A B-Box Transcription Factor CoBBX24 from Camellia oleifera Delays Leaf Senescence and Enhances Drought Tolerance in Arabidopsis
Round 1
Reviewer 1 Report
The manuscript is about the functional characterization of the transcription factor CoBBX24 from Camellia oleifera. It is very well-written and only some minor amendments are required before acceptance. Please refer below for the specific comments.
1) Line 19-20. "The root length ... acid." Please rewrite to improve clarity of the statement.
2) Line 21. The expression of genes related to leaf senescence and chlorophyll breakdown (e.g. SAG12, SAG29, NYC1, NYE1, and NYE2) was downregulated in OX-CoBBX24 lines.
3) Line 185. Refer to the wrong Fig.2A, should be Fig. 2C.
4) Fig. 2A, what does "CK" refer to? Fig.5B, what does "MOCK" refer to? Please relabel appropriately.
5) Line 204-205. Add (Fig. 2A) after the sentence.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The presented manuscript Y. Liu et al. “A B-Box Transcription Factor CoBBX24 from Camellia oleifera delays Leaf Senescence and Enhances Drought Tolerance in Arabidopsis” demonstrated a number of the results about functional role of CoBBX24 from Camellia oleifera in drought tolerance and leaf senescence. Here some remarks to make more clear reviewed manuscript:
INTRODUCTION
1) Please add more information about the function of the BBX24 protein. Which of the BBX24 proteins from the different species have been analysed in the past? How close are they functionally to the proteins of this family in Arabidopsis thaliana, which you use as model organism? At present, this part is mainly described in the discussion. I would like to see a focus on the data in the context of drought and senescence
2) Could you be more specific about the purpose of your work? Why did you choose to analyse this particular protein found in Camellia oleifera? There is a similar study on the CmBBX22 protein. It came to almost the same conclusions as you did with the CoBBX24 protein. Could you have a discussion on these results and a comparison of the functional activity of these proteins?
METHODS
Is the genome sequence of the Camellia oleifera available? Please provide a resource (link) or citation that contains this information.
Line 107: “2.3. Isolation and Sequence Analysis of CoBBX24”
Here I lacked the following information. How many BBX24 homologues represent in Camellia oleifera? Could you be sure that the only one copy of this gene in genome? Did you perform the 3′- and 5′-RACE PCR to assemble full-length cDNA of a single gene? Was the resulting sequence sent to GeneBank? Usually, all confirmed results (complete sequence of DNA, RNA or CDS) should be accessed to GeneBank before submission. Also experimental evidence of identification and isolation of full-length gene including sequence is required as a supplemental material.
Line 116: Specify source of pORE-R4-35SAA plasmid and selective
Line 125: Clarify what pCL means. Is it AT3G46640 (PCL1) cloned to pGBKT7 and the design is pGBKT7-pCL?
RESULTS
Line 165: As mentioned above, I am missing information on the homology to BBX4 Arabidopsis.
Figure 1 caption requires CoBBX24 protein accession number.
Line 185: Here you are referring to Figure 2A, but it is actually Figure 2C.
Line 203: Results should be signed as shown in Figure 2A.
Also here, it should be noted that these results of BBX24 transcript level under drought conditions are not fully described and clearly explained. Day 15 is not explained. I would also like to see the expression of a marker genes here. In general, I think a more careful study of gene expression during ontogenesis and day is needed. It may be necessary to take data on a daily basis every 6 h for 24 h. What happens to gene expression after the plant comes out of drought? Does it dependent on light ON/OFF? What about promoter activity of CoBBX24 and regulatory elements in it? What about CoBBX24 gene expression in the presence of ABA or fluridone?
Line 206: Experimental data demonstrating the homozygosity of CoBBX24-overexpressing Arabidopsis T3 transgenic lines should be provided in the supplementary data. Please provide the level of CoBBX24 expression in each transgenic line.
What is the effect of CoBBX24 overexpression on the germination rate of Arabidopsis seeds in the presence of ABA or fluridone or PEG?
Line 254: It would be interesting to see the relationships of genes involved not only in senescence but also in drought. For example, DREB2A and RCD1.
