Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

Xuexia Wu; Shengmei Zhang; Xiaohui Liu; Jing Shang; Aidong Zhang; Zongwen Zhu; Dingshi Zha

doi:10.1371/journal.pone.0226537

. 2020 Apr 17;15(4):e0226537. doi: 10.1371/journal.pone.0226537

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

Xuexia Wu ^1,^#, Shengmei Zhang ^1,^#, Xiaohui Liu ¹, Jing Shang ¹, Aidong Zhang ¹, Zongwen Zhu ¹, Dingshi Zha ^1,^*

Editor: Muthamilarasan Mehanathan²

PMCID: PMC7164647 PMID: 32302307

Abstract

Enzymes of the chalcone synthase (CHS) family participate in the synthesis of multiple secondary metabolites in plants, fungi and bacteria. CHS showed a significant correlation with the accumulation patterns of anthocyanin. The peel color, which is primarily determined by the content of anthocyanin, is an economically important trait for eggplants that is affected by heat stress. A total of 7 CHS (SmCHS1-7) putative genes were identified in a genome-wide analysis of eggplants (S. melongena L.). The SmCHS genes were distributed on 7 scaffolds and were classified into 3 clusters. Phylogenetic relationship analysis showed that 73 CHS genes from 7 Solanaceae species were classified into 10 groups. SmCHS5, SmCHS6 and SmCHS7 were continuously down-regulated under 38°C and 45°C treatment, while SmCHS4 was up-regulated under 38°C but showed little change at 45°C in peel. Expression profiles of key anthocyanin biosynthesis gene families showed that the PAL, 4CL and AN11 genes were primarily expressed in all five tissues. The CHI, F3H, F3’5’H, DFR, 3GT and bHLH1 genes were expressed in flower and peel. Under heat stress, the expression level of 52 key genes were reduced. In contrast, the expression patterns of eight key genes similar to SmCHS4 were up-regulated at a treatment of 38°C for 3 hour. Comparative analysis of putative CHS protein evolutionary relationships, cis-regulatory elements, and regulatory networks indicated that SmCHS gene family has a conserved gene structure and functional diversification. SmCHS showed two or more expression patterns, these results of this study may facilitate further research to understand the regulatory mechanism governing peel color in eggplants.

Introduction

Eggplant (S. melongena L.) is one of the most important thermophilic vegetables produced in many tropical and temperate regions around the world. The optimum growth temperature for eggplant is between 22 and 30°C. After subjected to high temperature treatment, eggplants may exhibit to stagnation of growth, abortion of flower buds, and pollen viability rate and fruit set decrease, and the peel’s color will turn light when the temperature is over 35°C. High temperature severely reduces the yield and affects the appearance quality of eggplant. However, the molecular mechanism of heat stress response in eggplants has not been thoroughly elucidated.

Anthocyanins are plant secondary metabolites and are among the most abundant natural pigments, that are responsible for the characteristic colors in flowers, fruits and vegetables plant tissues. The anthocyanin biosynthesis pathway has been studied in numerous plant species and most of the genes involved in this process have been identified. Moreover, anthocyanins play an important role in plant survival under stressful environmental conditions. High temperatures are known to reduce anthocyanin accumulation and have discoloration effects in many plant tissues, causing drastic effects in colored flowers [1, 2], and affecting the skin of such fruits as grape berries, apples and eggplant [3–7].

It is well known that CHS is the gatekeeper of the anthocyanin pathway [8]. Enzymes of chalcone synthase (CHS) are member of the plants-specific type III polyketide synthase (PKS) [9, 10], family and catalyze the first committed step of the branch of the phenylpropanoid pathway, which leads to the synthesis of flavonoids [11, 12]. Flavonoids are well known as a group of plant secondary metabolites that comprise several different classes of compounds, such as chalcones, flavones, flavonol isoflavones and anthocyanins. Flavonoids have a wide variety of biological functions in flower pigmentation, protection against UV radiation, pathogen defense, auxin transport and pollen fertility [13–15]. CHS also showed a significant correlation with synthesis of flavonoid compounds during heat stress defense. Heat stress responsive element in bread wheat (Triticum aestivum L.) has been found in the promoter of Chs-D1 gene [16]. High-temperature stress had a large impact on the expression of CHS7, CHS8 in both seeds and pods of Soybean [17]. The transcript levels of CHS decreased in apple peel and rose flower after heat treatment [1, 4]. In cork oak, CHS gene expression exhibited an increase under 45°C, but showed a decreased expression at 55°C [18]. The emergence of CHSV and CHSVII is important for the development of fungal heat stress tolerance and pathogenicity in pathogenic fungi. [19]. In addition, CHS (Sme2.5_00283.1_g00002.1) was up-regulated, and the other two CHS gene members were down-regulated under heat stress in peel of eggplant [7].

The product of the CHS reaction is a pivotal precursor for a large array of secondary metabolites derived from malonyl-CoA and p-coumaroyl-CoA. CHS exists as homodimeric iterative PKS (monomer size of 42–45 kDa) with two independent active sites that catalyze a series of decarboxylation, condensation, and cyclization reactions [10, 20]. Member of the CHS superfamily share high similarity in their amino acid sequence, which contains the structurally conserved catalytic center consisting of four residues, Cys-His-Asn-Phe, and most of the genes contain two exons and one intron [21]. However, the CHS gene family has not been characterized in eggplants to date.

In the current study, all SmCHS family members were identified in eggplant. A comprehensive analysis of members was performed, including gene structures, the biochemical characteristics of putative CHS protein, promoter cis-elements, phylogenetic relationships among members in other relative species, and their expression profiles in various organs/tissues under high temperature stress. The findings of the present study may facilitate functional studies on eggplant SmCHS family genes.

