Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

Round 1

Reviewer 1 Report

Dear Editor of the

Agronomy Journal

I have reviewed the manuscript titled “Preparation of polyclonal antibody against ZmBT1 protein and its application in hormone regulated starch synthesis”. The manuscript describes the isolation and cloning of the ZmBT1 cDNA encoding a putative protein transporter involved in starch accumulation during maize kernel development. A 171 bp nucleotide sequence of ZmBT1, designated cDNa-C, was amplified by PCR and ligated to the GST DNA sequence; the resulting fused DNA sequence was then cloned into the vector pGEX-6p- and expressed in the E. coli strain BL21 (DE3). The resulting recombinant protein was purified and used to produce rabbit polyclonal antibodies. The resulting antibodies were used to perform the immunoprecipitation and Western blot of the ZmBT1 protein in developing seeds or endosperm of the inbred line Mo17 of maize, and to evaluate in a response-time, the effect of the addition of different plant hormones on the expression of ZmBT1. The manuscript is interesting and shows novel results that are undoubtedly important for the scientific community working in the molecular and biochemical areas of seed development and metabolism, because it potentially adds a new biochemical tool, if the ZmBT1 antibody could be made available to the community.

However, I would like to recomend that the manuscript be accepted only after authors have corrected some of the data they have throughout the manuscript, and add additional information on materials and methods to clarify some of the results, they obtained.

Some of my main concerns are related to the inconsistency in the information provided or not provide by the authors in the materials and methods or in the figures, for example, the complete ZmBT1 cDNA sequence of 1314 bp encodes a protein product of 48.1 kDa, but they said in text 39-44 kDa, why or how?. Another of my concerns is related to the inconsistency in the information provided for the construction of the PGEx-6p-ZmBT1-C (382-437 aa), the authors said that they amplified and fused to GST a 171 bp product (encoding a product of 57 aa) plus the size of GST (data not provided in text or materials and methods); however, the authors wrote that they cloned a C-terminal a cDNA sequence encoding only 55 aa (382-437 aa), please check and clarify or correct the data.  

Another of my concerns is the size of the protein encoded by the vector PGEx-6p-ZmBT1-C (382-437 aa) which express only 55 amino acids of ZmBT1 fused to the largest GST, my question is if they used all the fused protein to produce the polyclonal antibody or if they split the fused protein and used only the ZmBT1-C-terminal (~6.2 kDa) protein to produce the antibody. They have to clarify.

Also, kDa sizes in Figures 4 and 5 must be added with respect to the Western blot and immunoprecipitation analysis, including those of the plant hormone treatments. Also add the kDa for b-actin.

Minor details are related to missing letter or extra words throughout the manuscript. I Have added a list for the authors and enlightened some of those comments in the manuscript.

Minor suggested corrections

Line 71: delete the in the sentence …..gene was integrated the into the cell membrane……..

Line 72: E. coli must be in italics

Line 122: E. coli must be in italics

Line 140: Split 50ug/ml and use “µ” instead “u”

Line 141: Split 50ug/ml and use “µ” instead “u”

Line 153: Split 50ug/ml and use “µ” instead “u”

Line 154: Use “p” in lower case

Line 172: Add the letter “B” to Bradford

Line 175: Describe the TBS salts and pH composition

Line 259: Zea mays must be in italics

Line 273: Figure F has inconsistency because it says 381-437 aa

Line 289: Clarify what is the meaning of of GST-SPI-C fusion protein

Line 361: E. coli must be in italics

My last question is whether the authors have followed a protocol to produce the primary antibody with ethical animal care.

The English throughout the text is fine with only a few mistakes; however if authors make the corrections suggested by the reviewer, then they must write a new text, according to the changes. 

Author Response

Reviewer 1

The manuscript is interesting and shows novel results that are undoubtedly important for the scientific community working in the molecular and biochemical areas of seed development and metabolism, because it potentially adds a new biochemical tool, if the ZmBT1 antibody could be made available to the community.

Author response: We thank the reviewer for reading our paper carefully and giving the above positive comments.

Reviewers Comments to the Authors:

1. The complete ZmBT1 cDNA sequence of 1314 bp encodes a protein product of 48.1 kDa, but they said in text 39-44 kDa, why or how?