DISSCUSION
The discussion is very short and mainly cites already published data, there is little clear explanation of the obtained results. I would have liked to see more discussion of the data that has already been obtained in relation to BBX24 in other organisms.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
In the present manuscript the authors describe the characterization of the B-box (BBX) transcription factor CoBBX24 cloned from Camellia oleifera through overexpression in Arabidopsis thaliana. The overexpression of this gene confers tolerance to drought in Arabidopsis and affects the sensitivity to exogenous ABA application. Through an expression analysis of senescence-associated and chlorophyll catabolic genes, the authors conclude that CoBBX24 overexpression delays leaf senescence, resulting in a certain degree of resistance to drought. I consider that certain points about this manuscript could be improved.
1. Lines 91-92: “Water was provided before the drought treatment, and then watering was stopped for the subsequent 15 d”. To specify the age of the Arabidopsis plants when you stopped watering the plants. To mention if fertilizer was used, what type and in what concentration, before and after the dry period. Also, in these types of experiments it is recommended to measure at least the amount of water in the substrate to get a better idea of the level of drought experienced by the plant.
2. Lines 93-95: “To detect water loss in the OX-CoBBX24 transgenic lines and WT plants, whole rosettes of 21-day-old plants were cut from the base and weighed at 0, 2, 4, 6, 8, 10 a.m. and 12 p.m. The experiment was repeated three times”. Specify the number of plants measured in each of the three times the experiment was repeated. Specify the conditions in which the measurements were made (which is very important), and how the percentage of water loss was calculated.
3. Line 104: “Each sample was run in three technical replicates”. How many biological replicates were analyzed? and How was a biological replicate constituted?
4- Lines 105-106: “All primers are listed in Supplemental Table S1”. It is not clear if the primers used in this work have been previously reported or were designed during this work. I consider that when the primers are reported for the first time, it is necessary to show information regarding to the efficiency of the primers. Since this information is very important if the 2−ΔΔCt method is used to calculate the changes in the expression levels of the analyzed genes.
5. Lines 108-109: “The open reading frame (ORF) of CoBBX24 from oil tea trees was cloned by PCR using the primers CoBBX24-ORF-F/R”. To mention which vector was used
to clone the gene.
6. Lines 115-117: “The amplicons and pORE-R4-35SAA vector were digested using EcoRI and NotI, and the products were ligated using a T4 DNA ligase (TaKaRa, Tokyo, Japan) to generate pORE-R4-35SAA-CoBBX24 fusions”. The pORE-R4-35SAA vector is the same one that is later referred to as 35S::GFP-CoBBX24. If so, homogenize the name, if they are different, the second one is not mentioned in the M&M section.
7. Lines 121-132: In order to the section 2.5. Transactivation Activity Analysis of CoBBX24 is better understood, it is necessary to mention that Yeast two-hybrid assays using the GAL4 system were used where the pGBKT7 vector contains the GAL4 binding domain.
8. Lines 139-140: “Semiquantitative PCR detected the expression of CoBBX24 in the transformants of the T3 generation using the primers RT-CoBBX24-F/R”. No evidence is shown that the transformant lines that were selected overexpress the CoBBX24 gene and at what level is this overexpression, to determine if there is a correlation between the level of overexpression and the level of tolerance to drought.
9. Lines 144-145: “Detached leaves were taken from four-week-old plants and incubated in water or 20 mL of 100 μM ABA solution (Solarbio, Beijing, China) under low light.” What does low light mean?
10. Lines 146-148: “For ABA treatment of attached leaves, four-week-old plants were sprayed on the leaves with 100 μM ABA, and samples were collected after 24 h”. It is not clear if this experiment was to collect the samples from which the expression levels of the different genes were analyzed.
11. Line 185: “(Fig. 2A)” Actually refers to Fig. 2C.
12. Lines 179-205: Section “3.2. Subcellular Localization, Transcriptional Activation, and Transcriptional Profiling of CoBBX24”. It is not clear which were the reasons for selecting this gene, to decide to overexpress it or hypothesize that this gene could be related to tolerance to drought stress.