Materials and methods

Plant materials and RNA extraction

The eggplant cultivar ‘Tewangda’ is a cold-tolerant cultivar with blackish purple skin. This cultivar grows vigorously and has good fruit setting. The fruit size was about 27.6 cm in length, 5.4 cm transverse diameter and a 209 g single fruit weight on average. ‘Tewangda’ fruits were grown at the same growth stage and were randomly selected. These plants were grown 144 days after sowing, and then placed inside incubators set at 27°C (CK), 38°C or 45°C for 3 or 6 h (three plants per treatment). For each treatment, the tissue samples of root, stem, leaf, flower and peel were obtained and immediately frozen in liquid nitrogen and stored at -80°C for RNA extraction and other analyses. All plant materials examined in this study were obtained from Shanghai Academy of Agricultural Sciences. Total RNA was extracted from each tissue sample using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. The extracted total RNA was stored at -80°C. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Identification of the CHS family members in the eggplant genome

The whole protein sequence of Solanum melongena L. (eggplant) were obtained from the Eggplant Genome DataBase (http://eggplant.kazusa.or.jp) [22], and those of Solanum tuberosum L. (potato, http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml) [23], Solanum lycopersicum (tomato, https://solgenomics.net/organism/Solanum_lycopersicum/genome) [24], Solanum penellii (wild tomato, https://www.plabipd.de/project_spenn/start.ep) [25], Capsicum annuum L. (pepper, http://peppergenome.snu.ac.kr) [26], Petunia axillaris (https://solgenomics.net/organism/Petunia_axillaris/genome) [27], Petunia inflate (https://solgenomics.net/organism/Petunia_inflata/genome) [27], and Nicotiana tabacum (common tobacco, https://www.ncbi.nlm.nih.gov/nuccore/AYMY00000000) [28]. The profiles of CHS (PF00195 and PF02797) were downloaded from the Pfam protein family database (http://pfam.xfam.org/), and these profile sequences were used as queries to perform BLASTP searches against the protein sequence data of all the species mentioned above with a maximum E-value of 1×10⁻³, respectively [29]. To further verify the exact copy number of CHS and remove redundant sequences, the Pfam database and Genome websites were also searched using “chalcone synthase” as keywords. All CHS sequences were submitted to EXPASy (https://web.expasy.org/protparam/) to calculate the number of amino acids, molecular weights and theoretical isoelectric points (pI).

Structural characterization

The locations and intron numbers of CHS were acquired through the genome website. All of the acquired protein sequences were first aligned by ClustalX software with the default parameters [30]. An unrooted maximum-likelihood phylogenetic tree was constructed using MEGA6 software with a Bootstarp value of 1000 times [31]. The MEME program (Version 5.0.5, http://meme-suite.org/tools/meme) was used to identify the conserved motif of the CHS sequences with the following parameters: any number of repetitions, maximum of 10 misfits and optimum motif width of 6–200 amino acid residues. The WoLF PSORT program was used to predict the subcellular localization information of CHS proteins (https://www.genscript.com/wolf-psort.html) [32].

Analysis of cis–acting elements in SmCHS

The upstream sequences (2 kb) of the SmCHS coding sequences in eggplant were retrieved from the genome sequence and then submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify regulatory elements [33].

Phylogenetic analysis of CHS genes

The full-length protein sequences of all eight species in Solanaceae were used for phylogenetic analysis. All of the protein sequences were first aligned by ClustalX software with the default parameters [30]. The phylogenetic tree was generated with MEGA6 software with a bootstrap test of 1000 times. The final tree was viewed and modified in Evolview software [34]. The CHS genes were classified into different groups according to the topology of the phylogenetic tree.

Expression analysis of antyocyanin biosynthetic genes and construction of the mRNA regulatory network

The RNA-seq results were obtained by our lab [35]. Gene expression level was estimated from mean FPKM (fragments per kilobase of exon model per million reads mapped) values for each treatment, and showed the expression patterns in heatmap.Significant differentially expressed genes (fold change ≥ 2 and p-value ≤ 0.05) were used to calculate the Pearson correlation coefficient between CHS genes and other genes. The TBtools program was used to elucidate the Gene Ontology (GO) functional classification for the mRNAs with correlation coefficients greater than 0.9 [36]. The top 5 regulatory mRNAs annotated by GO enrichment for the genes associated with anthocyanin biosynthesis were collected to construct the regulatory network. The network was visualized using Cytoscape [37].

qRT-PCR analysis

Expression level of anthocyanin biosynthesis genes, including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarateCoA ligase (4CL), CHS, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), dihydroflavonol4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), Anthocyanins11 (AN11), and most transcription factors, such as myeloblastosis (MYB), basic helix-loop-helix (bHLH) and metadata authority description schema(MADS1), were analyzed. First-strand cDNA was synthesized from 1 μg total RNA from 5 tissues (root, stem, leaf, flower and peel) using a Prime Script RT Reagent Kit (Takara, Dalian, China). The qRT-PCR reactions were performed in 96-well plates using the ABI 7500 fast Real-Time PCR system (Applied Biosystems, USA) with the QuantiFast SYBR Green PCR Kit (Qiagen, Duesseldorf, Germany). The qRT-PCR parameters were as follows: 95°C for 5 min, then 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The relative mRNA expression levels were calculated using the 2^-ΔΔCT method [38]. PGK(JX154676) was used as an internal control to normalize the data. For each sample, three biological repeats were performed, the relative expression levels were calculated using the standard curve and normalized by the control’s expression, the results were display by heatmap. The primer sequences are listed in S3 Table.

Results

Identification of CHS genes and sequence analysis in Solanaceae species

A total of 7 CHS (SmCHS1-7) genes in eggplant were identified after being verified by protein sequence analysis and BlAST search using the eggplant genome annotation database (S1A Table). The length of SmCHS protein ranged from 327 to 396 amino acids (Table 1, S2 Table). The PKS type III active sites of the enzymes and Phe215 connected with CoA binding are conserved among all SmCHS (S1 Fig). In addition, 66 CHS genes were characterized from 7 other Solanaceae species. The subfamily numbers of CHS genes ranged from 6 (Solanum penellii) to 13 (Petunia axillaris) (Table 1, S1B–S1H Table). The length for the other 7 Solanaceae species (Solanum tuberosum L., Solanum lycopersicum, Solanum penellii, Capsicum annuum L., Petunia axillaris, Petunia inflate and Nicotiana tabacum) proteins ranged from 156 to 431 amino acids (S1A–S1G Table). The average number of amino acids was calculated, and all the amino acids of each species are arranged in same order to form a data set. The correlation coefficients among the above data set were all greater than 0.99. This finding suggests that CHS genes are conserved in Solanaceae species.

Table 1. Features of SmCHS genes identified in eggplant.

