Expression of the VvMYB60 Transcription Factor Is Restricted to Guard Cells and Correlates with the Stomatal Conductance of the Grape Leaf

Round 1

Reviewer 1 Report

 Expression of the VvMYB60 transcription factor is restricted to guard cells and correlates with the stomatal conductance of the grape leaf.

Stomatal regulation during water stress plays an important role in stress tolerance. However, the molecular mechanism of stomatal control is yet to be completely understood. In this direction, the study is very important in understanding the mechanism of stomatal control mechanism. However, the following points may be addressed by the authors.

1. The material and method section says that the genotype Cabernet Sauvignon is also used along with 4 rootstocks. However, there is no data on Cabernet Sauvignon in the manuscript. Entire study was done only on 4 rootstocks. Therefore, the reference to Cabernet Sauvignon may be removed.
2. How the plants were raised? Is it from cuttings or from seeds? This may be mentioned in the manuscript. Seedlings are not true to type, hence don’t represent the mother plants.
3. Number of days of stress before taking the observations on stomatal conductance need to be mentioned.
4. The age of leaf or the leaf number from top which is taken for recording stomatal conductance and stomatal density need to be mentioned
5. Give more explanation on the role of MYB60 and SIRK in stomatal control. Since they are transcription factors, they may have other roles. What are the downstream genes regulated by these transcription factors? What is the interaction between these two genes?
6. If stomatal control is not involved in the tolerance, then what is the explanation by the authors for the tolerance mechanism in the tolerant genotypes? Is it the root mass which is involved or the cellular level tolerance?

Statistical analysis is enough and the replications are also fine. But need to mention about the number of times the observations were recorded for stomatal conductance.

Author Response

Reviewer1

Stomatal regulation during water stress plays an important role in stress tolerance. However, the molecular mechanism of stomatal control is yet to be completely understood. In this direction, the study is very important in understanding the mechanism of stomatal control mechanism. However, the following points may be addressed by the authors.

- We thank the Reviewer for his positive evaluation of our work and for his suggestions. Please find a point-by-point response to the Reviewer’s comments.

1) The material and method section says that the genotype Cabernet Sauvignon is also used along with 4 rootstocks. However, there is no data on Cabernet Sauvignon in the manuscript. Entire study was done only on 4 rootstocks. Therefore, the reference to Cabernet Sauvignon may be removed.

- We employed the cv Cabernet Sauvignon in the ice-blending and laser-microdissection experiments aimed at purifying the stomatal fraction from intact leaves. We clarified this issue in the Materials and Methods section (lines 100-101). We also amended the main text, in the Results section (line 174).

2) How the plants were raised? Is it from cuttings or from seeds? This may be mentioned in the manuscript. Seedlings are not true to type, hence don’t represent the mother plants.

- This is an important issue, which we did not address in the original manuscript. All the genotypes employed in the study were propagated by cuttings. We included this information in the Material and Methods section (lines 103-104).

3) Number of days of stress before taking the observations on stomatal conductance need to be mentioned.

- As indicated in the Results section, g_s measurements were performed “following three consecutive days at 50% or 30% FC” (lines 230-231). In line with the Reviewer’s indication, we also detailed this information in Materials and Methods (lines 112-113).

4) The age of leaf or the leaf number from top which is taken for recording stomatal conductance and stomatal density need to be mentioned.

- We included the missing information in the Material and Methods section (lines 119-120)

5) Give more explanation on the role of MYB60 and SIRK in stomatal control. Since they are transcription factors, they may have other roles. What are the downstream genes regulated by these transcription factors? What is the interaction between these two genes?

- In the Introduction, we highlighted the role of MYB60 transcription factor in the negative regulation of guard cell-related LYPOXIGENASE (LOX) genes, and its role in the synthesis of oxylipins in stomata (lines 71-75). In the Result section, we detailed the role of VvSIRK and its relation with the Arabidopsis guard cell-specific K⁺ channel KAT2 (lines 187-189).

6) If stomatal control is not involved in the tolerance, then what is the explanation by the authors for the tolerance mechanism in the tolerant genotypes? Is it the root mass which is involved or the cellular level tolerance?

- Investigating the developmental and physiological details of the tolerance mechanisms of the drought resistant rootstocks is clearly beyond the scope of the manuscript. Yet, as suggested by the Reviewer, it is important to acknowledge this relevant issue. We reported data from Alsina et al (2011) which clearly indicate that 1103P develop increased root biomass under stress, compared to the drought-sensitive rootstock 101.14. We also included data from Meggio et al (2014), which demonstrate the enhanced biochemical and physiological responses to water stress depicted by M4 root systems, as compared with roots from 101.14 (lines 300-304)

7) Statistical analysis is enough and the replications are also fine. But need to mention about the number of times the observations were recorded for stomatal conductance.

- We added the requested information in Materials and Methods (lines 114-115).

Reviewer 2 Report

In my opinion, the manuscript contains new and very interesting data.

I have only a few suggestions for improving statistical analysis.
In the title, discussion, and Figure 3 the Authors proved that expression of VvMYB60 correlates with the stomatal conductance. Therefore, I strongly suggest carrying out the statistical analysis of correlation coefficient and linear regression for the relationship between these parameters and presenting them on the graph. In my opinion, this analysis in a clear way will demonstrate these findings.

Author Response

Reviewer2

In my opinion, the manuscript contains new and very interesting data. I have only a few suggestions for improving statistical analysis. In the title, discussion, and Figure 3 the Authors proved that expression of VvMYB60 correlates with the stomatal conductance. Therefore, I strongly suggest carrying out the statistical analysis of correlation coefficient and linear regression for the relationship between these parameters and presenting them on the graph. In my opinion, this analysis in a clear way will demonstrate these findings.

- We thank the Reviewer for his positive response and for his relevant suggestion. We provided the linear regression analysis in Supplementary Figure 3 A and B. We included proper reference to Figure S3 in the main text along with the calculated coefficient of determination (R²) (lines 265 and 273).