13. Lines 203-205: “qRT-PCR was performed to investigate the transcriptional profile of CoBBX24 under drought stress. After 12 d of drought stress, the expression of CoBBX24 transcript decreased”.
a) Figure 2A is not quoted.
b) It is not mentioned to what extent the expression of the gene decreased.
c) If the overexpression of the CoBBX24 gene confers tolerance to drought, it would be expected that the expression of this gene would be induced under drought conditions. However, in the expression analysis the opposite effect was observed. How is this behavior explained?
13. Figure 3A shows two pots of each overexpressing line analyzed in the drought experiment. In the case of the OX-CoBBX24-1 line, no plant was recovered in one of the pots, while in the second pot all the plants recovered. This behavior suggests that the amount of water in the substrate was not the same when end of the drought period, which is a serious problem with this type of experiment if the water content in the substrate is not monitored.
14. Lines 220-221: “Fig. 3(B) Survival rates of CoBBX24 transgenic and WT plants after 7 d of re-watering following a 15-d drought treatment. Three independent experiments were performed”. The data shown in the graph is the average of the three experiments or only one of the three experiments and how many plants were analyzed.
15. Lines 227-229: “To assess whether the drought tolerance of overexpression lines was related to ABA, the root lengths of WT and OX-CoBBX24 plants were measured after outside ABA treatment”. To specify if only main or primary root was measured.
16. Lines 246-247: “Compliance with these visible phenotypes, the chlorophyll content in OX-CoBBX24 leaves was higher than that in WT leaves”. If ABA facilitates the breakdown of chlorophyll and according to the root experiment, the overexpression lines are more sensitive to ABA, what would be expected is that the overexpression lines have less chlorophyll instead of more chlorophyll compared to the WT. How is this discrepancy explained?
17. Figure 6A and 6B: The authors mention that ABF4 directly and positively regulates the expression of SAG29, so it would be expected that the lower the expression of ABF4, the lower the expression of SAG29. However, the expression analysis shows the opposite effect in the lines overexpressing CoBBX24, when comparing the data shown in Figures 6A and 6B. How is this behavior explained?
18. Figure 6C: When performing relative expression studies; regularly, the expression of the gene in the control condition is normalized to 1 and the expression in the other conditions are relative to that normalization in the control. However, to cite an example, for the SAG12 gene, it is not clear how the data were normalized since the expression of the gene at time 0 of the wt plant is greater than 2. Describe in more detail in the M&M section how to they calculated and normalized the expression levels of the genes in the different conditions.
19. Lines 284-285: “Additionally, Arabidopsis overexpressing CoBBX24 showed increased tolerance to drought stress”. This statement is repetitive of the previous one.
20. Lines 303-304: “The transcript level of ABF4, SAG12, SAG29, NYE1, NYE2, and NYC1 was decreased in OX-CoBBX24 plants under ABA treatment”. If the experiment in yeast suggests that this TF is an activator, how is it explained that in the overexpression lines there is less expression of the genes analyzed? Add this to the discussion section.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
All changes are accepted.
Author Response
Dear Reviewer,
Thanks a lot for your good suggestion. We find that the comments are of great help for the improvement of our manuscript. All of the comments have been considered in the revised manuscript, and all of the revisions are marked in yellow in the revised version.
Below we have provided in detail the point-by-point replies to the reviewer’s comments. Now we are submitting the revised manuscript to Horticulturae, and sincerely hope that it can be finally recommended for publication in this journal after the further evaluation.
Best wishes,
Yours sincerely,
Sicheng Ye
1. Lines 108-110: “The OX-108 CoBBX24 transgenic lines and WT plants were exposed to air drying at room temperature, samples were harvested 0, 6 h after the water deficit”.
This sentence does not make sense with respect to the rest of the paragraph.
Response: Thank you for your suggestion. We have deleted this sentence.