Gene Name	Gene ID	Number of amino acids
SmCHS1	Sme2.5_01077.1_g00016.1	333
SmCHS2	Sme2.5_02154.1_g00001.1	389
SmCHS3	Sme2.5_13923.1_g00001.1	389
SmCHS4	Sme2.5_00283.1_g00002.1	392
SmCHS5	Sme2.5_01039.1_g00002.1	327
SmCHS6	Sme2.5_00346.1_g00019.1	396
SmCHS7	Sme2.5_05261.1_g00004.1	383

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Structure and conserved motif analysis of SmCHS

The 7 SmCHS genes were distributed on 7 scaffolds. To better understand the evolution of SmCHS genes, an unrooted maximum-likelihood tree was constructed based on the 7 SmCHS protein sequences, and the SmCHS were classified into 3 clusters (i, ii and iii) (Fig 1). Among the SmCHS genes, only one SmCHS7 had three exons, and the others had two exons (Fig 1) based on information available from the genome annotation. These results suggest the potential diversity of the biological functions of the SmCHS genes in eggplants, previous studies also have similar conclusion [39, 40].

The phylogenetic tree was on the left of the figure and showed that the SmCHS genes were classified into three clusters (i, ii and iii). The exon/intron organization of SmCHS is shown on the right of the figure. For SmCHS gene organization, yellow boxes represent exons, black lines represent introns, and green boxes indicate upstream/downstream regions. The lengths of the exons and introns are drawn to scale.

To understand the functional diversification of SmCHS, the conserved motifs of these 7 protein sequences were identified by the MEME program, and 10 conserved motifs were detected in eggplant (Fig 2, Table 2). The Chal_sti_synt_C domain and Chal_sti_synt_N domain were included in motifs 1 and motifs 2, respectively. For all 7 eggplant SmCHS proteins, Motif 1 and motif 2 exist in all of them, motif 3 is only absent in SmCHS5, and motif 4 and motif 5 are only absent in SmCHS1. The N-terminal domain (PF00195) of the CHS protein contained motif 1 and the combination of motifs 3, 4, 6, 7 and 9. The C-terminal domain (PF02797) of the CHS protein contained motif 2 and the combination of motifs 5, 8 and 10. Therefore, the motif configuration of the SmCHS reflects the conservation and diversity of the CHS family. To further investigate the subcellular localization information of SmCHS proteins, the WoLF PSORT program was used to predict the localization of SmCHS protein [31]. SmCHS7 was predicted to localize in the nucleus, whereas SmCHS4 and SmCHS6 were predicted to localize in the chloroplast. The others SmCHS proteins were predicted to localize in the cytoplasm. The different compositions of the domains and subcellular localization may indicate functional diversity.

Fig 2 — Ten conserved motifs are indicated in differently colored boxes.

Table 2. List of the putative motifs of CHS proteins.

Motif	Length (amino acids)	Best possible match
Motif 1	167	IKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITAVGFRGPSETHPDSLVGQA
Motif 2	57	DWNSJFWIAHPGGPAILDQVELKLGLKPEKLRATRQVLSDYGNMSSACVLFILDEMR
Motif 3	56	RLCDKSMIKKRYMHLTEEILKENPNLCEYMAPSLDARQDIVVVEVPKLGKEAAQKA
Motif 4	38	QRAEGPATILAIGTATPSNCVDQSTYPDYYFRITNSEH
Motif 5	27	TTGEGLDWGVLLGFGPGLTIETIVLHS
Motif 6	11	LIEAFEPLGIS
Motif 7	8	MVTVEEVR
Motif 8	6	FCEKLI
Motif 9	7	QNIGKVN
Motif 10	7	ELKEKFK

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Stress-related cis-elements in SmCHS promoters

To further study the potential regulatory mechanisms of SmCHS during abiotic stress responses, the 2 kb upstream sequences from the translation start sites of SmCHS were used to identify the cis-elements (Fig 3A). The results showed that all SmCHS had common upstream promoter elements, including TATA-box and CAAT-box, which occurred more than 100 times; therefore, these sequences were presumed to be the promoter sequences (Fig 3B). The elicitor response element (ERE) and myeloblastosis binding cis-elements (MYB) occurred more than 10 times in the SmCHS upstream sequences. Research has shown that an increase in CHS activity causes a high accumulation of flavonoids that inhibits polar auxin transport [8, 41, 42]. Two cis-acting elements (ABRE, involved in abscisic acid responsiveness; AuxRR, involved in auxin responsiveness) were found in the upstream regions. MYB and myelocytomatosis (MYC) binding sites have also been identified, which may greatly influence plant stress tolerance. Cluster analysis of cis-element number showed that 7 SmCHS genes were divided into 3 groups (Ⅰ, Ⅱ, Ⅲ), and SmCHS1, SmCHS2 and SmCHS3 had similar regulatory pattern (Fig 3A). Five cis-elements (CARE, GCN4-motif, GT1-motif, MRE and TCT-motif) exist only in group Ⅰ, GARE-motif only exist in group Ⅲ. STRE exist in group Ⅱ and Ⅲ. These results showed that SmCHS is activated by a wide range of environmental and developmental stimuli, and there are many complex means of regulating SmCHS activity in eggplants.

Fig 3 — (A) Frequency of cis-element occurrence in upstream sequences. (B) Predicted *cis*-elements in *CHS* gene promoters. The scale bar indicates the length of promoters.

Phylogenetic analysis of CHS genes in Solanaceae

To analyze the evolutionary relationships of CHS genes in Solanaceae, an unrooted phylogenetic tree was constructed using full-length amino acid sequences. All 73 CHS genes were classified into 10 groups (Fig 4, Table 3), and the number of CHS gene groups ranged from two to eleven. The 7 SmCHS were categorized into 6 groups (groups Ⅰ, Ⅱ, Ⅶ, Ⅷ, Ⅸ and Ⅹ), and group Ⅱ contained SmCHS1 and SmCHS2. Groups Ⅰ, Ⅱ, Ⅸ and Ⅹ exist in all eight species, and groups Ⅲ, Ⅳ and Ⅴ were absent in Solanum melongena L., Solanum penellii, Solanum lycopersicum and Solanum tuberosum L.. The group Ⅵ is absent in Capsicum annuum L., Nicotiana tabacum, Petunia inflate and Petunia axillaries (Table 3). The Ⅷ, Ⅸ and Ⅹ groups are distinguished from other groups mainly depends on the position 1–164 amino acids, GroupsⅠ, Ⅱ and Ⅲ are relatively conservative at the position 260–360 amino acids, in which the other groups are very diverse(S1 Fig). These results suggested that the CHS were conserved, but small variations existed among the eight species in Solanaceae and showed that SmCHS1, SmCHS2 and SmCHS3 were more conserved than SmCHS4 according to the phylogenetic tree.

Fig 4 — The colored region is associated with 10 groups of proteins (Group Ⅰ to Ⅹ).

Table 3. Distribution of CHS genes in the phylogenetic tree.

