Characterization of Vascular plant One-Zinc finger (VOZ) in soybean (Glycine max and Glycine soja) and their expression analyses under drought condition.

Vascular plant one-zinc-finger (VOZ) transcription factors regulate plant growth and development under drought conditions. Six VOZ transcription factors encoding genes exist in soybean genome (both in Glycine max and Glycine soja). Herein, GmVOZs and GsVOZs were identified through in silico analysis and characterized with different bioinformatics tools and expression analysis. Phylogenetic analysis classified VOZ genes in four groups. Sequence logos analysis among G. max and G. soja amino acid residues revealed higher conservation. Presence of stress related cis-elements in the upstream regions of GmVOZs and GsVOZs highlights their role in tolerance against abiotic stresses. The collinearity analysis identified 14 paralogous/orthologous gene pairs within and between G. max and G. soja. The Ka/Ks values showed that soybean VOZ genes underwent selection pressure with limited functional deviation arising from whole genome and segmental duplication. The GmVOZs and GsVOZs were found to express in roots and leaves at seedling stage. The qRT-PCR revealed that GmVOZs and GsVOZs transcripts can be regulated by abiotic stresses such as polyethylene glycol (PEG). The findings of this study will provide a reference to decipher physiological and molecular functions of VOZ genes in soybean.

Introduction

Wild soybean (Glycine soja) is an ancestor of cultivated soybean (Glycine max L.). Soybean is an important source of edible oil, protein, food, and feed [1]. Owing to intensive domestication and selection for higher yield, G. max has lost considerable genetic diversity through continuous genetic bottleneck. G. soja can be a promising source of new genes for G. max breeding required in response against climatic changes. The genomes of G. max and G. soja have been sequenced which enable whole genome analyses of gene families in both the species of soybean [2, 3].

Isolation and genome-wide characterization of plant transcription factors (TFs) have critical importance [4-6]. TFs play their role in stress tolerance by regulating stress responsive genes. Hence, expression of genes plays important roles in plant responses to different stimuli. Vascular plant one-zinc-finger (VOZ) TF family is present in many plant species. The VOZ family contains ~635 members and there homologous in 166 plant species [7]. The VOZs play protective role against ever changing environmental stresses and regulate plant growth and development. The VOZ proteins are plant specific TF family with two members, VOZ1 and VOZ2, exclusively present in vascular plants [11]. Mitsuda et al. [8], identified the first VOZ protein which binds to the GCGTNx7ACGC palindromic sequence in the regulatory region of the Arabidopsis thaliana V-PPase gene (AVP1) using yeast one-hybrid screening [9-11]. This VOZ plays important role in regulating flowering in crop plants. The VOZ1/2 phytochrome B-interact with TF and regulate the vegetative growth to reproductive stage by up-regulating flowering locus T and down-regulating flowering locus C expression in A. thaliana [12]. The VOZ1/2 promote flowering mainly by modulating and interacting with CONSTANS (CO), though this is independent of FLC in A. thaliana [11]. Besides flowering, transcriptional regulators inhibits plant tolerance to abiotic stress factors such as drought, high and low temperature. For example, over-expression of VOZ2, damaged the tolerance of plants against drought and low temperature stresses in A. thaliana but improved resistance against fungal pathogen [13]. In A. thaliana voz1/voz2 (dual mutants) showed enhanced tolerance to low temperature and water stresses. VOZ1 and VOZ2 suppressed the expression of DREB2C or DREB2A [14, 15]. During salt stress, VOZ also response likewise in low temperature and water stress conditions. According to some reportsthe genes, responsible for salt stress tolerance, are being controlled directly or indirectly by VOZ1/2 [16, 17].

At first, VOZ gene family was characterized in A. thaliana based on its expression and function. Afterwards this gene family was reported in various crop plants [10, 16, 17]. Research on the characterization of VOZ in other crop species is still nascent. In this study, 12 members of VOZ family were characterized in G. max and G. soja. Phylogenetic analysis, sequence logos, sub-cellular localization, gene duplication chromosomal distribution, Ka/Ks ratio, promoter cis-element, and exon/intron structure were predicted. Moreover, sub-cellular localization and transcript abundance of GmVOZs and GsVOZs were also measured under drought stress conditions. The results of this study will provide a reference for future research on regulatory roles of VOZ transcription factors in soybean.

