Auxin-Induced Adventitious Rooting in Pepper Involves CaLBD16: Functional Evidence from Tomato Overexpression

Comments 1: The main question is why the authors include pepper in the title, although only one experiment was conducted on it. All other experiments, transformations, etc., were conducted on tomato plants.

Response 1: Thank you for pointing this out. I agree with this comment. First, we investigated the role of a pepper gene. However, the genetic transformation system for pepper itself is not well established. Although relevant methods have been published, no study on pepper has yet utilized materials derived from stably inherited, homologous genetic transformation in pepper. Tomato, as a plant belonging to the same Solanaceae family with a more mature genetic transformation system, is often used to validate phenotypes observed in pepper. Therefore, we heterologously overexpressed the pepper gene in tomato to observe its phenotype.

Comments 2: In the "Results" chapter, the authors repeatedly use the words "significantly more," "significantly increased," "significantly activates," etc. (Lines 73, 88, 121, 150, 158, 174). Numerical concepts are necessary.

Response 2: Agree. I have added the fold difference information in the manuscript to make the results clearer and easier to understand. Relevant changes have been made in lines 71, 94, 126, 146, and 169.

Comments 3: The discussion is very sparse and does not provide insight into the extent to which this issue has been addressed for pepper.

Response 3: Agree. We thank you for your guidance on our writing. We have revised the final paragraph to clarify the significance for pepper, which can be found in line 246.

Comments 4: Line 250. The tissues from which the extraction was carried out are not indicated.

Response 4: Agree. We appreciate your correction. We apologize for any confusion caused by our oversight; the treatment and sampling sites have been corrected in line 271.

Comments 1: The title is confusing because the authors used pepper, tomato, and tobacco in the research.

Response 1: Thank you for pointing this out. Because the gene we are studying is from pepper, which lacks a stable transgenic system, phenotypic studies related to pepper are typically conducted by overexpressing the gene in tomatoes to observe the phenotype. Tobacco, on the other hand, is used for certain experiments due to its greater convenience, making it a common approach in such studies.

Comments 2: The authors conducted a large number of experiments, performed a precise analysis, and applied molecular biology methods. However, authors did not apply spatial cell biology methods and did not describe a real mechanism. https://doi.org/10.1111/plb.70178. This is a limitation of the current study, which needs to be mentioned in the results/discussion/ conclusions.

Response 2: We agree with the reviewer on this point. In line 193, we reiterate that the difference between the endogenous auxin concentration gradient and the transient pulse effect of exogenous IAA is a limitation of our study. As for the spatial cell biology methods, we did not conduct sufficient in-depth validation of this phenotype.

Comments 3: L 20 –“The formation of adventitious roots plays a key role in plant asexual reproduction” – I am not sure. Rooting is a secondary process, after de novo shoot induction.

Response 3: Adventitious roots refer to roots that originate from the stems or leaves of plants. In the process of asexual reproduction (such as cuttings and layering), it is precisely by inducing the stems or leaves to produce adventitious roots that new independent plants can be formed. Therefore, the formation of adventitious roots indeed plays a crucial role in asexual reproduction

Comments 4: key genes ¿? Not all auxin biosynthesis pathways affect AR. There are 11 auxin biosynthesis genes that differ in location and roles in development

Response 4: Auxin synthesis is indeed an important factor in inducing adventitious root formation. The reviewer's comment suggests that not all auxin synthesis genes promote adventitious root formation. This is indeed the case; in the literature we cited later, AtYUC2 and AtYUC6 regulate adventitious root formation, whereas not all YUC genes possess this function.

Comments 5: L47- 50- it seems some confusion. WOX5 marked the stem cell (QC) after establishing de novo auxin gradients and rather serves as a readout of auxin gradients. WOX11 may be linked with specific auxin synthesis, indeed.