Plant Pecies	Number	Phylogenetic Group
Plant Pecies	Number	Ⅰ	Ⅱ	Ⅲ	Ⅳ	Ⅴ	Ⅵ	Ⅶ	Ⅷ	Ⅸ	Ⅹ
Solanum melongena L.	7	1	2	0	0	0	0	1	1	1	1
Solanum penellii	6	1	1	0	0	0	0	1	1	1	1
Solanum lycopersicum	7	1	1	0	0	0	1	1	1	1	1
Solanum tuberosum L.	10	2	1	0	0	0	1	3	1	1	1
Capsicum annuum L.	9	1	1	0	2	0	0	2	1	1	1
Nicotiana tabacum	12	2	2	0	0	1	0	3	0	2	2
Petunia inflate	9	2	1	1	1	1	0	0	1	1	1
Petunia axillaris	13	1	1	3	1	3	0	0	1	1	2

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Expression profile of key anthocyanin biosynthesis genes in eggplants under heat stress

Using the RNA-seq data of eggplant peel, a heatmap of 96 key anthocyanin biosynthesis genes (PAL, C4H, 4CL, CHS, CHI, F3H/F3’H, F3’5’H, DFR, ANS, 3GT, MYB1, MYB2, bHLH1, AN11, MADS1) was established under heat stress (Fig 5). The expression of anthocyanidin synthase (ANS) and MYB2 was not identified during this sampling period (the undetected genes were also color-coded for 0 in Fig 5). For seven SmCHS genes, expression of three genes(SmCHS5, SmCHS6, and SmCHS7) were not identified, and the other four SmCHS genes were divided into two groups according to their expression patterns. Three of those four SmCHS genes (SmCHS1, SmCHS2, and SmCHS3) were continuously down-regulated under 38°C and 45°C treatment compared with the CK. However, SmCHS4 was up-regulated under 38°C, but showed little change under 45°C in peel. These phenomena have also been observed in some other key gene families associated with anthocyanin biosynthesis. According to the RNA-seq results of 96 anthocyanin biosynthesis key genes in eggplant peel, SmCHS4 showed the highest expression level at the 38°C-3h along with eight other genes (Sme2.5_03336.1_g00008.1_PAL, Sme2.5_00041.1_g00017.1_4CL, Sme2.5_00283.1_g00002.1_smCHS4, Sme2.5_00298.1_g00002.1_F3H, Sme2.5_02066.1_g00012.1_F3H, Sme2.5_04260.1_g00001.1_F3H, Sme2.5_15970.1_g00001.1_F3H, Sme2.5_00670.1_g00012.1_DFR, Sme2.5_00747.1_g00013.1_AN11) (Fig 6). In particular, Sme2.5_03336.1_g00008.1_PAL expression level under 38°C doubled but was down-regulated at 45°C compared with CK; Sme2.5_00670.1_g00012.1_DFR, Sme2.5_00747.1_g00013.1_AN11 expression level increased 3–4 fold and 7–10 fold under 38°C, respectively. These results suggest that these genes may have tissue-specific or functionally differentiation.

Fig 5 — The color box from blue to red indicate an increased expression level.

Fig 6 — These genes have the highest expression level at 38°C-3h in eggplant peel. The error bars represent the standard error of the means of three biological replicates.

mRNA regulatory network associated with anthocyanin biosynthesis in eggplant

Pearson correlation coefficient was calculated between all the mRNAs and anthocyanin biosynthesis related genes, the mRNAs with correlation coefficient greater than 0.9 are considered to be genes that are co-expressed with the anthocyanin biosynthesis genes. A total of 4928 mRNA correlation coefficients were more than 0.9, and all of these mRNAs were functionally categorized in the GO database. The top 20 GO enrichment results of biological processes are shown in Table 4. The genes involved in the regulation of biological processes (GO:0050789), regulation of cellular metabolic processes (GO:0031323) and regulation of gene expression (GO:0010468) were collected and filtered to construct a regulatory network. In totally, 67 anthocyanin biosynthesis key genes and 146 regulatory mRNAs were included in this regulatory network (S2 Fig). These GO enrichment results suggest that the anthocyanin biosynthesis pathway may be regulated by a wide range of environmental and developmental stimuli.

Table 4. Top 20 GO enrichment results of biological processes.

GO term	GO ID	P value
cellular biosynthetic process	GO:0044249	0
cellular nitrogen compound biosynthetic process	GO:0044271	0
cellular response to chemical stimulus	GO:0070887	0
cellular response to stress	GO:0033554	0
regulation of biological process	GO:0050789	0
regulation of cellular macromolecule biosynthetic process	GO:2000112	0
developmental process	GO:0032502	0
regulation of RNA biosynthetic process	GO:2001141	0
regulation of cellular metabolic process	GO:0031323	0
cellular component organization	GO:0016043	0
response to organic substance	GO:0010033	1.11E-16
protein metabolic process	GO:0019538	1.11E-16
regulation of nitrogen compound metabolic process	GO:0051171	1.11E-16
regulation of gene expression	GO:0010468	1.11E-16
response to stimulus	GO:0050896	1.11E-16
regulation of nucleobase-containing compound metabolic process	GO:0019219	1.11E-16
response to chemical	GO:0042221	1.11E-16
cell communication	GO:0007154	1.11E-16
response to stress	GO:0006950	1.11E-16
oxoacid metabolic process	GO:0043436	2.22E-16

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Expression pattern of anthocyanin biosynthesis key genes in different tissues under heat stress

Using the qRT-PCR data, a heatmap of 20 key anthocyanin biosynthesis genes was established in different tissues under heat stress (Fig 7). The qRT-PCR results showed a high consistency with the RNA-seq data, which suggested that the RNA-seq data were credible. Most of the CHS genes were expressed in peel and were expressed at low levels in other tissues. The PAL, 4CL and AN11 genes were mainly expressed in all five tissues. The CHI, F3H, F3’5’H, DFR, 3GT and bHLH1 genes were expressed in flower and peel. MADS1 was expressed in stems, leaves, flowers and peels. Under heat stress, cluster i (cluster show in Fig 2) was continuously downregulation, cluster ii was up-regulated 4 times under 38°C compared with CK in peel, and cluster iii was not detected in most eggplant tissues.