Materials and methods

Sequence identification

Arabidopsis VOZs protein sequences has been used as query sequences to identify VOZ transcription factors and proteins in different plant species such as G. max, G. soja, M. truncatula, G. raimondaii, S. melongena, P. trichocarpa, P. patens, A. comosus, O. sativa, Z. mays and B. rapa. To confirm the retrieved VOZ protein sequences, Interproscan 63.0 (IPR039277) (http://www.ebi.ac.uk/InterProScan/) and SMART (http://smart.embl-heidelberg.de/) tools were used. The identified VOZ members were confirmed by using Batch Web CD search tool CDD (Conserved Domains Database) search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Biophysical properties such as protein length, isoelectric point, molecular weight were computed using ExPASy ProtParam tool (http://us.expasy.org/tools/protparam.html).

Sequence alignment and phylogeny

To create sequence logos for conserved amino acid residues (AARs) multiple sequence alignment of G. max and G. soja was performed independently using CLUSTAL X 2.0 and sequence logos were created from WEBLOG [18]. For phylogenetic analysis, protein sequences of VOZs from different plant species were aligned and phylogenetic tree was made in MEGA V 6 (https://www.megasoftware.net) by using maximum likelihood (ML) method [19]. Bootstrap method with thousand replicates was used to determine the reliabilities of the groups.

Sub-cellular localization

Sub-cellular localization of GsVOZs and GsVOZs were predicted using the online tool CELLOv.2.5 (http://cello.life.nctu.edu.tw/). The sub-cellular localization was also observed in Nicotiana benthamiana leaf cells having vectors with the coding regions of GmVOZ1 and GsVOZ1 and GFP as a control, were transferred into N. benthamiana leaves through Agrobacterium tumefaciens (GV3101) mediated transformation and spotted 4 days after incubation at 23°C in a photoperiod of 6 h dark and 16 h light. Oligonucleotide sequences and enzymes used in present study are provided in S1 Table.

Exon/intron and cis-element analysis

GmVOZs and GsVOZs were initially aligned by CLUSTAL X 2.0 followed by MEGA V6 to create ML tree. The resultant bed-file was subjected to Gene structure display server 2.0 (GSDS), which was used to identify the exons and introns [20]. For cis-element analysis of GmVOZs and GsVOZs promoter regions ⁓2 kb upstream of ATG were considered as promoter region and subjected to PlantCARE Database [21] and predicted cis-element were categorized on the basis to their functional significance.

Collinearity and Ka/Ks ratio analysis

For collinearity analysis, paralogous/orthologous data were obtained as descried by Yang et al. [22] and then figure was produced using CIRCOS package [23]. Non-synonymous (Ka) and synonymous (Ks) deviation level ratios were computed by aligning duplicated gene pair protein sequences in CLUSTAL X 2.0, after which they were translated into cDNA sequences using PAL2NAL program [24]. At the end, Ka and Ks values were computed with the help of CODMEL program using the PAML package [25].

Gene expression profiling of GmVOZs and GsVOZs

G. max cv. William-82 and G. soja cv. W05 were used as plant material to determine the tissue specific expression pattern under normal as well as under drought stress conditions. To analyze the expression of GmVOZs and GsVOZs genes in different tissue and development stages, soybean plants were grown at research farm of MNS University of Agriculture, Multan, and all samples were collected under field conditions with standard agronomic practices. To explore the expression under drought conditions, William-82 and W05 were grown in a 3:1 (w/w) mixture of soil and sand (day/night temperature 22/20°C), germinated and irrigated with Hoagland solution once in every two days. After 28 days, the seedlings were germinated with 20% Poly Ethylene Glycol 6000 (PEG). Root samples from control and treated seedlings were taken at 0h, 1h, 3h and 6h after treatment. RNA was extracted using RNAprep Pure Plant Kit (TIANGEN, China). PrimeScript®RT reagent kit (Takara, China) was used to synthesize cDNA. For qRT-PCR, Premix Ex TaqTM II (Takara) was used and PCR amplification was performed on CFX connect Real-Time PCR detection system (Hercules, California 94547, U.S.A). The housekeeping genes, Actin and GAPDH, were used as internal control in G. max and G. soja, respectively. Each experiment was conducted in triplicates. Oligonucleotide sequences used in current work are listed in S1 Table.

