PIN3 from Liriodendron May Function in Inflorescence Development and Root Elongation

Round 1

Reviewer 1 Report

The authors identified LhPIN3 found in timber tree. It has been shown that the function is quite similar to PIN1 in Arabidopsis, but more research is needed. Although it requires a genome-wide analysis, it seems to have been oversimplified.

Fig6A , the number of flowers in the LhPIN3 overexpressing line increased compared to WT. However, in the complemented line with pin1 KO, does the 35S promoter only have the same number of flowers as WT? An explanation for this should be added. If a promoter such as Ubqintin was used instead of 35S in the complement experiment, Figure 6B can be understood.

Fig5, I know the phenotype of the pin1 mutant, but it would be better to put the picture for the LhPIN3 OE line on the same floor and take a picture to clearly show the difference. Although the authors have included a scale bar, it should be easier to show how it differs from Col-0.

In Fig. 4, A and B, why are the root lengths different for WT at 20 days? Rather than distinguish between A and B, it seems necessary to show the data of the same experiment.

Fig3, it would be better to show the photos grown vertically on the same MS medium at once. Of course, there is no problem to show the difference between each individual, but I think it is right to show the developmental difference more clearly.

Fig2A, It seems convenient to attach a name for each tissue rather than using a number. In C, what is the zero point of expression of LhPIN3? How about using the data that actually did qRT-PCR in Fig2? It seems that the data now can be used for backup.

In Fig1, the figure legned seems to lack explanation. Please add as much detail as possible to explain it.

Author Response

Response to Reviewer 1 Comments

 

Point 1: Fig6A, the number of flowers in the LhPIN3 overexpressing line increased compared to WT. However, in the complemented line with pin1 KO, does the 35S promoter only have the same number of flowers as WT? An explanation for this should be added. If a promoter such as Ubqintin was used instead of 35S in the complement experiment, Figure 6B can be understood.

Response1: Thanks for the comments. In Arabidopsis, the expression pattern of ProPIN3:PIN3-GFP in pin1 mutant was comparable to that of wild-type and occasional ectopic expression of PIN3-GFP had no effect on the pin1 phenotype (Guenot et al., 2012, Plant Physiology, doi: 10.1104/pp.112.200402), indicating that PIN3 cannot rescue the lack of PIN1 function. In addition, ProPIN1:PIN3 in pin1 mutant led to abnormally shaped and often increased number of petals in Arabidopsis (Zhang et al., 2020, Science Advance, doi: 10.1126/sciadv.abc8895), indicating that PIN3 cannot rescue the pin1 defects in the floral organ. Consistently, we also found that abnormal number of petals and sepals existed in 35S:LhPIN3 pin1 planets (new Supplementary Materials). Overall, we suggested that 35S:LhPIN3 can increase the flower number in Arabidopsis wild-type, likely independently of AtPIN1. Thus, it can increase the flower number of pin1 mutant not too much but fail to rescue the defects in flower organ development, such as numbers of petals. We also included this discussion in the revised manuscript (Page 11, Line 359-366).

 

Point 2: Fig5, I know the phenotype of the pin1 mutant, but it would be better to put the picture for the LhPIN3 OE line on the same floor and take a picture to clearly show the difference. Although the authors have included a scale bar, it should be easier to show how it differs from Col-0.

Response 2: Thanks for the comments. Flowers differed little between wild-type and overexpressing plants, so we added pictures of petals to show the difference, and a description of the difference in the Results and Discussion sections, citing literature that can testify to our result.

 

Point 3: In Fig. 4, A and B, why are the root lengths different for WT at 20 days? Rather than distinguish between A and B, it seems necessary to show the data of the same experiment.

Response 3: Thanks for the comments. There are some differences in average root length of two Arabidopsis wild-type (4.8cm and 5.4cm), thus, we conduct ANOVA analysis using the data of two group and the result showed that the root length of WT has not significant difference(p=0.212). We believe that the difference is due to random error caused by a combination of factors, since the two transgenic trials were not conducted simultaneously. Another reason could be the difference of the range of Y-axis of the two graphs, and we show off our data below.