Discussion

It is well-known that the CHS gene family plays a significant role in the growth and development of plants. In many species, multigene families of CHS have been identified. For example, six CHS genes have been described in turnip [43]. In maize, 14 complete CHS genes have been identified [39]. A total of 27 CHS genes were found in rice [40]. These studies showed that CHS members were divided into two or more subclasses according to phylogenetic analysis. Generally, genes grouped into the same subclasses shared similar evolutionary features, and obtained the same expression pattern. In our study, the identified sequences showed a high level of coding sequence similarity (above 90%). The SmCHS were classified into three clusters based on the results of the maximum-likelihood tree. At 35°C, previous studies showed that SmCHS1 and SmCHS3 (Sme2.5_01077.1_g00016.1, Sme2.5_13923.1_g00001.1) were down-regulated in peels of eggplant [7], which is in keeping with our results, other two clusters CHS genes show different expression patterns. These results suggest the functional diversification of SmCHS.

Flavonoids have numerous functions and contribute to pigments, signaling molecules, and protectants against biotic and abiotic stresses. The flavonoid biosynthetic pathway is one of the most intensively investigated pathways for applied biological and genetic processes, as well as for understanding gene regulation, characterizing transposable elements and producing of agronomically stress-tolerant plants and natural dietary antioxidants. Biosynthesis of anthocyanins responds to environmental stress factors, such as light, nutrient depletion, and temperature change. The peel color determined by the content of anthocyanin is a majority economically important trait for eggplant, and this color is modulated by the genes in the flavonoid biosynthesis pathway. Compared with other tissues, SmMYB1 and all anthocyanin biosynthetic key genes (SmCHS, SmCHI, SmF3H, SmDFR) except SmPAL were dramatically up-regulated in the fruit skin of the purple cultivar [44]. The full length cDNA of SmCHS, SmCHI, SmF3’5’H, and SmDFR were isolated from eggplants by Jiang (2016) [45]. These genes have the highest expression levels in peels except for SmF3H, which was detected in stems [45]. The expression profiles of these key gene families under heat stress were investigated in our study. ‘These anthocyanin biosynthesis key genes (PAL, 4CL, AN11, CHI, F3H, F3’5’H, DFR, 3GT and bHLH1) show tissue specific expression, suggesting that these genes respond at the late stage of the anthocyanin pathway and directly regulate the color of fruit skin and flower.

Heat stress reduced the anthocyanin content and the enzyme activities of CHS, DFR, ANS and 3GT/UFGT in eggplant peel and strengthened the activity of PAL [35, 46]. When the temperature exceeds 35°C, the eggplant will be dehydrated and shrink, and the peel’s color will lighten. CHS is a key enzyme of the flavonoid biosynthesis pathway. Most of the genes associated with flavonoid biosynthesis were down-regulated under heat stress. In this study, the genes of the flavonoid biosynthesis pathway showed tissue-specificity, and genes expressed in different phases and tended to change over time (Fig 7). Under heat stress, SmCHS4 and some anthocyanin biosynthesis related genes show different expression profiles at 38°C-3h (Fig 6), suggest that these co-up-regulated genes contribute to protect the eggplant at beginning of heat stress defense. In addition, 52 gene expression levels were reduced under heat stress, which was similar to Lv’s (2019) results [7], while 35 gene expression levels were not identified. These results suggest that some key anthocyanin biosynthesis genes help to protect the eggplant from damage to heat stress. Moreover, these gene families exhibited two or more expression patterns and performed multiple genetic functions to regulate anthocyanin content. Combined with regulatory networks, it is possible to further understand the regulatory mechanism of peel color in eggplants.

Conclusions

In this study, a genome-wide analysis of the SmCHS gene family in eggplants was performed. The CHS protein biochemical characteristics, phylogenetic relationships, gene structures, cis-regulatory elements, regulatory network and functional predictions of the smCHS gene family members were examined. The SmCHS gene family has conserved gene structure and functional diversification. CHS plays important roles in the anthocyanin biosynthesis pathway, exhibits two or more expression patterns and executes multiple functions to regulate anthocyanin content in eggplant peels under heat stress. The result of this study may contribute to the production of heat-resistant eggplant for further research on the functions, regulation and evolution of the CHS family.

Supporting information

S1 Table. CHS protein sequences of Solanum species.

(XLSX)

Click here for additional data file.^{(26.8KB, xlsx)}

S2 Table. Features of CHS genes identified in Nicotiana tabacum.

(XLSX)

Click here for additional data file.^{(15.9KB, xlsx)}

S3 Table. Primers used for real time PCR analysis.

(DOCX)

Click here for additional data file.^{(17.9KB, docx)}

S1 Fig. Sequence alignment of all 73 CHS proteins of Solanum specie.

Color bars on the left represent the 10 groups in Fig 4. Active site residues are highlighted in yellow, malony-CoA binding sites are highlighted in blue and other conserved sequence are shown in green.

(JPG)

Click here for additional data file.^{(9.4MB, jpg)}

S2 Fig. Interaction network key to anthocyanin biosynthesis in eggplant.

The pink labels represent the CHS gene family.

(PDF)

Click here for additional data file.^{(24.4KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

In addition, the authors have declared this work was supported by the Agricultural Committee Basic Project (Shanghai Agricultural word (2015) No 6-2-3), the National Key Technology R&D Program during the 13th Five-Year Plan Period (2017YFD0101904) and the China Agriculture Research System (Grant No. CARS-25). And the funding bodies supporting this work did not play a role in the design of the study and the collection, analysis, and interpretation of data or in the composition of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0226537.r001

Decision Letter 0

Muthamilarasan Mehanathan

27 Jan 2020

PONE-D-19-33168

Genome-wide analysis of chalcone synthase (CHS) family from eggplant (Solanum melongena L.) in flavonoid biosynthetic pathway and expression pattern in response to heat stress

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Reviewer #1: In this manuscript entitled ‘Genome-wide analysis of chalcone synthase (CHS) family from eggplant (Solanum melongena L.) in flavonoid biosynthetic pathway and expression pattern in response to heat stress’ the authors have identified CHS-encoding genes of eggplant, performed their in silico characterization and analysed the expression pattern of key genes associated with anthocyanin biosynthesis pathways. Here are the following comments on the research for the authors:

1. The manuscript need to be revised for its English, grammar and flow of information. For example in L130: ‘The profiles of CHS (PF00195 and PF02797) wwere downloaded from the Pfam’- wwere need to be corrected to were. Line 198: ‘the other 7 Solanaceae species ranged from 17.3 to 47.4, the length of protein ranged’ kDa need to be added to the molecular weights mentioned here.

2. It would be more informative for the readers if authors will provide a comparative mapping of CHS genes in the genome of other related plants where sequence information are available.