Author response: Thank you for pointing this out. ZmBT1-1 encodes a protein with a predicted molecular mass of ca. 47 kDa [1]. In maize endosperms, ZmBT1-1 is present as three 39, 40, and 44 kDa proteins [36-38], ZmBT1 belongs to the mitochondrial carrier family (MCF) and contains three evolutionarily conserved mitochondrial carrier protein domains. Due to the presence of 6 transmembrane domains, the expression of ZmBT1 in microorganisms is challenging. Sullivan et al. (1995) successfully prepared antibodies against BT1 by using the gene sequence encoding 56 amino acids at the C-terminal of BT1 protein [38]. Thus, in this study the C-terminal structure was used to construct a prokaryotic expression vector pGEX-6p-ZmBT1-C (382-437aa) containing a GST tag. Since the samples are recombinant proteins and are not full-length sequences, then the band size must be inconsistent with the predicted size; According to the landing of ZmBT1 (GRMZM2G144081) in the Uniprot database, there are 5 homologs of ZmBT1 (A0A1D6H3J2, A0A1D6H3J1, A0A1D6H3J0, A0A1D6H3I8, A0A1D6H3J3), In ZmBT1, there are two transit peptide cleavage sites, located at 40 amino acid residues upstream of the KTGGL motif and at 13 amino acid residues, respectively. During the transcription and translation of a gene, different forms of proteins can be produced by splice variants; heterodimers.  The same gene may express different protein isoforms in different tissues or developmental stages. These are common reasons why ZmBT1 may show different bands, but the specifics may vary depending on factors such as study methods, experimental conditions and sample sources. Further experiments and studies can help determine the specific origin and significance of these bands.

[1] Sullivan TD, Strelow LI, Illingworth CA, Phillips RL, Nelson OE Jr. Analysis of maize brittle-1 alleles and a defective Suppressor-mutator-induced mutable allele. Plant Cell. 1991;3(12):1337-1348. doi:10.1105/tpc.3.12.1337.

[36] Gizak A, Duda P, Wisniewski J, Rakus D. Fructose-1,6-bisphosphatase: From a glucose metabolism enzyme to multifaceted regulator of a cell fate. Adv Biol Regul. 2019; 72:41-50. doi:10.1016/j.jbior.2019.03.001

[37] Bahaji A, Muñoz FJ, Seguí-Simarro JM, et al. Mitochondrial Zea mays Brittle1-1 Is a Major Determinant of the Metabolic Fate of Incoming Sucrose and Mitochondrial Function in Developing Maize Endosperms. Front Plant Sci. 2019; 10:242. doi:10.3389/fpls.2019.00242

[38] Li HM, Sullivan TD, Keegstra K. Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein. J Biol Chem. 1992;267(26):18999-19004.

2. Comment from Reviewer 1 related to the inconsistency in the information provided for the construction of the PGEx-6p-ZmBT1-C (382-437 aa), the authors said that they amplified and fused to GST a 171 bp product (encoding a product of 57 aa) plus the size of GST (data not provided in text or materials and methods); however, the authors wrote that they cloned a C-terminal a cDNA sequence encoding only 55 aa (382-437 aa), please check and clarify or correct the data.  

Author response: Thank you for underlining this deficiency. For the construction of PGEx-6p-ZmBT1-C (382-437 aa) vector, we designed primers using maize endosperm cDNA as the template and PCR amplified a 171 bp fragment of the C-terminus of ZmBT1 gene, which was consistent with the theoretical molecular weight (Figure 3D). The PGEX-6p-ZmBT1-C vector was successfully constructed by enzymatic digestion and ligation reaction. The recombinant plasmid PGEX-6p-ZmBT1-C was verified by double digestion with BamH I and EcoR I, and a fragment with a band size of 171bp was obtained, which was consistent with the expected results (Figure 3E)；As for the size of the product of the constructed fusion expression protein PGEx-6p-ZmBT1-C (382-437 aa), we described in result 3.3（Recombinant plasmid construction and prokaryotic expression） of the main text. After identification by 12%SDS-PAGE, a considerable number of recombinant ZmBT1 protein bands with molecular weights ranging from 25kDa to 35 kDa were obtained. This is consistent with the expected theoretical molecular weight (approximately 32 kDa) (Figure 3G).

3. They have to clarify if they used all the fused protein to produce the polyclonal antibody or if they split the fused protein and used only the ZmBT1-C-terminal (~6.2 kDa) protein to produce the antibody.

Author response: Thank you for pointing this out. We are purifying the fusion proteins after induction according to the GST-tag protein purification kit (catalog number: p2262, Beyotime) used to purify the recombinant fusion proteins without using proteases to split the fusion proteins, so we are using all the fusion proteins to produce polyclonal antibodies.

4. kDa sizes in Figures 4 and 5 must be added with respect to the Western blot and immunoprecipitation analysis, including those of the plant hormone treatments. Also add the kDa for β-Actin.

Author response: Thank you for the above suggestion. According to your suggestion, our immunoprecipitation analysis in Figure 4 and Figure 5 and Western blot of hormone treatment in figure 6 increased the size of ZmBT1 and β-Actin.

