does not lead to an increase in plant resistance to drought. This fact is explained by the influence of various factors, as well as by the fact that important enzymes are not decisive in the morphological growth of plant tissues. This work will be of interest to readers and may be accepted in its current format.

Author Response

In their article, the authors focused on studying the effect of heat shock proteins on the resistance of the Arabidopsis model culture to drought. The schemes and design of the experiment are logical and standards. Modern approaches of molecular biology are used. The toolkit of analytical methods for verifying the results of an experiment is beyond doubt. An interesting fact has been established that the increased expression of heat shock proteins in Arabidopsis culture does not lead to an increase in plant resistance to drought. This fact is explained by the influence of various factors, as well as by the fact that important enzymes are not decisive in the morphological growth of plant tissues. This work will be of interest to readers and may be accepted in its current format.

Reviewer 5 Report

Heat shock proteins (HSPs) play important roles in various stress conditions. In this study, CmHSP90.5, whose expression is induced by heat and salt, was cloned from chrysanthemum (Chrysanthemum morifolium) ‘Jinba’ and expressed in Arabidopsis. Then several experiments looking at gene structure, localisation, and response to salt and heat conditions were performed.

In general the experimental work was carried out logically and methodically and the conclusions drawn matched the results observed in Arabidopsis.  I think this study would be strengthened if the authors justified why they used Arabidopsis as a model organism?  While Arabidopsis maybe easy to cultivate and transform, that doesn't mean that the results observed in this study can be extrapolated to other plants per se without some sort of justification.  The authors should also mention that actual experimentation in Chrysanthemum itself needs to be performed to make any lasting conclusions about heat or salt tolerance qualities of CmHSP90.5. 

Author Response

Heat shock proteins (HSPs) play important roles in various stress conditions. In this study, CmHSP90.5, whose expression is induced by heat and salt, was cloned from chrysanthemum (Chrysanthemum morifolium) ‘Jinba’ and expressed in Arabidopsis. Then several experiments looking at gene structure, localisation, and response to salt and heat conditions were performed.

In general, the experimental work was carried out logically and methodically and the conclusions drawn matched the results observed in Arabidopsis.  I think this study would be strengthened if the authors justified why they used Arabidopsis as a model organism?  While Arabidopsis maybe easy to cultivate and transform, that doesn't mean that the results observed in this study can be extrapolated to other plants per se without some sort of justification.  The authors should also mention that actual experimentation in Chrysanthemum itself needs to be performed to make any lasting conclusions about heat or salt tolerance qualities of CmHSP90.5. 

We have emphasized that CmHSP90.5 as a target/tool for increasing the resistance of horticultural plants to stress in the text. Please refer to page3, line99; page17, line495.

Round 2

Reviewer 2 Report

The MS is ok now, the only concern is the small size of figures.