3. Fig. 7. represents key anthocyanin biosynthesis gene expression profiles in response to heat stress. A bar diagram with proper error bars for individual gene will be the most appropriate way for presentation of this result. The authors are advised to prepare the same for readers convenient.

4. Authors have performed the phylogenetic classification of CHS gene in Solanaceae and found that 73 CHS genes have classified into 10 groups. However, it has not been mentioned or discussed that on what basis the CHS are classified and how one group differs with the others.

5. As authors have mentioned in the text about some of the cis regulatory element present on all the SmCHS promoter, similarly they should mention the unique cis element for each SmCHS genes. Not all CHS genes had shown similar expression pattern, thus a probable correlation of cis regulatory element on promoter and expression pattern of the gene should be mentioned in the discussion.

Reviewer #2: The manuscript entitled “Genome-wide analysis of chalcone synthase (CHS) family from eggplant (Solanum melongena L.) in flavonoid biosynthetic pathway and expression pattern in response to heat stress” by Wu et al. reports the in silico analysis of chalcone synthase (CHS) gene family in eggplant. In brief, the current manuscript presents biochemical characteristics, phylogenetic relationships, gene structures, cis-regulatory elements, regulatory network and functional predictions of the CHS gene family members.

The current quality of the manuscript leaves a number of questions to be answered, which are outlined below. The exposition of the results and its discussion in the manuscript needs a major revision including careful English usage and style edition.

1. The manuscript title starts with ‘Genome-wide analysis’; however, most of the analysis has been performed on CHS proteins. The work performed in the manuscript does not justify the title.

2. The ‘Introduction’ section of the manuscript is not coherent. Also, it does not describe the relevance of studying CHS gene family in proper manner.

3. One of the major concerns is, authors have given RNA-Seq data in the result section. However, the source of this data is not described in the “Material and Method” section.

4. Also, authors have not provided the proper statistics of the validation experiment. Statistical analysis should be done to evaluate the significance of qRT-PCR data. It is recommended to include statistical analysis in the ‘Materials and methods’ section.

5. Writing in Subheadings 4 to 7 under the ‘Results’ section is very technical. The data is not described properly. Authors have chosen to explain the dataset in the ‘Discussion’ section. The outcome of the experiments should be described in the ‘Results’ section and interpretation in consistence with the previous studies should be given in the ‘Discussion’ section.

6. In Figure 3A, the abundance of cis-elements is given in all the CHS genes in eggplant. How the regulation of all the genes can be shown together?

7. In Figure 5, there is no downregulation value. How the data has been calculated? What control has been taken and how the data has been normalized?

8. Figure 6, network is not very clear. A clear image has to be provided.

9. In ‘Material and Method’ section, what is CK? Why the samples harvested at 28º C are termed as CK and all the subsequent data is compared to CK?

10. In Figure 7, all the genes are showing high expression at 3 h CK. Do authors have any explanation of these results? Also, this data has been recorded from which tissue?

11. The other major issue is that this manuscript requires a thorough language editing since there are numerous grammatical errors including dropped articles, split infinitives, improper word usage etc. It is advisable that the manuscript must be edited by an English-speaking personal.

12. Reference section has to be rechecked. For example, in few places journal name is abbreviated (J Exp Bot) and in few places it is not (Plant physiology). In few references, journal name is capitalized and in others it is not.

13. Biochemical analysis of CHS proteins (pI, molecular weight etc.) is not relevant to the manuscript. This data can be removed from the draft.

Overall, the manuscript is not of adequate quality. The manuscript should be revised thoroughly for data presentation, result interpretation, description and language.

**********

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Reviewer #1: Yes: ROSHAN KUMAR SINGH

Reviewer #2: No

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PLoS One. 2020 Apr 17;15(4):e0226537. doi: 10.1371/journal.pone.0226537.r002

Author response to Decision Letter 0

5 Mar 2020

Dear Reviewers

Thank you and anonymous reviewers very much for your kind and useful comments for our manuscript “Genome-wide analysis of chalcone synthase (CHS) family from eggplant (Solanum melongena L.) in flavonoid biosynthetic pathway and expression pattern in response to heat stress” (PONE-D-19-33168). We have read and seriously considered the comments very carefully, checked and revised the manuscript many times. Major revisions throughout the revised manuscript have been made as followings.

1. The title name have been changed to “Chalcone synthase (CHS) family analysis from eggplant (Solanum melongena L.) in flavonoid biosynthetic pathway and expression pattern in response to heat stress”

2. The ‘Introduction’ section has been modified according to the comments

3. The results of all 73 CHS proteins sequence alignment, conserved residues and sequence diversity were analyzed.

4. We corrected some other grammatical errors and words spelling mistakes.

The followings are responses to the comments point-by-point.

Review Comments to the Author

1. The manuscript need to be revised for its English, grammar and flow of information. For example in L130: ‘The profiles of CHS (PF00195 and PF02797) were downloaded from the Pfam’- wwere need to be corrected to were. Line 198: ‘the other 7 Solanaceae species ranged from 17.3 to 47.4, the length of protein ranged’ kDa need to be added to the molecular weights mentioned here.

Answer: Thank you for pointing out the mistakes in the manuscript. We have corrected these issues in the revised manuscript according to comments, and checked the manuscript many times. Please refer to line 163 in revised manuscript with track changes.

2. It would be more informative for the readers if authors will provide a comparative mapping of CHS genes in the genome of other related plants where sequence information are available.

Answer: Chromosomal location of CHS genes were added in Table S1. Sequence alignment of all 73 CHS proteins of Solanum specie showed in the Fig S1. The Ⅷ, Ⅸ and Ⅹ groups are distinguished from other groups mainly depends on the position 1-164 amino acids, GroupsⅠ, Ⅱ and Ⅲ are relatively conservative at the position 260-360 amino acids, in which the other groups are very diverse(S1 Fig). Please refer to line 325-328.

Answer: Thanks for your suggestion. Error bars have been added in the Fig7.

Answer: The results of all 73 CHS proteins sequence alignment were added in S1 Fig. Conserved residues and sequence diversity of 73 CHS proteins were analyzed. Please refer to line 240-244 and 325-328

Answer: I'm sorry we miss the point. We have added this to the ‘result’ section refer to line 303-307.

1. The manuscript title starts with ‘Genome-wide analysis’; however, most of the analysis has been performed on CHS proteins. The work performed in the manuscript does not justify the title.

Answer: We changed the manuscript title as to ‘Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress’.

