MaHAK5, a Potassium Transporter of Banana, Enhanced Potassium Uptake in Transgenic Arabidopsis under Low Potassium Conditions
, Ganjun Yi 1,2,3, Hong Liu 4, Chunyu Li 1,2,3,* and Siwen Liu 1,2,3,*
Round 1
Reviewer 1 Report
Dear authors
Carefully read and address my comments
Manuscript title: MaHAK5, a potassium transporter of banana, enhanced potassium uptake in transgenic Arabidopsis under low potassium conditions
Manuscript Number: horticulturae-1957898
Dear Editor,
Please find the evolution report of the manuscript entitled “MaHAK5, a potassium transporter of banana, enhanced potassium uptake in transgenic Arabidopsis under low potassium conditions”.
I went thoroughly through this manuscript, and my comments are mentioned below.
1. The authors well addressed the role of banana HAK5 under LK conditions. The experimental design and manuscript writing was done well. Here I mentioned my major comments on this manuscript. Therefore, my recommendation is to accept the manuscript after addressing my comments.
-How did the authors confirm the T3 transgenic lines, they have solid data like southern blotting? How they generated the T3 transgenic lines?? Please explain properly in the methodology section
-
The manuscript needs serious description in the methodology section and description. There is no proper explanation in the discussion section.
-How authors measured chlorophyll content and fresh weight??
-Is there any data related to silencing or knockout of HAK5 and uptake capacity under LK?? If you add this to the current manuscript. Other explain why you didn’t analyze parallel with WT and OE lines??
Author Response
Reviewer 1：
Dear authors
Carefully read and address my comments
Manuscript title: MaHAK5, a potassium transporter of banana, enhanced potassium uptake in transgenic Arabidopsis under low potassium conditions
Manuscript Number: horticulturae-1957898
Dear Editor,
Please find the evolution report of the manuscript entitled “MaHAK5, a potassium transporter of banana, enhanced potassium uptake in transgenic Arabidopsis under low potassium conditions”.
I went thoroughly through this manuscript, and my comments are mentioned below.
- The authors well addressed the role of banana HAK5 under LK conditions. The experimental design and manuscript writing was done well. Here I mentioned my major comments on this manuscript. Therefore, my recommendation is to accept the manuscript after addressing my comments.
Answer: We appreciate the reviewer’s recognition of our work. Thanks for the valuable suggestions for our manuscript. Accordingly, we modified 3 figures (Figure 1, 4 and 5) and added Supplementary Table 1. Now we have submitted the revised manuscript (including both a clean version and a version with changes highlighted) and point-to-point responses to the review comments below. And the mentioned line number below are based on the clear version.
-How did the authors confirm the T3 transgenic lines, they have solid data like southern blotting? How they generated the T3 transgenic lines?? Please explain properly in the methodology section
Answer: We have added the relative information on obtaining the T3 transgenic lines in methodology. See line 93-102 in new manuscript. ‘Transformation and Identification of MaHAK5 OE Arabidopsis: To generate the Ma-HAK5-overexpressing line (MaHAK5 OE), the MaHAK5 coding sequence was cloned into the pBI121 vector with 35S promoter and was transformed into Arabidopsis Col-0 plants. The construct was introduced into Arabidopsis through floral-dip method [22]. Transgenic seedlings were selected on MS medium (Sigma M-5519) containing 50 μg ml-1 kanamycin. Genomic DNA from kanamycin-resistant plants’ leaves were extracted and a PCR with gene specific primers (Supplementary Table 1) was performed for secondary screeding. T1 plants positive with PCR were grown to harvest T2 seeds. Similarly, T3 seeds from homozygous lines were harvested following the primary screening by 50 μg ml-1 kanamycin and secondary with PCR to ensure stable transformation’
The manuscript needs serious description in the methodology section and description. There is no proper explanation in the discussion section.
-How authors measured chlorophyll content and fresh weight??
Answer: We have added the information on measurement of chlorophyll content and fresh weight. See line 149-155. ‘For determination of chlorophyll content, shoots of two-week-old Arabidopsis WT and transgenic plants were collected and weighed (n > 40). The chlorophyll was extracted in 80% acetone in the dark as described by Feng et al. [25]. Chlorophyll content was calculated using MacKinney’s-specific absorption coefficients in which chlorophyll a = 12.7 (A663) − 2.69 (A645) and chlorophyll b = 22.9 (A645) − 4.68 (A663) [26]. The total chlorophyll content was shown as mg of chlorophyll per gram of fresh shoots. At least three biological replicates were performed per experiment.’
-Is there any data related to silencing or knockout of HAK5 and uptake capacity under LK?? If you add this to the current manuscript. Other explain why you didn’t analyze parallel with WT and OE lines??