Results

Identification of VOZ gene family members

In silico approach was used to identify the 47 VOZ gene members in 12 different species including 2 each from Arabidopsis thaliana, Medicago truncatula, Brachypodium distachyon, Ananas comosus, and Solanum melongena, 3 each from Psychomitrella patens, and Gossypium raimondii, 4 from Populus trichocarpa, 6 each from Zea mays, Glycine max, Glycine soja and 9 from Brassica rapa. We noticed that all the selected species have a minimum of two VOZ genes with Z. mays, G. max, G. soja and B. rapa have a greater number of members and M. truncatula, P. paten have only two and three members of VOZ genes, respectively, showing that this gene family was subjected to expansion in higher plants. The biophysical properties of GmVOZ and GsVOZ genes including locus ID, gene length, coding sequences (CDs), protein length, molecular weight (MW), isoelectric point (pI), chromosome position and sub-cellular localization of all GmVOZs and GsVOZs were determined (S2 Table). Fewer variations were observed in GmVOZs and GsVOZs in terms of coding sequences, proteins length and molecular weights. However, the predicted sub-cellular localization results indicated that all GmVOZs and GsVOZs are localized in nucleus (S2 Table).

Sequence logos

Sequence logo was generated to check the extent of conserveness of VOZ proteins family in G. max and G. soja (Fig 1). We observed that sequence logos among two species were highly conserved across the N and C termini such as I (1), L (3), C (7)], P (8), F (9), L (10), W (26), H (32), F (55), and so forth (Fig 1). Beside this, no compositional bias of any pattern of conserved AARs was observed in sequence logos of G. soja and G. max. Sequence logos provides a defined depiction of sequence similarity as compared to consensus sequences, significant features of alignment, pattern in sequence conservation, and assist to discover and analyze those proteins [18]. Hence, VOZ sequence logos in G. max and G. soja will help to discover, examine and assess the pattern of VOZ protein sequence conservation in other plant species.

Fig 1

Vascular plant one-zinc-finger sequence logo of G. max and G. soja.

Amino acid pattern depicts that they are highly conserved.

Sub-cellular localization for GmVOZ and GsVOZ was initially predicted by using CELLOv.2.5 (http://cello.life.nctu.edu.tw/) and were found to be localized in nucleus. For confirmation, GmVOZ1-GFP and GsVOZ-GFP fusion proteins were expressed in tobacco leaves. GmVOZ1 and GsVOZ1 was specifically localized in the nucleus (Fig 2).

Fig 2

Sub-cellular localization of GmVOZ1 and GsVOZ1.

Brightfield, fluoresence (GFP) and merged images of tobacco leaves infiltrated with Agrobacterium.

Evolutionary and phylogenetic relationship of VOZ protein

Phylostratum analysis of VOZ genes identified the primitive ancestry as VOZ genes were present in P. patens (Bryophyte)- one of the early plant pedigree (Fig 3A). VOZ genes were existing in A. comosus (angiosperm), A. thaliana, M. truncatula G. raimondaii, B. rapa, S. melongena, G. max, G. soja and P. trichocarpa (dicotyledons), Zea mays and B. distachyon (monocotyledons). These results suggested that VOZ genes originated from early land plants’ phlyostratum and probable orthologous genes of VOZ are existing throughout plant kingdom. We also constructed ML tree to explore the deeper relationship of VOZ genes of 12 species. The prefixes, At, Gm, Gs, Pp, Pt, Gr, Sm, Zm, Br, Bd, Mt and Ac were used before the names of VOZ genes from A. thaliana, G. max, G. sjoa, P. patens, P. trichocarpa, G. raimondaii, S. melongena, Z. mays, D. distachyon, M. truncatula and A. comosus, correspondingly. The ML tree displayed that all 47 VOZ genes from 12 different species were classified into 7 clades. The clades were differentiated with different color schemes (Fig 3B). In this study, clade-1 (light-green color) and clade-3 (red-color) contained 13 genes each, followed by clade-7 (brown-color) having seven VOZ genes, clade-6 (orange-color) contained 2 while clade-4 (dark-green color) and clade-5 (purple-color) contained one each gene of VOZ. Clade-1/2 one contains genes from angiosperms and dicotyledons, clade-3 contains genes from dicotyledons, clade-5/6 contains genes from bryophytes and clade-4/7 contains genes form monocotyledons. We also found that VOZ genes of monocots and dicots were closely grouped to each other. Gene enlargement in G. max and G. soja can be detected in our phylogenetic analysis, as their eight pairs out of 12 were closely grouped to each other (Fig 3B). The paralogous pairs showed that VOZ genes underwent duplication event in their evolution resulting in gene family expansion.