5. Whether the authors have followed a protocol to produce the primary antibody with ethical animal care.

Author response: Thank you for pointing this out. Rabbits were maintained in the Animal Core Facility following procedures approved by the Animal Care and Use Committee of Sichuan Agricultural University (no 20160320, Chengdu, China).

Minor suggested corrections

Line 71: delete the in the sentence …..gene was integrated the into the cell membrane……..

Author response: We have modified the sentence according to the previous comment.

Line 72: E. coli must be in italics

Author response: “E. coli” is changed to “E. coli” (Line 72).

Line 122: E. coli must be in italics

Author response: “E. coli” is changed to “E. coli” (Line 122).

Line 140: Split 50ug/ml and use “µ” instead “u”

Author response: “u” is changed to “µ” (Line 140).

Line 141: Split 50ug/ml and use “µ” instead “u”

Author response: “u” is changed to “µ” (Line 141).

Line 153: Split 50ug/ml and use “µ” instead “u”

Author response: “u” is changed to “µ” (Line 153).

Line 154: Use “p” in lower case

Author response: “P” is changed to “p” (Line 154).

Line 172: Add the letter “B” to Bradford

Author response: Corrected. “radford” has been changed to “Bradford” (Line 172).

Line 175: Describe the TBS salts and pH composition

Author response: Thank you for the suggestion. We have added the information required as explained above for TBS salts and pH composition (Lines 175).

Line 259: Zea mays must be in italics

Author response: “Zea mays” was changed to “Zea mays” (Line 259).

Line 273: Figure F has inconsistency because it says 381-437 aa

Author response: Thank you for the suggestion. We have modified Figure 3F.

Line 289: Clarify what is the meaning of of GST-SPI-C fusion protein

Author response: This is a mistake in writing. We apologise for the errors and have made the suggested. “GST-SPI-C” was changed to “GST-BT1-C”.

Line 361: E. coli must be in italics

Author response: “E. coli” was changed to “E. coli” (Line 361).

We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have

Thanks!

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

The manuscript is well written and the topic is relevant. I do recommend its acceptance. I just have few questions regarding the methods.

Whenever the authors mention seeds, they are referring to the cariopses fruit? This is important to verify. Or does the authors removed the pericarp?

Figure 5. The authors conducted analysis off BT1 expresso in seeds and endosperm. Did they try some tissue print of the cariopses?

Figure 6. The results are related to hormonal expression in what plant tissue?

Author Response

Reviewer 2

The manuscript is well written and the topic is relevant. I do recommend its acceptance.

Author response: We thank the reviewer for reading our paper carefully and giving the above positive comments.

Reviewers Comments to the Authors:

1. Whenever the authors mention seeds, they are referring to the cariopses fruit? This is important to verify. Or does the authors removed the pericarp?

Author response: Thank you for pointing this out. Verifying this will be of great help to our future research. Corn is an annual herb of the grass family with a complete fruit and seed structure. The corn kernel is the fruit of corn. The corn fruit belongs to the caryopsis and contains only one seed. At maturity, the pericarp and seed coat come together and cannot be separated and are closed, and a fully mature corn kernel can germinate and grow. Therefore, corn kernels are also seeds. Previous studies have found that ZmBT1 is mainly expressed in the seeds and endosperm. For maize seeds, we took intact kernels from 3-30 d after self-pollination without removing the pericarp, and for maize endosperm, we took seeds from 6-30 DAP but removed the seed coat and embryo.

2. The authors conducted analysis off BT1 expresso in seeds and endosperm (figure 5).  Did they try some tissue print of the cariopses?

Author response: Thank you for pointing this out. Our work focused on the key role of ZmBT1 in starch accumulation during maize seed development, mainly involving maize seeds and endosperm, but also some analysis of other maize tissues (embryo, root, stem, and leaf), limited to tissue expression analysis, and of course, for the analysis of some tissue print of the cariopses as you suggested, we did not consider, which gives us some ideas for future research. For example, phosphorylation of ZmBT1 may affect its localization in its cell (data not published), so we can isolate maize endosperm protoplasts to explore the localization of ZmBT1.

3. The results are related to hormonal expression in what plant tissue? Figure 6.

Author response: Thank you for underlining this deficiency. Plant hormones are critical regulators of various stages of plant growth and development, including endosperm starch synthesis. The ZmBT1 is mainly expressed in the seeds and endosperm, so the direction of our study was the effect of hormones on ZmBT1 expression in maize seeds. To study the response of ZmBT1 to plant hormones, the 10 DAP middle seed of corn cob was used and treated with different hormones (0h,6h,12h,24h) and analyzed. The detail is also included in figure legend (line 354-355).

We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have

Thanks!

Author Response File: Author Response.pdf