Response 5: Previous studies (doi:10.1104/pp.16.01067) have indicated that auxin accumulation induces the expression of WOX11 and WOX5, with WOX11 directly activating WOX5 expression. However, the protein accumulation of WOX11 and WOX5 occurs as a continuous process; by the time WOX5 protein accumulation reaches a sufficiently high level, WOX11 protein accumulation has already declined, suggesting that these two factors play distinct roles in different stages of cell differentiation.

Comments 6: L56 -Where is the tomato? A large piece of work is a tomato.

Response 6: Although the tissue we used for phenotypic validation was tomato, the gene function actually originates from pepper, with tomato serving merely as a vehicle. Due to the lack of a relevant experimental system in pepper, this represents a major limitation in pepper research.

Comments 7: L 88 – cell type? “was significantly upregulated”??

Response 7: We thank the reviewer for the question. Here, we refer to the expression pattern in the hypocotyl in response to exogenous IAA treatment, rather than the expression pattern within the whole plant. Due to unclear description in the methods section, this has now been corrected in line 268.

Comments 8: L106 - (B) CaLBD16 is localized to the nucleus ?? =(B) CaLBD16 is localized to the nucleus in differentiated tobacco cells. Scale bar?

L125 - (B) CaARF6 is localized to the nucleus. = (B) CaARF6 is localized to the nucleus in differentiated tobacco cells. Scale bar??

Response 8: We are grateful to the reviewer for the valuable comments. We have revised the manuscript accordingly at lines 110, 132, and 170. The scale bars have also been added to the figure legends. The scale bars in the images are located in the lower right corner and are very small, as they were automatically generated by the computer imaging system and thus cannot be modified.

Comments 9: L128 “35S:CaARF6 with Pro- CaLBD16:LUC in N. benthamiana leaves” – overexpression in an artificial differentiated system.

Response 9: We thank the reviewer for pointing out the limitations of the experiment. We have clarified in lines 125 and 166 that this experiment was conducted in an artificial differentiated system and involved transient overexpression.

Comments 10: L143 - (C) Morphological observation of AR formation – I saw significant differences of shoot structure in transgenic lines = more shoot-derived auxin per seedlings = more AR. Please, clarify.

Response 10: We thank the reviewer for the insightful comments. According to the literature, ARF transcription factors are auxin response factors that function downstream of the auxin signaling pathway, a finding we also verified through exogenous IAA treatment (Figure 3A). Whether ARF6 participates in a positive feedback loop with auxin was not addressed in our study. All experimental materials were sown simultaneously, and lines with similar growth vigor were selected for experimental validation. However, given that the seedling stage is a period of rapid plant development, we observed in preliminary experiments that faster rooting led to quicker nutrient absorption, resulting in differences in hypocotyl length and overall growth. Therefore, shoot structure was not specifically examined in this study. Nevertheless, the phenotype of IAA-activated ARF6 promoting adventitious rooting remains consistent.

Comments 11: L152 – there is no “expression pattern of CaLBD” . Which cell type? Phloem? Epidermis? Glandular trichomes?

Response 11: We apologize for the misunderstanding caused by the lack of labeling. This refers to the upregulation of CaLBD16 expression in response to IAA treatment, and we have now added a reference to the figure.

Comments 12: L178 (B) Morphological observation of AR formation in CaWOX11-overexpress- - significantly more developed shoots here. Please, explain whay do you think that wox11 has a direct effect on AR only.

Response 12: We thank the reviewer for the comments. In fact, during the selection of seedlings for mechanical injury treatment, we did not observe significant differences in shoot morphology. Prior to mechanical injury treatment, the plants were in a vigorous growth phase, and their morphology varied from day to day. After treatment, due to the earlier onset of rooting, the hypocotyl length of lines that rooted earlier temporarily showed some inconsistency, and at this stage, the newly formed adventitious roots were very short. However, this difference eventually disappeared. This suggests that the earlier rooting may have influenced shoot development, rather than the shoots affecting the hypocotyl. Furthermore, overexpression affects the entire plant, and our focus is specifically on its role in adventitious root formation along the hypocotyl. As the reviewer noted, the plant functions as an integrated whole, making it indeed difficult to isolate its specific role. However, based on more detailed observations and reports from previous studies, we believe that this represents a direct regulation of adventitious root formation.