Fig 3

Phylogenetic analyses of vascular plant one-zinc-finger (VOZ) from 12 different species.

(A) The phylostartum analyses of VOZ gene family. (B) Phlyogenetic tree divided all 47 genes into seven clades. Bootstrap values were also mentioned near the node of each branch.

Exon/intron pattern in a single gene is related to its biological role. Gene structure analysis along with phylogenetic tree displayed that all GmVOZs and GsVOZs have three introns and four exons (Fig 4) and similar genes grouped close to one another in the same groups. We observed that all GmVOZs and GsVOZs exhibited conserved patterns of exon/intron structure even through gene duplication occurred a long time ago during evolution. Expression of genes is governed by promoter and by the binding of TF to cis-element present in the upstream region. PlantCARE database was used, to determine the cis-acting element in the upstream region of GmVOZs and GsVOZs gene. The result showed that GmVOZs and GsVOZs gene carried stress related cis-acting element in their promoter such as LTR (for low temperature), MYB, MBS (for stress and defense), ABRE and CGTCA-motif (for different stress response) and W-Box (fungal elicitor) repeats (Fig 5) [4-6].

Fig 4

Gene structure display of G. max and G. soja.

Red-box represents untranslated and blue-box represents exon regions.

Fig 5

Putative cis-acting elements in a 2.0 kb 5’ flanking region upstream from the start codon of GmVOZs and GsVOZs.

Expression profiling of GmVOZs and GsVOZs

Gene expression profiling foretells the biological role of a gene; hence we investigated the expression of GmVOZs and GsVOZs in Williams-82 and W05, respectively. All genes were used to assess the tissue specific transcript abundance levels by qRT-PCR in roots and leaves at seedling stage, green pods, flower, seed 10, 21, and 35 days after flowering. These results indicated that VOZ might play functional roles in different developmental stages of soybean. Moreover, all studied genes had relatively higher transcript level in roots revealing their role with limited functional divergence during evolution (Fig 6). Analyses of GmVOZs and GsVOZs gene expression under PEG6000 was also performed in this study. The responses of all studied genes were observed at different time points i.e., 0h, 1h, 3h, 6h after treatment via qRT-PCR. All studied genes had higher transcript level as compared to control (0h) at various time intervals for PEG6000 treatment (Fig 7). Together with these observations, GmVOZs and GsVOZs showed obvious resistance against PEG6000 treatment advocating that these genes are possible candidates for breeding drought resistant soybean cultivars.

Fig 6

Expression analysis of GmVOZs and GsVOZs genes at different developmental stages in specific organs.

Leaves (L), Roots (RT), Green Pods (GP), Flower (FL), Seed 10, 21, 35 days after flowering.

Fig 7

Expression pattern analyses of GmVOZs and GsVOZs under PEG6000 treatment estimated by qRT-PCR analyses.

The error bars show standard deviations.

Chromosomal distribution, gene duplication and synteny analysis

The studied GmVOZs and GsVOZs were mapped to their corresponding chromosomal loci. The results exhibited that the chromosomes contained only single GmVOZ and/or GsVOZ gene. The uneven distribution denotes that the gene loss might have taken place during evolution.