Answer: We have added the previous report about the effect of HAK5 on the potassium uptake under LK. See line 235-236. ‘Previous studies have shown that knockout of HAK5 in Arabidopsis resulted in a chlorotic phenotype and low K+ content under LK conditions [26].’
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article describes the cloning and functional characterization of a potassium transporter (MaHAK5) from banana (Musa acuminate). A yeast strain lacking a K+ uptake system was transformed with MaHAK5 and the results indicate that this protein has K+ uptake activity in vivo. The expression of the gene is analyzed in different tissues and growing conditions. The overexpression of MaHAK5 in Arabidopsis plants enhanced the ability of the plant to uptake K+ under low potassium stress.
The manuscript is, in general, clear and well structured. The results obtained are very interesting but some clarifications and modifications are needed to support the conclusions of the authors.
Major issues:
Q1. The phylogenetic tree shown in figure 1c needs to be improved. Some of the branches are not well resolved at the base. The tree could be easily improved by adding an outgroup, for instance, HAK proteins from distant groups of monocots (OsHAK1, OsHAK17). For an example see Ruiz-Lau et al 2016.
Q2. One of the main conclusions of the article is that MaHAK5 was upregulated in roots under low K+ (LK) stress (Abstract, lines 251-252, lines 277-288). However, the experiments shown in Figure 4 do not support this conclusion.
The two figure panels (Figure 4a and b) show that, 8 hours after the treatment, the expression of the gene increases compare to the expression at 2h. The results are the same in both panels and therefore are not specific of K+ deficient conditions.
The experiments need to be combined to analyze the effect with and without the treatment (sufficient and deficient K+) and they need to be shown in the same histogram.
Figure 4 shows comparative expression level independently for each panel. It not possible to claim that MaHAK5 have lower or higher expression in the two growth conditions essayed.
Q3. The experiment design of the data shown is Figure 4 is not explained in the manuscript. What is the meaning of the time in the x-axis (2h, 8h, 14h and 20h) in Fig4a panel? Why is MaHAK5 induced after 8h under K+ sufficient conditions? What means 8h in this experiment? Where from have been transferred the plants?
Q4. Figure 5f. To be consistent with the previous panels, the two transgenic lines should be included in this experiment (K+ content analysis). Results obtained in single line are not conclusive in this type of experiments.
Minor issues:
M1. Line 84. Growth conditions where bananas were grown need to be specified.
M2. Lines 87-90. The description of the overexpression of MaHAK5 in Arabidopsis plants seems to be misplaced within the plant material section. The use of the abbreviation “WT” in line 88 should be avoided, and replaced by the species (Arabidopsis). The promoter used in the construct is not written. In general, this particular methodology is insufficiently described.
M3. Legend figure 1. The legend should include an explanation of the abbreviations used in the figure that identify the species of the proteins (Zm, Zea mays, etc).
M4. Figure 5a. The number of cycles used in the semiquantitative RT-PCR needs to be included for each gene.
Author Response
Reviewer 2：
The article describes the cloning and functional characterization of a potassium transporter (MaHAK5) from banana (Musa acuminate). A yeast strain lacking a K+ uptake system was transformed with MaHAK5 and the results indicate that this protein has K+ uptake activity in vivo. The expression of the gene is analyzed in different tissues and growing conditions. The overexpression of MaHAK5 in Arabidopsis plants enhanced the ability of the plant to uptake K+ under low potassium stress.
The manuscript is, in general, clear and well structured. The results obtained are very interesting but some clarifications and modifications are needed to support the conclusions of the authors.
Answer: We appreciate the reviewer’s recognition of our work. Thanks for the valuable suggestions for our manuscript. Accordingly, we modified 3 figures (Figure 1, 4 and 5) and added Supplementary Table 1. Now we have submitted the revised manuscript (including both a clean version and a version with changes highlighted) and point-to-point responses to the review comments below. And the mentioned line number are based on the clear version.
Major issues:
Q1. The phylogenetic tree shown in figure 1c needs to be improved. Some of the branches are not well resolved at the base. The tree could be easily improved by adding an outgroup, for instance, HAK proteins from distant groups of monocots (OsHAK1, OsHAK17). For an example see Ruiz-Lau et al 2016.
Answer: Thank you for your suggestion. We have modified the tree by adding OsHAK1 as outgroup. See the new figure 1 at line 181.
Q2. One of the main conclusions of the article is that MaHAK5 was upregulated in roots under low K+ (LK) stress (Abstract, lines 251-252, lines 277-288). However, the experiments shown in Figure 4 do not support this conclusion.
The two figure panels (Figure 4a and b) show that, 8 hours after the treatment, the expression of the gene increases compare to the expression at 2h. The results are the same in both panels and therefore are not specific of K+ deficient conditions.
The experiments need to be combined to analyze the effect with and without the treatment (sufficient and deficient K+) and they need to be shown in the same histogram.