2. The ‘Introduction’ section of the manuscript is not coherent. Also, it does not describe the relevance of studying CHS gene family in proper manner.

Answer: We have added more information about CHS involve in synthesis of flavonoid compounds during heat stress defense. Please refer to line 70-80.

3. One of the major concerns is, authors have given RNA-Seq data in the result section. However, the source of this data is not described in the “Material and Method” section.

Answer: The overview of RNA-seq result from our lab was published (Zhang S et al, BMC Plant Biol. 2019 (1): 1-13). We therefore added a brief description of data source about RNA-seq in the“Material and Method” section. Please refer to line 196-198.

Answer: we have added the statistical analysis information of qRT-PCR in the ‘Materials and methods’ section. Please refer to line 223-225.

Answer: We agree and have revised the ‘results’ and ‘discussion’ accordingly. Please refer to line 351-361.

6. In Figure 3A, the abundance of cis-elements is given in all the CHS genes in eggplant. How the regulation of all the genes can be shown together?

Answer: We changed the histogram of figure 3A into a table to help readers understand the results.

7. In Figure 5, there is no downregulation value. How the data has been calculated? What control has been taken and how the data has been normalized?

Answer: Some errors have been corrected in line 351 of manuscript. Three SmCHS genes (SmCHS1, SmCHS2, and SmCHS3) were continuously downregulated under 38 ℃ and 45 ℃ treatment compared with the CK (27 ℃). Gene expression level was estimated by RNA-seq from mean FPKM (fragments per kilobase of exon model per million reads mapped) values for each treatment, and showed the expression patterns in heatmap. Each treatment had three biological replicates.

8. Figure 6, network is not very clear. A clear image has to be provided.

Answer: Is the network image you referred to Figure S2? We're sorry that there are too many genes in the network image. The picture can be expanded up to 6400%, so that the above words can be read clearly.

9. In ‘Material and Method’ section, what is CK? Why the samples harvested at 28º C are termed as CK and all the subsequent data is compared to CK?

Answer: The optimum growth temperature for eggplant is between 22 and 30°C. Eggplants subjected to high temperature may exhibit to stagnation of growth, abortion of flower buds, and decrease of pollen viability rate and fruit set, and the peel’s color will turn light when the temperature is over 35°C. Therefore, samples of 27 °C were termed as CK.

10. In Figure 7, all the genes are showing high expression at 3 h CK. Do authors have any explanation of these results? Also, this data has been recorded from which tissue?

Answer: Under heat stress, SmCHS4 and some anthocyanin biosynthesis related genes show different expression profiles at 38 ℃-3h. These results suggest that these co-up-regulated genes contribute to protect the eggplant at beginning of heat stress defense. These data were recorded from the eggplant peels, which is indicated in the Figure 5 and Figure 6.

Answer: We checked and revised the manuscript many times. Some grammatical errors and words spelling mistakes have been modified by English editing company of American Journal Experts (AJE).

Answer: This issue has been corrected.

13. Biochemical analysis of CHS proteins (pI, molecular weight etc.) is not relevant to the manuscript. This data can be removed from the draft.

Overall, the manuscript is not of adequate quality. The manuscript should be revised thoroughly for data presentation, result interpretation, description and language.

Answer: Biochemical analysis of CHS proteins was removed.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(22.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0226537.r003

Decision Letter 1

Muthamilarasan Mehanathan

23 Mar 2020

PONE-D-19-33168R1

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

PLOS ONE

Dear Dr. Wu,

We would appreciate receiving your revised manuscript by May 07 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Muthamilarasan Mehanathan, Ph.D.

Academic Editor

PLOS ONE

[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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The manuscript entitled “Chalcone synthase (CHS) family member analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress” by Wu et al. reports the in silico analysis of chalcone synthase (CHS) gene family followed by the expression analysis in eggplant.

First, I would appreciate the authors that they have addressed all the queries stated in the previous review. The quality of the manuscript has improved a lot. However, there are still few questions unanswered. Also, there are so many grammatical errors in the manuscript that altogether changes the meaning of the study. I have highlighted all the comments in the manuscript itself.

Please revise the manuscript once again for the grammatical corrections and other queries stated in the manuscript pdf file.

**********

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: No

Reviewer #2: No

Attachment

Submitted filename: PONE-D-19-33168_R1.pdf

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Attachment

Submitted filename: Review_March 2020.doc

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PLoS One. 2020 Apr 17;15(4):e0226537. doi: 10.1371/journal.pone.0226537.r004

Author response to Decision Letter 1

31 Mar 2020

The followings are responses to the comments point-by-point.

1. line 20. “isprimarily”

Answer: a space was added between the two words.

2. line 30-31. “occurrence of the duplicated words, “Under heat stress”

Answer: we removed duplicate words.

3. line 32. Wrong presentation of “at 38 ℃3h”

Answer: we have corrected.

4. line 33. “CHS protein biochemical characteristics”, deleted content in the result.

Answer: We have used evolutionary relationship instead of biochemical characteristics.

5. line 36-37. “SmCHS showed two or more expression patterns and performed multiple functions to regulate anthocyanin content. Combined with analysis of regulatory networks, to the results of this study may facilitate further research to understand the regulatory mechanism governing peel color in eggplants.”. Grammatical errors.

Answer: We have revised the sentences. We are sorry about our errors.

6. line 42-43. “subject to high”. Grammatical error.

Answer: We have corrected according to comments.

7. line 44. “decrease of pollen”.

Answer: We have revised it.

8. line 47. “molecular mechanism governing high temperature stress”

Answer: We have rewritten the sentence.

9. line 66. “for instance”

Answer: We have deleted the two words.

10. line 68-69. “in bread wheat”.

Answer: We have revised the manuscript.

11. line 72. Full-width comma is behind of the word “oak”.

Answer: We have revised it.

12. line 74. “CHSV and CHSVII”

Answer: We have changed italics to non-italics.

13. line 101-102. 'fruit shape has a' is not sounding proper.

Answer: We have revised the manuscript.

14. line 103. This line is not meant for the materials. It can be written into the Introduction section.

Answer: We have deleted the sentence.

15. line 141. “boostarp test”, Grammatical error.

Answer: We have used “Bootstarp value” instead of “bootstrap test”.

16. line 186. 1 μg of what? Please mention.

Answer: We have revised the manuscript according to the suggestion above.

17. Line 208. “The the length”, “the” repeated.

Answer: We have deleted the duplicate word according to the comment.

18. Line 209-210. Which other solanacae species?

Answer: We have added some details according to the comment.