To explore the locus relationships among GmVOZs and GsVOZs genes, we identified the orthologous/paralogous gene pairs for the cultivated and wild soybean. Synteny analysis showed that several gene loci are highly conserved (Fig 8). A total of 14 orthologous/paralogous gene pairs were identified, of which all studied genes were attributed to segmental or whole genome duplication (WGD) to form paralogous gene pair between both species. During the process of evolution, gene pairs can undergo functional divergence which can result in non-functionalization (when two duplicated genes acquires a mutation in regulatory or coding regions that renders the gene non-functional), sub-functionalization (directs specific function of gene expression), or neo-functionalization (when one of the two duplicated genes acquires a mutation in regulatory or coding regions resulting in novel and useful function of gene). Ka/Ks values were calculated to determine the extent and nature of selection pressure on these duplicated gene pairs. It has been established that Ka/Ks = 1 indicates pseudogenes because of neutral selection, Ka/Ks > 1 represents positive selection of accelerated evolution, while the ratio of Ka/Ks < 1 exhibits the ability for genes duplication for purifying selection. In this study, we found that all GmVOZs and GsVOZs genes showed Ka/Ks values less than 1 (S3 Table), anticipating that soybean VOZ genes were subjected to purifying selection pressure with limited functional deviation because of WGD and segmental duplication. The Ka and Ks divergence values exhibited that for 14 duplicated gene pairs.

Fig 8

Gene duplication among GmVOZs and GsVOZs gene.

Blue line indicates orthologous/paralogous pairs. Gm01 to Gm20 indicates the chromosomes of G. max and Gs01 to Gs20 represent the chromosome of G. soja.

Discussion

Present yield grain trends in important crops are insufficient to meet the requirements of global population of ~ 9 billion by 2050. This is further challenged by climate change which is anticipated to get worse with extreme weather events and it has been well established that, water stress limits growth, development, and productivity in crop plants [1]. The present genomics landscape of crop plants has been revolutionized, courtesy next generation sequencing technologies which offer sequencing information up to many manifolds. The availability of genome assemblies for soybean [1] creates new opportunities for more precise genome-wide studies in soybean. Several experiments have been performed to explore the role and biological function of VOZ genes in different crop plants [8, 10–12, 15, 16]. However, no systematic analyses of VOZ genes in G. soja in comparison with G. max, has been reported till date. In present study, we conducted analyses of VOZ genes in cultivated (G. max) and wild (G. soja) species of soybean. Ours is the first study to investigate the evolutionary relationships, gene duplication and selection pressure on GmVOZs and GsVOZs. Recent advancement in soybean genome sequencing made it possible to conduct present exploration [1, 17]. This study will be helpful in laying a foundation for further work.

GmVOZs and GsVOZs genes show evolutionary conservation

We classified VOZ genes from bryophytes, angiosperms, dicots and monocots. Phylostratum analysis of VOZ gene family revealed that the primitive plant pedigree as VOZ genes were present in P. paten (bryophytes), suggested that VOZ genes originated from non-vascular plants phylostratum and potential orthologous genes are present throughout kingdom plantae [26]. The existence of VOZ genes in each selected species, with nine VOZ genes from B. rapa, six VOZ genes from G. max and G. soja, and only two genes from P. patens, revealed the evolutionary conserveness of VOZ genes and these genes experienced expansion in higher plants. Furthermore, GmVOZ and GsVOZ genes represented close relationship, strengthening the fact that both species shared common ancestor [2]. Sequence logos, for AARs were highly conserved in G. max and G. soja at N and C termini showing that VOZ genes are evolutionary conserved which might be helpful to know the outline of VOZ protein sequence conservation in other species.

Promoter sequences of GmVOZ and GsVOZ genes had nearly same distribution of cis-elements related to stress responses. The cis-elements such as LTR, MYB, MBS, ABRE, CGTCA-motif and W-Box were identified in both studied species of soybean. The presence of these cis-elements with specific features depicted the predicted functions in plant growth, development, biotic and abiotic stress resistance. Exon-intron structure is important that may be contributed by insertion/deletion events. Several studies reported extensive gain or loss of introns during eukaryotic diversification. Gene structure analysis exhibited that duplicated genes have similar exon-intron pattern, whereas, intron length differs among different genes showing that intron length may play crucial roles in the functional diversification of GmVOZ and GsVOZ genes. Introns also play important role for the evolution of different plant species [27]. Here, we found that all GmVOZ and GsVOZ genes had three introns showing that both G. max and G. soja were newly evolved with fewer introns, which supported previous studies that intron numbers decreased with the passage of time [28] and suggested that newly evolved species have fewer introns as compared with primitive ones [27]. Gene structure of GmVOZs and GsVOZs were extremely similar, showing that these genes were highly conserved. Moreover, introns experienced weak selection pressure and presence of a greater number of introns were thought to have gained novel functions during evolution.