 

 

 

Table 1: Root length of wild-type and transgenic plants

	Root length of 10d (cm)				Root length of 20d (cm)
	WT	OE-1	OE-2	OE-3	WT	OE-1	OE-2	OE-3
Fig 4 A	1.8	4	3.41	3.49	4.8	8.3	8.46	8.46
	1.86	5.9	5.16	4.23	5	7.6	8.46	9.54
	1.74	2.1	4.56	3.56	4.6	9	7.46	7.65
	1.95	3.8	4.02	4.16	6	8.14	9.46	10.54
	1.65	4.2	5.22	4.56	3.6	8.46	6.14	8.23
	1.79	4.9	3.36	3.16	5.13	9.46	10.87	9.77
	1.81	3.1	4.16	5.46	4.47	7.14	9.63	9.26

 

 

Table 2: Root length of wild-type、pin1 mutant and transgenic plants

	Root length of 10d (cm)					Root length of 20d (cm)
	WT	pin1 mutant	Line-1	Line-2	Line-3	WT	pin1 mutant	Line-1	Line-2	Line-3
Fig 4 B	1.8	0.6	1.8	1.74	1.59	4.8	2	5.5	5.56	6.79
	1.86	0.43	1.73	1.98	1.98	7.3	2.7	5.71	5.19	5.46
	1.74	0.77	1.87	1.59	2.16	4.6	1.3	5.29	5.63	5.16
	1.95	0.72	1.92	1.99	2.25	6	1.73	4.29	6.79	8.46
	1.65	0.48	1.68	2.36	1.26	5.6	2.27	6.71	5.49	5.46
	1.79	0.89	1.46	2.18	1.56	5.13	1.28	6.42	7.16	7.16
	1.81	0.31	2.14	1.49	1.79	4.47	2.72	4.58	4.56	5.43

 

Point 4: Fig3, it would be better to show the photos grown vertically on the same MS medium at once. Of course, there is no problem to show the difference between each individual, but I think it is right to show the developmental difference more clearly.

Response 4: Thanks for the comments. We conduct two experiments in Figure3, Figure A and Figure B is used to show the difference of Arabidopsis wild-type and LhPIN3-OE plants; and the results of another experiment is showed in the Figure C and Figure D. We believed that the pictures we show are sufficient to explain the results we obtained.

 

Point 5: Fig2A, It seems convenient to attach a name for each tissue rather than using a number. In C, what is the zero point of expression of LhPIN3? How about using the data that actually did qRT-PCR in Fig2? It seems that the data now can be used for backup.

Response 5: Thanks for the comments. Our transcriptome data has been published and can also be obtained on the NCBI website, cold and heat stress accession numbers were PRJNA679089 and drought stress accession number was PRJNA679101. The “COL” on the graph is the expression of Oh. In order to prevent misperceptions, we have modified it.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The author et al., cloned and characterized the auxin 14 transporter gene PIN-FORMED3 (PIN3) from timber tree hybrid. LhPIN3  was isolated from hybrid Liriodendron plants then sequence analysis was performed with comparison of Arabidopsis and other species which showed the conserved protein encoding membrane trans superfamily. Then the expression profile confirmed from different developmental stage of Liriodendron. Functional characterization was performed using overexpression of LhPIN3 in arabidopsis and complementation of Arabidopsis pin1 mutant allele.

Overall, the study is well presented, provide sufficient data, and offer new findings of molecular aspects in the regulation of PIN in Liriodendron. This article will contribute important finding in science community. I have no major comment about the research and manuscript. I have few comments and suggestion that author may consider.

1. In the introduction, the purpose of the study is mentioned in line 92 to understand the function of PIN3 in Liriodendron. However, the paragraph leading to this purpose not clearly wrote the clear background of selection of PIN3 among other PIN families. The current last paragraph is little bit hard to understand the flow. You may make it more concise or would be easier if author could possibly just explain more about PIN3. Or just removed the line 88-91.
2. In Figure 2B, it will be easier, in the legend you can put exact label or in the caption write it clearly (1, embryonic callus; 2, suspension culture; 3, somatic embryo… ect.). Too many number to track if using “respectively”.
3. It is interesting discussion from line 287-302 about the stress response of PIN. However, discussion about the expression pattern may be more in line with your data. Maybe start with expression pattern and the stress response will be last. Just consider this, which one is more suitable to your flow of the article.
4. While I agree that the expression in the inflorescense is one of the key organs regulated by PIN3. Still, the expression was highly in the roots, so maybe author can explain more root phenotype in results. Because in the discussion it is well discussed in line 308~ but very less in the result.
5. Please check the writing consistency of gene. Some you wrote it Upper case, Italic.

 

Author Response

Response to Reviewer 2 Comments

Point 1: In the introduction, the purpose of the study is mentioned in line 92 to understand the function of PIN3 in Liriodendron. However, the paragraph leading to this purpose not clearly wrote the clear background of selection of PIN3 among other PIN families. The current last paragraph is little bit hard to understand the flow. You may make it more concise or would be easier if author could possibly just explain more about PIN3. Or just removed the line 88-91.

Response 1: Thanks for the comments. We want to introduced the knowledge about Liriodendron in the last paragraph, but the logic seems confusing. Thus, we moved the line 88-91 to the beginning of the last paragraph of Introduction, and added the function of PIN3 among other PIN families.

