Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

Nithiwat Suntichaikamolkul; Lalida Sangpong; Hubert Schaller; Supaart Sirikantaramas

doi:10.1371/journal.pone.0260665

. 2021 Nov 30;16(11):e0260665. doi: 10.1371/journal.pone.0260665

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

Nithiwat Suntichaikamolkul ¹, Lalida Sangpong ¹, Hubert Schaller ², Supaart Sirikantaramas ^1,^3,^*

Editor: Frances Sussmilch⁴

PMCID: PMC8631664 PMID: 34847184

Abstract

Durian (Durio zibethinus L.) is a major economic crop native to Southeast Asian countries, including Thailand. Accordingly, understanding durian fruit ripening is an important factor in its market worldwide, owing to the fact that it is a climacteric fruit with a strikingly limited shelf life. However, knowledge regarding the molecular regulation of durian fruit ripening is still limited. Herein, we focused on cytochrome P450, a large enzyme family that regulates many biosynthetic pathways of plant metabolites and phytohormones. Deep mining of the durian genome and transcriptome libraries led to the identification of all P450s that are potentially involved in durian fruit ripening. Gene expression validation by RT-qPCR showed a high correlation with the transcriptome libraries at five fruit ripening stages. In addition to aril-specific and ripening-associated expression patterns, putative P450s that are potentially involved in phytohormone metabolism were selected for further study. Accordingly, the expression of CYP72, CYP83, CYP88, CYP94, CYP707, and CYP714 was significantly modulated by external treatment with ripening regulators, suggesting possible crosstalk between phytohormones during the regulation of fruit ripening. Interestingly, the expression levels of CYP88, CYP94, and CYP707, which are possibly involved in gibberellin, jasmonic acid, and abscisic acid biosynthesis, respectively, were significantly different between fast- and slow-post-harvest ripening cultivars, strongly implying important roles of these hormones in fruit ripening. Taken together, these phytohormone-associated P450s are potentially considered additional molecular regulators controlling ripening processes, besides ethylene and auxin, and are economically important biological traits.

Introduction

Durian (Durio zibethinus L., see S1 Fig), well known as the king of fruit, is an important economic fruit crop in Southeast Asia. Besides having rich custard and almond flavors, the aril flesh also contains a number of different bioactive compounds that are beneficial to human health [1, 2]. In terms of global trade, Thailand has been the top durian exporter with 65.9% market share of the 2019 global production, comprising more than 1.47 billion USD for export value (https://www.tridge.com/, search in 2021). However, this climacteric fruit has a short post-harvest life, posing problems to extending the marketing period and export to distant markets [2]. Fruit organoleptic properties such as flavor, odor, and color appearance are the main factors affected by developmental and ripening processes. Traditional breeding is often used to improve yields and fruit quality. However, this is difficult because of the long cultivation, slow reproductive cycle, and quality traits complexity of durian [2]. Therefore, in-depth knowledge of the genetic and biochemical basis of such characteristics is strongly needed. In 2017, the durian genome was sequenced [3], and since then advances in ‘omics’ techniques provided a better understanding of the molecular mechanisms underlying fruit ripening, including those related to sulfur metabolism [1, 4] and transcription factors [5, 6]. However, biosynthetic pathways and their regulatory mechanisms governing important traits, such as aril flesh softening, the synthesis of pigment and bioactive compounds, sugar metabolism, and phytohormone crosstalk, are still unknown in durian and should be further investigated.

Cytochrome P450-dependent monoxygenases (P450s), heme-thiolate proteins, comprise a large superfamily of enzymes that respond to environmental stimuli and developmental stages. The general mechanism of this enzyme involves the addition of an oxygen atom regiospecifically and stereospecifically [7, 8]. Plant P450s have evolved into diverse families, which usually exhibit biochemically conserved functions. Families with essential functions, such as hormone metabolism or the synthesis of biopolymers, typically show a low copy number, whereas families with adaptive functions have expanded in certain taxa [9]. Most plant P450s are anchored to the cytoplasmic surface of the endoplasmic reticulum by a hydrophobic peptide present at the N-terminus, possibly forming a transmembrane segment and targeting some P450s to plastids or mitochondria [10]. Recently expanded P450 families might have new ecological functions [7], but these are more difficult to predict than the functions of conserved P450 families. Therefore, few P450s have been functionally characterized. Mining the plant cytochrome P450 complement (CYPome) in various crops such as soybean [11], rice [12], tomato [13], and grapevine [14, 15], has revealed significant information for plant quality improvement. For example, CYP78A predominantly enlarges the pericarp and septum tissues of tomato fruits [16]. Further, the fruit ripening process is controlled by various biosynthetic pathways involved in the action of P450s. Therefore, it is hypothesized that any P450 enzyme expressed during the course of fruit development and ripening has a considerable potential to act as a key catalyst for the biosynthesis of bioactive metabolites implied in that process.