GmVOZs and GsVOZs gene expression are regulated by drought stress

The results of ours study demonstrated that GmVOZs and GsVOZs have higher transcript abundance in leaves and roots at seedling stage, showing the positive role, that these genes may play in plant growth and root development. VOZ genes are likely to respond in an ever-changing environment and are involved in plant growth and development [8, 17, 29]. AtVOZ1 and AtVOZ2 redundantly support flowering in photoperiod pathway [10]. In rice, OsVOZ1 and OsVOZ2 were shown to be rapidly induced in response to water stress [31] Similar results were obtained by Li et al. and Dey et al. [17, 30]. However no systematic study of VOZ genes in G. soja has been reported till to date. The expression of GmVOZ and GsVOZ genes can considerably be upregulated by abiotic stress such as PEG. Together with these outcomes, we conclude that GmVOZ and GsVOZ genes might be instrumental for breeding drought tolerant soybean.

Gene duplication and selection pressure

Tandem duplication events happen when two or more genes are positioned on same chromosome, whereas, segmental or WGD events occurr between the chromosomes. Tandem duplications support increment in introns and can generate novel genes [31] however, we did not find evidence of tandem repeat in this work. All gene pairs were associated with segmental or WGD. Many gene families expanded more in plants as compared to other eukaryotes, indicating that these expansions were associated with environmental and selection pressures. The sequence based tools assist to study the organization, evolution and syntenic relationships of genomes. By using linkage maps, synteny of various species can be compared and in general, closely related species shows higher synteny level and this syntenic level decreases with growing phylogenetic distance [32]. Fourteen paralogous/orthologous gene pairs within and between G. max and G. soja were identified. The Ka/Ks values showed that soybean VOZ genes underwent strong selection pressure with limited functional deviation arising from whole genome and segmental duplication. To estimate the environmental and selection pressure, Ka and Ks substitution rates were calculated. In general, Ka/Ks < 1, Ka/Ks > 1 and Ka/Ks = 1 indicate purifying selection, positive selection and neutral evolution, correspondingly [4, 6]. In current work, we observed that Ka/Ks values of GmVOZ and GsVOZ genes were < 1 suggesting that GmVOZ and GsVOZ genes experienced strong purifying selection pressure.

Conclusion

In this study, six genes each of GmVOZs and GsVOZs were investigated and were further split into four major groups on the basis of phylogenetic analyses. Sequence logos analyses between G. max and G. soja amino acid residues showed higher conservation. Presence of abiotic stress cis-elements in the promoter regions of GmVOZs and GsVOZs clearly showed their role in tolerance against environmental stresses. Collinearity analysis revealed that the studied genes underwent purifying selection pressure. Gene expression experiment results showed the studied genes differentially expressed under normal and drought stress conditions. The present work was the first systematic analysis and comparison between G. max and G. soja. These outcomes provide a foundation for further exploring the functions of GmVOZs and GsVOZs in plant abiotic stress responses.

Oligonucleotide sequences and enzymes used in present study.

(DOCX)

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Biophysical properties of GmVOZ and GsVOZ.

(DOCX)

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Gene orthologous/paralogous and Ka/Ks value estimation.

(DOCX)

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31 Mar 2021

PONE-D-21-06451

Characterization of Vascular Plant One-Zinc Finger in Soybean (Glycine max and Glycine soja) and their Expression Analyses Under Drought Condition

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Reviewer #1: Abstract

1. Line 27-28. “and response against”, replace with under

2. Line 29. “less data”, replace with fewer information

3. Line 31. “Into” replace with in

4. Few sentences require author attention for clarity of message for readership community.

5. Kindly rephrase the abstract make it more concise.

Keywords

Kindly revise keywords, add keywords other than in title.

Introduction

Kindly remove the short paragraphing. The introduction part better consist of 3 to 4 paragraphs.