19. Line212-214. What exactly as done after taking an average of the amino acids? It is still not clear

Answer: According the comment, we have rewritten the sentence.

20. Line 226-227. Is there any literature available citing this functional diversity for SmCHS 7?

Answer: We have added two references according the comment.

21. Line 250. Please replace 'and' with 'whereas'

Answer: We have corrected the error.

22. Line 252. 'cytoplasm' instead of 'cytoplasmic'

Answer: We have corrected the error.

23. Line 255 Table 2. 'Width' or "Length', please check once. Also, please add the unit. For example, in this case, it must be 'amino acids'

Answer: We have used “Length” instead of “Width” and have added the unit.

24. Line 257. Please describe Figure 3A first. Please try to maintain the sequence throughout the manuscript.

Answer: We have adjusted the order of the two subgraph (Fig3A and Fig3B) in Fig 3. we also have revised the manuscript.

25. Line 266-277. Please add 'element' after 'elicitor response'. Binding site or what? Please specify.

Answer: We have corrected them according the comments.

26. Line 293. Please add space before 'is'

Answer: We have revised the manuscript according the comment.

27. Line 300. Why SmCHS4 is in italics, but not the others. Moreover, the author is talking about protein sequences in this line. Do we really need to italicize it?

Answer: We have revised it.

28. Line 304. 'Colored' instead of 'color'

Answer: We have corrected the error.

29. Line 309. This RNAseq data was obtained from which tissue. Please specify.

Answer: We have added details of tissue.

30. Line 312. Why authors have chosen to write the name of these to genes in the list of genes not identified in the study?

Answer: We want to show that the CHS gene families may have tissue-specific or functionally differentiation. We have added this inference to the end of this paragraph.

31. Line 313-314. In this line, it is written that few of the SmCHSs were not identified. However, in the heatmap figure, expression data of all the CHS gene is given. How is that possible?

Answer: In order to show all genes related to anthocyanin biosythesis, the expression level of undetected genes were set to 0. These undetected genes were also shown in the heat map. We have rewritten these sentences.

32. Line 319. “at 45 ℃”

Answer: We have revised the manuscript.

33. Line 345. Please rewrite this line. It is not making any proper sense. Please add few lines in this paragraph to describe the link between this paragraph and SmCHSs.

Answer: We have added some details in this paragraph to describe the link between this paragraph and SmCHSs.

34. Line 350. The function can not be involved in anything. Please rewrite this line.

Answer: We have rewritten this sentence according the comment.

35. Line 398. Please capitalize 'biosynthesis'. You may write 'stress factors' instead of 'stressors'.

Answer: We have corrected the errors.

36. Line 405. Please cite the reference properly with the year.

Answer: We have corrected it.

37. Line 422. Please remove full stop.

Answer: We have removed the full stop.

38. Line 424. What is this reference? Please describe.

Answer: We have revised the manuscript.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(21.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0226537.r005

Decision Letter 2

Muthamilarasan Mehanathan

2 Apr 2020

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

PONE-D-19-33168R2

Dear Dr. Wu,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Muthamilarasan Mehanathan, Ph.D.

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0226537.r006

Acceptance letter

Muthamilarasan Mehanathan

6 Apr 2020

PONE-D-19-33168R2

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

Dear Dr. Wu:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Muthamilarasan Mehanathan

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. CHS protein sequences of Solanum species.

(XLSX)

Click here for additional data file.^{(26.8KB, xlsx)}

S2 Table. Features of CHS genes identified in Nicotiana tabacum.

(XLSX)

Click here for additional data file.^{(15.9KB, xlsx)}

S3 Table. Primers used for real time PCR analysis.

(DOCX)

Click here for additional data file.^{(17.9KB, docx)}

S1 Fig. Sequence alignment of all 73 CHS proteins of Solanum specie.

(JPG)

Click here for additional data file.^{(9.4MB, jpg)}

S2 Fig. Interaction network key to anthocyanin biosynthesis in eggplant.

The pink labels represent the CHS gene family.

(PDF)

Click here for additional data file.^{(24.4KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(22.2KB, docx)}

Attachment

Submitted filename: PONE-D-19-33168_R1.pdf

Click here for additional data file.^{(3.8MB, pdf)}

Attachment

Submitted filename: Review_March 2020.doc

Click here for additional data file.^{(31.5KB, doc)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(21.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress

Xuexia Wu

Shengmei Zhang

Xiaohui Liu

Jing Shang

Aidong Zhang

Zongwen Zhu

Dingshi Zha

Roles

Abstract

Introduction

Materials and methods

Plant materials and RNA extraction

Identification of the CHS family members in the eggplant genome

Structural characterization

Analysis of cis–acting elements in SmCHS

Phylogenetic analysis of CHS genes

Expression analysis of antyocyanin biosynthetic genes and construction of the mRNA regulatory network

qRT-PCR analysis

Results

Identification of CHS genes and sequence analysis in Solanaceae species

Table 1. Features of SmCHS genes identified in eggplant.

Structure and conserved motif analysis of SmCHS

Fig 1. Phylogenetic relationship and gene structure analysis of SmCHS genes.

Fig 2. Motifs conserved across all CHS proteins in eggplant.

Table 2. List of the putative motifs of CHS proteins.

Stress-related cis-elements in SmCHS promoters

Fig 3. Cis-elements in CHS family gene promoters.

Phylogenetic analysis of CHS genes in Solanaceae

Fig 4. Phylogenetic tree of CHS genes in Solanaceae species.

Table 3. Distribution of CHS genes in the phylogenetic tree.

Expression profile of key anthocyanin biosynthesis genes in eggplants under heat stress

Fig 5. Heatmap of 96 key anthocyanin biosynthesis genes expression level in eggplants peel under heat stress.

Fig 6. Expression profiles of SmCHS4 and eight anthocyanin biosynthesis genes in response to heat stress.

mRNA regulatory network associated with anthocyanin biosynthesis in eggplant

Table 4. Top 20 GO enrichment results of biological processes.

Expression pattern of anthocyanin biosynthesis key genes in different tissues under heat stress

Fig 7. Expression profiles of 20 key anthocyanin biosynthesis genes in different tissues.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Muthamilarasan Mehanathan

Roles

Author response to Decision Letter 0

Decision Letter 1

Muthamilarasan Mehanathan

Roles

Author response to Decision Letter 1

Decision Letter 2

Muthamilarasan Mehanathan

Roles

Acceptance letter

Muthamilarasan Mehanathan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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