2. Lines 215-221: “After 12 d of drought stress, the expression of CoBBX24 transcript decreased 5.6-fold relative to untreated plants (Fig. 2A). For assays on the effects of water deficit, plants were exposed to air drying by placing them on dry paper at room temperature. The expression levels of CoBBX24 were decreased at 3 and 12-h points (Fig. S2A). DREB2A as one of the main regulators of drought, the expression of CoDREB2A transcript increased after 12 d of drought stress (Fig. S2B). The expression levels of CoBBX24 were decreased at 6- and 12-h points by ABA treatment (Fig. S2C).”
This paragraph needs to be rewritten.
It is better, first, describe the expression profile at short times and then at long times.
It is not clear why the expression of DREB2A was analyzed.
It is not clear why CoBBX24 expression was analyzed in an ABA treatment.
Response: Thanks a lot for your good suggestion! We have added the description in the new revision (lines 192 to 196).
The drought treatment of plants in the soil lasted for a long time, and DREB2A as one of the main regulators of drought, can be used as a marker gene for drought stress.
Due to the sensitivity of transgenic Arabidopsis to ABA was detected, the expression level of CoBBX24 under ABA treatment was analyzed.
3. Lines 256-260: “Growth on a medium containing 3 μM ABA resulted in more severe inhibition of root growth in OX-CoBBX24 plants compared to WT plants (Fig. 4B). The seed germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment (Fig. S4), indicating that overexpression of CoBBX24 in Arabidopsis increases ABA sensitivity.”
The text says that 3 micromolar was used and figure S4 indicates 0.2 micromolar
Response: Thank you for your suggestion. I’m sorry for the confused expression. We added the concentration of ABA in the new revision (lines 303 to 305).
4. Lines 258-259: “The seed germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment (Fig. S4), ”
The authors do not show quantitative data indicating that the germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment.
Response: Thank you for your suggestion. We have added the descriptions and figure in the new revision (lines 213 to 217, Fig. S4A).
5. Lines 284-285: “Additionally, Arabidopsis overexpressing CoBBX24 showed increased tolerance to drought stress”. This statement is repetitive of the previous one.
Response: Thank you very much! We have deleted this sentence.
The sentence was not deleted.
Response: Thank you very much. We have deleted this sentence in line 249.
6. Lines 314-316: “The amounts of SsBBX24 transcript decreased in leaves of 2-week-old phytotron-grown S. tuberosum plants in water-deprived plants [49]. ”
It is confusing that S. tuberosum is mentioned and the gene is called SsBBX24.
Response: Thank you for your suggestion. “S. tuberosum” changed to “S. sogarandinum”
7. The data in Figures 2 and S2B indicate that in Camellia oleifera when CoBBX24 is repressed by drought at 12 days, CoDREB2A is induced by drought at the same time. However, in Arabidopsis when CoBBX24 is overexpressed, AtDREB2A is also induced by drought (Fig. S5A).
How is this different behavior explained?
Add this explanation in the discussion.
Response: Thanks a lot for your good suggestion! We have added this to the discussion section (lines 288-292).
8. In the discussion section, mention if there is a correlation between the expression level of CoBBX24 in the Arabidopsis overexpression lines and their level of tolerance to drought and in the expression levels of the genes analyzed, compare this behavior with other studies.
Response: Thank you very much! We have added this to the discussion section (lines 261-269).
Drought-induced leaf senescence happens gradually, previous studies have shown that some genes are involved in senescence and also play a crucial role in stress response [52,53]. Overexpression of GhTZF1 enhanced drought tolerance and delayed drought-induced leaf senescence through regulate the expression of antioxidant genes and SAGs in transgenic Arabidopsis [54]. Overexpression of GhWRKY91 delayed leaf senescence and improved drought tolerance in transgenic Arabidopsis [55]. NtNAC028 loss-of-function tobacco plants showed delayed leaf senescence and increased tolerance to drought stress [56]. It has been hypothesized that these genes integrate the different signaling pathways and play important roles between stress responses and senescence.
References:
Guo YF, Gan SS. Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments. Plant Cell Environ. 2012 Mar;35(3):644-55.
Jan S, Abbas N, Ashraf M, Ahmad P. Roles of potential plant hormones and transcription factors in controlling leaf senescence and drought tolerance. Protoplasma. 2019 Mar;256(2):313-329.