In this study, we subjected the durian genome to a comprehensive search for genes encoding P450s. Information on durian P450s was then compared to that of other fully sequenced plant genomes. Taken together with transcriptome libraries, we identified all cytochrome P450s in durian (DzP450s) and determined their expression profiles throughout five development and post-harvest ripening stages of the Monthong cultivar, a slow post-harvest ripening cultivar. Large-scale expression analyses were performed for selected DzP450 genes using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To validate ripening-associated P450s, we determined the expression levels of the candidate DzP450s in the aril of fruit treated with the ethylene inhibitor (1-methylcyclopropene) and ethylene releaser (ethephon). We also compared gene expression during the ripening stages to that of another popular Thai cultivar, Phuangmanee, a quick post-harvest ripening cultivar. Finally, we investigated potential cultivar-dependent P450s that preferentially expressed in four different cultivars exhibiting different ripening behaviors. As a result, we found that the remarkable expression of P450s is potentially involved in the metabolism of phytohormones during the ripening stages of durian fruit, suggesting possible crosstalk between phytohormones that regulate fruit ripening.

Materials and methods

Plant materials

Durian fruit of slow post-harvest ripening cultivars (Monthong and Kanyao) and quick post-harvest ripening cultivars (Chanee, and Phuangmanee) were harvested for independent biological replicates (one fruit, separate trees) from a commercial plantation located in eastern Thailand (12°40′39.2″N 102°05′35.2″E). The number of durian fruits used depends on the individual experiments listed in the next sections. Fruit samples of a similar size and weight (~3–4 kg each) were harvested at different stages, specifically preharvest stages (immature 1 and immature 2), harvest stage (mature), and postharvest stages (midripe and ripe). To ensure that samples of the different cultivars were compared at the same ripening stage, the fruit firmness of each sample was measured as previously described [5]. Briefly, the firmness values of the immature 1, immature 2, mature, midripe, and ripe stages were in the ranges of 36–44 N, 44–47 N, 47–54 N, 2–6 N and 1–2N, respectively. For immature1 stage, fruit samples were collected and peeled at 70 days (Chanee and Phuangmanee) and 85 days (Monthong) after anthesis. For immature2 stage, fruit samples were collected and peeled at 80 days (Chanee and Phuangmanee) and 95 days (Monthong) after anthesis. For the mature stage, fruit samples were collected and peeled at 90 days (for Chanee and Phuangmanee) and 105 days (for Monthong) after anthesis. For the midripe stage, fruit samples at the mature stage were kept at 30°C for 2 days (for Chanee and Phuangmanee) and 3 days (for Monthong) and then peeled. For the ripe stage, fruit samples at the mature stage were kept at 30°C for 3 days (for Chanee and Phuangmanee) and 5 days (for Monthong and Kanyao) and then peeled. All peeled samples were immediately frozen in liquid nitrogen and stored at −80°C for transcriptome and RT-qPCR analysis.

Ethephon and 1-MCP treatments

In total, 15 independent biological replicates (one fruit, separate tree) of the Monthong cultivar (mature stage) were used, which were then randomly separated into three treatment groups, with five fruits for the control (natural ripening), five fruits for ethephon treatment, and five fruits for 1-methylcyclopropene (1-MCP) treatment, as previously described by Khaksar et al. [5]; for the control group, the fruit samples were left at 30°C. For ethephon treatment, the cut surface of the fruit stalk was brushed with aqueous 52% (w/v) ethephon (2-chloroethylphosphonic acid, Bangkok, Thailand), at approximately 1 mL/stalk, and dried at 30°C. For 1-MCP treatment, each fruit sample was placed in a 76 L sealed container and treated with 1-MCP gas for 12 h at 30°C. 1-MCP gas was generated by adding water to a 1-MCP tablet (Xianfeng, China) at a final concentration of 1% (w/w), and 38 mL of the 1-MCP solution was immediately placed in a beaker. This resulted in a final concentration of 0.5 mL L⁻¹ of 1-MCP in the container. The fruit samples were stored at 30°C and 85–90% relative humidity. Fruit in all groups were peeled after 5 days (ripe stage) and immediately stored at −80°C for further analysis.