Figure 4 shows comparative expression level independently for each panel. It not possible to claim that MaHAK5 have lower or higher expression in the two growth conditions essayed.
Answer: Thank you for your constructive suggestion. We have attenuated the function of MaHAK5 in banana under LK stress response by describing it only seem to be upregulated at the early stage of potassium starvation. Accordingly, we have modified the abstract (line 22-24: qRT-PCR analysis showed that MaHAK5 was upregulated in roots and leaves under early stage of low K+ (LK) stress.), result (line 214-221: Expression levels of MaHAK5 in various tissues under different levels of K+ stress were analyzed using qRT-PCR. As shown in Fig. 4, MaHAK5 was widely expressed in all examined tissues. After transfer to LK condition for 8 h, the MaHAK5 expression rose significantly in roots and leaves, while the transcript levels did not significantly change after 14 h. We also measured the transcript levels of MaHAK5 in pseudostem at different time, and showed that MaHAK5 was not induced by LK stress in banana pseudostem. These results indicated that MaHAK5 may function in K+ uptake in banana roots at the early stage of potassium-deficient conditions.), and discussion (line 271-277: Here, we demonstrated that MaHAK5 showed a higher expression pattern in banana roots when plants were exposed to LK conditions for 8 h (Fig. 4), a result that was consistent with those for OsHAK5[17] and ZmHAK1[18]. In addition, the AtHAK5 transcript in-creased for the time points also for a longer starvation period of 7 d, showing a continuing induction of AtHAK5 transcripts [30]. However, we determined that MaHAK5 transcript levels were only induced at banana roots and leaves for 8 h potassium starvation, while remained normal for further deficient K+ treatment (Fig. 4).)
Q3. The experiment design of the data shown is Figure 4 is not explained in the manuscript. What is the meaning of the time in the x-axis (2h, 8h, 14h and 20h) in Fig4a panel? Why is MaHAK5 induced after 8h under K+ sufficient conditions? What means 8h in this experiment? Where from have been transferred the plants?
Answer: we have specified this experiment in Materials and Methods section. See line 84-92: Uniform banana plantlets with 4 fully expanded leaves were transferred to a 10 L container supplied with full nutrient Hoagland solution (K+-sufficient) or Hoagland solution without potassium (K+-deficient). Each treatment had three biological replicates, and each treatment of plants were divided into roots, pseudostems and leaves. Samples were collected at different time points. After collecting, the samples were immediately frozen in liquid nitrogen and stored at -80°C for further RNA extraction. Nicotiana benthamiana was grown in a growth chamber at 25°C. Arabidopsis wild-type Col-0 (WT) was cultivated in a temperature-controlled glasshouse (16 h light and 8 h dark cycle, at 22°C with 40% humidity).
Q4. Figure 5f. To be consistent with the previous panels, the two transgenic lines should be included in this experiment (K+ content analysis). Results obtained in single line are not conclusive in this type of experiments.
Answer: Thank you for your suggestion. We missed to measure the potassium content of MaHAK5 OE1, so we did this experiment again, which is the reason why we prolong the modification time. See the ne Figure 5 in line 246.
Minor issues:
M1. Line 84. Growth conditions where bananas were grown need to be specified.
Answer: Revised as suggested. See line 83-92.
M2. Lines 87-90. The description of the overexpression of MaHAK5 in Arabidopsis plants seems to be misplaced within the plant material section. The use of the abbreviation “WT” in line 88 should be avoided, and replaced by the species (Arabidopsis). The promoter used in the construct is not written. In general, this particular methodology is insufficiently described.
Answer: Revised as suggested. We have separated the Transformation and Identification of MaHAK5 OE Arabidopsis part alone. See line 93.
M3. Legend figure 1. The legend should include an explanation of the abbreviations used in the figure that identify the species of the proteins (Zm, Zea mays, etc).
Answer: Revised as suggested. See line 186-188: Ta, Triticum aestivum; Os, Oryza sativa; Zm, Zea mays; Ma, Musa acuminata; Mb, Musa balbisiana; Gm, Glycine max; Nt, Nicotiana tabacum; At, Arabidopsis thaliana.
M4. Figure 5a. The number of cycles used in the semiquantitative RT-PCR needs to be included for each gene.
Answer: Revised as suggested. See new Figure 5a in line 246.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors,
I accepted your revisions for this current manuscript. However, I found few grammatical mistakes in English language, please check this manuscript carefully.
For instance, Page No: 99, it should be screening not screeding.
Author Response
Thank you for your constructive suggestion for our work. We have checked throughout the paper and made some revisions.
Reviewer 2 Report
The authors have adequately addressed all my comments. Thank you for taking the time to consider my suggestions. The manuscript is much improved.
Author Response
Thank you for your hard work on our paper, we really appreciate it.