 

Point 2: In Figure 2B, it will be easier, in the legend you can put exact label or in the caption write it clearly (1, embryonic callus; 2, suspension culture; 3, somatic embryo… ect.). Too many number to track if using “respectively”.

Response 2: Thanks for the comments. The description of the material in this part is indeed too cumbersome. We have revised it according to your suggestion.

 

Point 3: It is interesting discussion from line 287-302 about the stress response of PIN. However, discussion about the expression pattern may be more in line with your data. Maybe start with expression pattern and the stress response will be last. Just consider this, which one is more suitable to your flow of the article.

Response 3: Thanks for the comments. We only considered the order of the discussion sections in the order of the results, but it is better to start with expression pattern and end with stress response to meet the research focus of the article.

 

Point 4: While I agree that the expression in the inflorescense is one of the key organs regulated by PIN3. Still, the expression was highly in the roots, so maybe author can explain more root phenotype in results. Because in the discussion it is well discussed in line 308~ but very less in the result.

Response 4: Thanks for the comments. The function of LhPIN3 in root length of Arabidopsis was studied by performing the experiment. Thus, we don’t think it is necessary to add other phenotypes. We have revised our result section to make it more specific and have supplemented some literatures about the function of PIN3 on lateral root development to enrich our discussion section.

 

Point 5: Please check the writing consistency of gene. Some you wrote it Upper case, Italic.

Response 5: Thanks for the comments. We have revised the article in strict accordance with the format of protein upper case, gene uppercase italics, and mutant lowercase italics.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript ID forests-1628484 titled "PIN3 from Liriodendron may function in inflorescence development and root elongation" aimed to decipher the function of PIN3 gene in the tree hybrid Liriodendron (Liriodendron chinense × L. tulipifera). Understanding the function of this gene is relevant as previous studies noticed by the authors revealed that it plays an important role in plant. From this point of view, this topic research is relevant. Based on this, the experimental design is scientifically sound leading to record significant results such as PIN3 expression quantification which showed high expression in the roots. Despite these significants acheivements, the paper needs major modifications such as:

1. the transformation protocol needs more description
2. the authors have to showed by PCR and Southern blot that the transfert gene is physically present into the transformed plant genome and show the copy number.
3. Based on this manuscript cannot be accepted for publication on its current form.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

Point 1: the transformation protocol needs more description

Response 1: Thanks for the commends. More details about transformation and screening of positive plants were added to the material and method section.

 

Point 2: the authors have to showed by PCR and Southern blot that the transfert gene is physically present into the transformed plant genome and show the copy number.

Response 2: Thanks for the comments. The transgenic T3 lines were selected using KAN resistance and then further verified by PCR using primers (Table S4 and Figure S3). Combing the fact that all transgenic T3 lines showed obvious different phenotypes compared to the control with no obvious differences among these transgenic T3 lines, we believed that these evidences were enough to confirm the transfer gene. Such method is broadly used to verify the transfer gene in Arabidopsis (Tiwari et al., 2020, Plant cell reports, doi: 10.1007/s00299-020-02620-1; Lu et al., 2019, 10.3389/fpls.2019.00299).

 

Point 3: Based on this manuscript cannot be accepted for publication on its current form.

Response 3: We listened carefully to the reviewers, added more details about transformation and screening of positive at method section, performed PCR assay to check positive, and more, adjusted the introduction of the paper to make it more logical, replaced some pictures, used average values to improve the results. We are delighted to receive positive feedback.

Round 2

Reviewer 1 Report

I hope that the figure in Fig6B is properly corrected.

Author Response

Dear Forests Editor,

RE: forests-919340-"PIN3 from Liriodendron may function in inflorescence development and root elongation"

 

We would like to thank you and the two reviewers for the thoughtful and constructive suggestions that substantially improved our manuscripts. We have modified the abscissa annotations in Figure 6B to make it more concise. We are delighted to receive positive feedback. All changes are marked using the track changes mode. Thanks for the commends.

 

 

 

 

 

On the behalf of the authors

Yours sincerely,

 

Prof Jin-Hui Chen

Corresponding author

Reviewer 3 Report

The manuscript is significantly improved after revision. All the suggestions were taken into account by the authors. 

Author Response

Dear Forests Editor,

RE: forests-919340-"PIN3 from Liriodendron may function in inflorescence development and root elongation"

 

We would like to thank you and the two reviewers for the thoughtful and constructive suggestions that substantially improved our manuscripts. It is very helpful for us to revise our manuscript, it makes our materials and methods more specific, and makes our results more credible, Thanks for the comments.

 

 

On the behalf of the authors

Yours sincerely,

 

Prof Jin-Hui Chen

Corresponding author

 

Author Response File: Author Response.docx