1. Line 48. “genetic bottlenecking”, replace with genetic bottleneck

2. Line 48-50. “G. soja can provide a promising source of new genes for G. max breeding required to respond to population urge is face of climatic changes”. Kindly rephrase the sentence for better understanding.

3. Line 50. “The genomes of G. max [2] and G. soja [3]” , place citations at proper place.

4. Line 52-54. Rephrase the sentence structure for better understanding the actual meanings.

5. Line 58. “Stress factor”, kindly elaborate which stress factor.

6. Line 63. “AVP1”, recheck the names of gene must be Italic.

7. Line 74. “thaliana but improved resistance to fungal pathogen”, kindly cite appropriate citation.

8. Line 74. “voz1voz2 (dual mutants)” must be voz1/voz2

9. Line 76. “DREB2A” make it Italic.

10. Line 77-78. “According to published reports”, cite the related articles.

11. Line 86, “Beside this”, replace with moreover

Materials and Methods

1. Line 134. “soybean plants grown under field conditions”, mention the location of field trail.

Results

1. Line 152-155. Rephrase sentence.

2. Line 165. “cultivated and wild soybean”, mention the G. max and G. soja.

3. There are few sentences which need rephrasing, carefully gone through the result section and make necessary modification.

Discussion

The discussion part is weak as less similar work has been cited by the authors. The above cited references have been referred in discussion part which need to revisit by authors to make discussion more comprehensive.

The authors needs to add the conclusion part to highlight the important outcome of present results.

References

Carefully follow the journal’s reference format.

Figures

Figure 1. Italic the scientific names.

Figure 2. Italic the gene names

Reviewer #2: The present article presented interesting information on VOZ transcription factors involved in drought stress tolerance. After carefully going through the article, I found that it can be accepted for possible publication. However, the authors must address the below-mentioned minor issues before official acceptance.

1. Please add a separate abbreviations list.

2. Line 25-26. “To attain sustainable production ……… for the genetic improvement”. Rephrase the following sentence.

3. Line 29. “At present, less data is available regarding GmVOZs and GsVOZs”. Rephrase the sentence.

4. Line 35. “G. max and G. soja”, please write full name on the first appearance, later utilize the abbreviated form throughout the manuscript.

5. Line 40. Add some more outcomes of the present investigation to strengthen the conclusion.

6. Line 48-50. “G. soja can provide a ….. face of climatic changes”. Rephrase the sentence.

7. Line 79. Provide a suitable reference.

8. Line 82. Replace “juvenile” with foundation

9. The present study's objectives are not clear, so there is a need to strengthen the objective part.

10. Gene names should be in Italic throughout the manuscript.

11. Line 159. Replace “Not too many” with few.

12. Avoid the repetition of information throughout the result section.

13. The discussion part is weak and needs improvement; cite some latest references.

14. Provide a separate conclusion section.

Decision

Accept with minor revision.

Reviewer #3: It is an interesting study. In general, the paper is well written and structured. However, in my opinion the paper has some shortcomings in regards to some text. I also suggested to cite more relevant. Given these shortcomings the manuscript requires minor revisions.

Reviewer #4: The manuscript presents an interesting study. However, in my opinion there are few shortcomings in the compilation of text.

1- The author should consider rearrangement of the paragraphs/sentences in the introduction to make it more clear to the reader and easy to follow. This will also help to generate association between the two sections i.e. introduction and discussion

2- I suggest adding literature citation at line 77-79;

3- Lines 256-258 can better be a part of discussion to support your results.

4- The author is advised to add references of the figures and tables of their own results in the discussion section, to illustrate a clear interpretation of their own results with the previous studies. This will increase a reader's understanding of what is new and what was lacking in the previous studies.

5- Line 279-280 must be supported with references 1 and 16.

6- I suggest addition of conclusion which will bring out the importance of work as the author states that it is the first investigation of it's type. Conclusions must be drawn appropriately based on the data presented data.

6- Scale is not provided with Figure No. 2. The caption do not explain how much the image has been edited or manipulated. It do not mention the microscope used to capture it and under what conditions. A figure's caption should provide it's complete stand alone explanation.

7- Reference Number 15 is repeated again at No. 32.