Zhou T, Yang XY, Wang LC, Xu J, Zhang XL. GhTZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic Arabidopsis. Plant Mol Biol. 2014 May;85(1-2):163-77.
Gu LJ, Ma Q, Zhang C, Wang CC, Wei HL, Wang HT, Yu SX. The Cotton GhWRKY91 Transcription Factor Mediates Leaf Senescence and Responses to Drought Stress in Transgenic Arabidopsis thaliana. Front Plant Sci. 2019 Oct 29;10:1352.
Wen CL, Liu T, Deng ZC, Zhang ZL, Wang Q, Wang WF, Li W, Guo YF. Characterization of NAC transcription factor NtNAC028 as a regulator of leaf senescence and stress responses. Front Plant Sci. 2022 Aug 15;13:941026.
9. Indicate the time in figure S2B.
Response: Thank you very much! We added the time in figure S2B.
10. Add title and figure captions to supplementary figures.
Response: Thanks a lot for your good suggestion! We added supplementary figure legends in the new revision (lines 563-585).
Author Response File: Author Response.docx
Reviewer 3 Report
The authors of the present manuscript have made substantial changes by responding to almost all the comments made in the first round of revision. However,there are some points that need to be clarified.
1. Lines 108-110: “The OX-108 CoBBX24 transgenic lines and WT plants were exposed to air drying at room temperature, samples were harvested 0, 6 h after the water deficit”.
This sentence does not make sense with respect to the rest of the paragraph.
2. Lines 215-221: “After 12 d of drought stress, the expression of CoBBX24 transcript decreased 5.6-fold relative to untreated plants (Fig. 2A). For assays on the effects of water deficit, plants were exposed to air drying by placing them on dry paper at room temperature. The expression levels of CoBBX24 were decreased at 3 and 12-h points (Fig. S2A). DREB2A as one of the main regulators of drought, the expression of CoDREB2A transcript increased after 12 d of drought stress (Fig. S2B). The expression levels of CoBBX24 were decreased at 6- and 12-h points by ABA treatment (Fig. S2C).”
This paragraph needs to be rewritten.
It is better, first, describe the expression profile at short times and then at long times.
It is not clear why the expression of DREB2A was analyzed.
It is not clear why CoBBX24 expression was analyzed in an ABA treatment.
3. Lines 256-260: “Growth on a medium containing 3 μM ABA resulted in more severe inhibition of root growth in OX-CoBBX24 plants compared to WT plants (Fig. 4B). The seed germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment (Fig. S4), indicating that overexpression of CoBBX24 in Arabidopsis increases ABA sensitivity.”
The text says that 3 micromolar was used and figure S4 indicates 0.2 micromolar
4. Lines 258-259: “The seed germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment (Fig. S4), ”
The authors do not show quantitative data indicating that the germination rate and the proportion of green cotyledon in OX-CoBBX24 plants were lower than WT under ABA treatment.
5. Lines 284-285: “Additionally, Arabidopsis overexpressing CoBBX24 showed increased tolerance to drought stress”. This statement is repetitive of the previous one.
Response: Thank you very much! We have deleted this sentence.
The sentence was not deleted.
6. Lines 314-316: “The amounts of SsBBX24 transcript decreased in leaves of 2-week-old phytotron-grown S. tuberosum plants in water-deprived plants [49]. ”
It is confusing that S. tuberosum is mentioned and the gene is called SsBBX24.
7. The data in Figures 2 and S2B indicate that in Camellia oleifera when CoBBX24 is repressed by drought at 12 days, CoDREB2A is induced by drought at the same time. However, in Arabidopsis when CoBBX24 is overexpressed, AtDREB2A is also induced by drought (Fig. S5A).
How is this different behavior explained?
Add this explanation in the discussion.
8. In the discussion section, mention if there is a correlation between the expression level of CoBBX24 in the Arabidopsis overexpression lines and their level of tolerance to drought and in the expression levels of the genes analyzed, compare this behavior with other studies .
9. Indicate the time in figure S2B.
10. Add title and figure captions to supplementary figures.
Author Response
Please see the attachment
Author Response File: Author Response.docx