Comments 13: “the basal parts of the hypocotyls” – but you used shoots, please, avoid confusion

Response 13: We apologize for any confusion caused by the terminology. After verifying the anatomical definitions, we confirm that the explants used in our study—specifically, the cuttings made below the cotyledons and above the root—are indeed hypocotyls, not shoots. Accordingly, both the adventitious root formation and the IAA treatment described in our manuscript occurred specifically on the hypocotyl.

Comments 14: what about carbohydrates? Maybe you used MS-salt, not MS medium?

It is very interesting that you used high N:P ratio (48:1) for rooting. It is well-known that such a ratio is far from optimal for rooting. Can you please explain your ideas? Please note that someone may have ignored such information; it does not mean you need to repeat it. Please, next time, be attentive.

Response 14: We thank the reviewer for pointing out the details in our description. Carbohydrates are indeed important, and we have revised the MS medium formulation accordingly in line 261. The reason for using MS medium is based on our method for obtaining overexpression in tomatoes, where the final rooting step involves MS medium supplemented with IBA.

Comments 15: L.236- 8000 lux = xxx mkmol/m2/sec.

Response 15: We thank the reviewer for the comments. Since we used a full-spectrum daylight lamp, the light intensity of 8000 lux corresponds to 144 μmol·m⁻²·s⁻¹., this has been corrected in line 267.

Comments 16: L237 – foliar spray can not be compared with medium addition.

Comments 17: L238 - Samples were harvested “  which? Have you mixed all the mRNA populations from whole seedlings or only from the hypocotyl vasculature? This is a key question here: it seems you measure some virtual “average” among” many cell populations, right? With each have own gene expression profile. Please consider that activation of gene expression in different cell files (such as mesophyll and vasculature, for example) may have a different meaning and effects. Even though they belong to “auxin signaling pathways”. Please consider this in the discussion.

Response 16 and 17: We apologize for the ambiguity caused by our wording. To clarify, this treatment was applied by spraying the hypocotyls for both treatment and sampling. This has been corrected in line 268.

Comments 18: L257 - = putative subcellular localization. Here, you used a very specific cell type and can not extrapolate this localization to a highly cytoplasmic-reach tomato hypocotyl cell.

Response 18: We thank the reviewer for the insightful comments, which have broadened our understanding of subcellular localization experiments. Constrained by experimental conditions, we employed the most conventional method—transient overexpression in tobacco leaves for subcellular localization observation—which is indeed not sufficiently rigorous.

Comments 19: L275 - = putative interaction. Here you used an artificial system, induced the expression of a gene in a specific cell, and claimed that they interact. Even you did not show localization (cell type) of original gene in tomato/pepper.

Response 19: We thank the reviewer for the comments. We agree that it would be more appropriate to validate protein-DNA interactions in plant native systems. However, the experimental system in pepper remains relatively underdeveloped, and our research group is currently working to improve this issue.

Response to Comments on the Quality of English Language

Point 1: L34 produce elite plants with desirable traits = multiply

Response 1: We appreciate the reviewer's suggestion for improving our wording and have revised it accordingly in line 32.

Point 2: auxin plays the most critical = endogenous auxin plays the key role.

Response 2: Thank you for pointing out this issue, it has been corrected to "exogenous auxin" in line 34.

Point 3: L38- High concentrations of IAA can stimulate the differentiation of phloem cells = Treatments with exogenous IAA can stimulate the differentiation of phloem cells, creating a way for unidirectional endogenous auxin canalization.

Response 3: We thank the reviewer for identifying the issues with our language expression and have revised it accordingly in line 37.