8- Reference No. 1 needs recheck citation.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-06451_ Comments.docx

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Submitted filename: Reviewer Report PlosOne.docx

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9 Apr 2021

Response to reviewer's have been uploaded.

Submitted filename: 5. PONE-D-21-06451-Response to Reviewer Comments.docx

Click here for additional data file.

5 May 2021

PONE-D-21-06451R1

Characterization of Vascular Plant One-Zinc Finger in Soybean (Glycine max and Glycine soja) and their Expression Analyses Under Drought Condition

PLOS ONE

Dear Dr. Fiaz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: I inform you that although your study addresses a current topic and presents potentially publishable contents, it is still required to make some minor modifications as suggested by the reviewer in order to be approved for publication in Plos One. Please have a thorough look into the manuscript to remove typos and other grammatical mistakes.

==============================

Please submit your revised manuscript by Jun 19 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Umair Ashraf

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The author/s has addressed all the comments/suggestions. There are further no comments from my side for author/s.

Reviewer #2: The authors have addressed all the comments, and the manuscript has been significantly improved. I have no more comments. The current version should be accepted for publication in Plos One.

Congratulations!!

Reviewer #3: (No Response)

Reviewer #4: Dear Author

1. The conclusion paragraph needs revision e.g. rearranging sentences, use of present tense.

2. Line 357 replace "six each genes of" to "six genes each of".

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Galal Bakr Anis

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

18 May 2021

Response to reviewer have been uploaded.

Submitted filename: 4. PONE-D-21-06451-Response to Reviewer Comments.docx

Click here for additional data file.

26 May 2021

PONE-D-21-06451R2

Characterization of Vascular Plant One-Zinc Finger in Soybean (Glycine max and Glycine soja) and their Expression Analyses Under Drought Condition

PLOS ONE

Dear Dr. Fiaz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: There are still some typos and grammatical mistakes. Few examples are: Line 282: "Present yield grain trends" which should be 'grain yield' and Line 267-68: "Gene expression experiment results" should be "Results of gene expression experiment". So I recommend that author should re-check the manuscript thoroughly and should proof read from a native English speaker to improve the overall write-up. Manuscript should be free from grammatical mistakes with correct sentence formation.

==============================

Please submit your revised manuscript by Jul 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Umair Ashraf

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

There are still some typos and grammatical mistakes. Few examples are: Line 282: "Present yield grain trends" which should be 'grain yield' and Line 267-68: "Gene expression experiment results" should be "Results of gene expression experiment". So I recommend that author should re-check the manuscript thoroughly and should proof read from a native English speaker to improve the overall write-up. Manuscript should be free from grammatical mistakes with correct sentence formation.

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Jun 2021

Response to Reviewers

We are thankful to reviewers for their valuable comments for the improvement of article. Moreover, we are also thankful to editor for his/her active role to communicate effectively at the different stages of the article. We have revised the whole manuscript in round 1 of review process and again modified the revised article in light of comments/suggestions provided during round 2 of review process. The point to point response for reviewer 4 comments and suggestions has been given below and highlighted in text in track changes for your ease.

Reviewer #4: Dear Author

1. The conclusion paragraph needs revision e.g. rearranging sentences, use of present tense.

2. Line 357 replace "six each genes of" to "six genes each of".

Response: Thank you for your valuable comment/suggestion. We have carefully revised the article and taken help from native speaker to modify English language as per Journal standard. The typo are fixed, grammatical mistakes are removed and needful sentence structure is improved.

Submitted filename: 5. PONE-D-21-06451-R3-Response to Reviewer Comments.docx

Click here for additional data file.

15 Jun 2021

Characterization of Vascular Plant One-Zinc Finger in Soybean (Glycine max and Glycine soja) and their Expression Analyses Under Drought Condition

PONE-D-21-06451R3

Dear Dr. Fiaz,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Umair Ashraf

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Jun 2021

PONE-D-21-06451R3

Characterization of Vascular Plant One-Zinc Finger (VOZ) in Soybean (Glycine max and Glycine soja) and their Expression Analyses Under Drought Condition

Dear Dr. Fiaz:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Umair Ashraf

Academic Editor

PLOS ONE