Identification and classification of durian P450 genes

All P450 sequences of Arabidopsis thaliana, cotton (Gossypium raimondii), and cacao (Theobroma cacao) were obtained from the Cytochrome P450 Homepage (https://drnelson.uthsc.edu/) [17]. These sequences were used as input queries to search against the durian genome database (PRJNA400310) using TBLASTN with an e-value cut-off of 1e–5. The other parameters were set at default values. All top blast hits were collected, redundancies were removed, and protein IDs and sequences were searched in the NCBI database. Possible missing sequences were also manually mined in the protein table for D. zibethinus (https://www.ncbi.nlm.nih.gov/genome/?term=Durio) by searching for “P450,” “CYP,” “hydroxylase,” and “monooxygenase” as keywords. All collected sequences were initially classified based on protein homology against well-nomenclature P450s from cotton or cacao (https://drnelson.uthsc.edu/); families share ≥ 40% identity and subfamilies share ≥ 55% identity [18].

Conserved motif analysis

Protein sequences of durian P450s were collected in fasta format and separated into two groups, A-type and non-A-type. MEME suit [19] was used to analyze conserved motif structures in these sequences using default parameters. The amino acid frequencies of each motif were generated in the individual profiles.

Phylogenetic analysis

All protein sequences of durian P450s were aligned using CLUSTALW [20] with default parameters. Phylogenetic trees were constructed using maximum likelihood as a statistical method in MEGAX software [21]. Tree topology was assessed by bootstrap analysis with 100 resampling replicates. The tree was visualized and colored using Figtree (http://tree.bio.ed.ac.uk/software/figtree/).

Transcriptome analysis of Musang king and Monthong cultivars

Expression analysis was performed to gain insight into the role of the identified DzP450 in various tissues. We used publicly available RNA-sequencing data from the MaGenDB webpage [22] to determine the gene expression of DzP450s in four different tissues, namely leaf, root, stem, and aril, of the Musang king cultivar (the sequenced genome). For the five ripening stages, we used our RNA-sequencing data of the Monthong cultivar [Project accession number: PRJNA683229 (Mature and Ripe stages) and PRJNA732556 (Immature1, Immature 2, and Midripe stages)]. Reference-based transcriptome analysis was performed using OmicsBox program (v1.4.1.1). The raw reads were filter using FASTQ quality check package of the program using default parameters. Then, the clean reads were aligned to the reference genome of durian cv. Musang King [3] using the STAR package (v2.7.8a) [23], and the expression value at the gene level was calculated using HTSeq (v 0.9.0) [24] with default parameters. The reads per kilobase of transcript and per million mapped reads (RPKM) method was used for normalization of the expression levels [25]. The expression profile of the genes at different stages was analyzed using time course expression analysis of maSigPro (v.1.58.0) package (P<0.05, R-squared cutoff 0.7). Thereafter, the normalized total read counts were used to generate a heatmap using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) [26].

RT-qPCR analysis

Total RNA was isolated from durian fruit aril samples using PureLink Plant RNA Reagent (Waltham, MA) following the manufacturer’s instructions. Genomic DNA was removed using DNase I (Waltham, MA). The quality and quantity of RNA samples were examined using agarose gel electrophoresis and an Eppendorf BioPhotometer D30 with A260/280 and A260/230 ratios from 1.8 to 2.0 and 2.0 to 2.2, respectively, following the standard guidelines described [27]. For reverse transcription, 1 μg of total RNA was used to generate cDNA using a RevertAid First Strand cDNA Synthesis Kit (Waltham, MA), following the manufacturer’s recommended protocol and the standard guidelines for reverse transcription [27]. The PrimerQuest online tool (https://www.idtdna.com/PrimerQuest/Home/Index) was used to design the primers used in this study, which are presented in S1 Table. RT-qPCR was performed in a total volume of 10 μL containing 1 μL of diluted cDNA, 5 μL of Luna Universal qPCR Master Mix (Ipswich, MA), and 200 nM of each gene-specific primer. A Bio-Rad CFX95 Real-time System was used under the following conditions: initial activation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. Three independent biological replicates were used for each RT-qPCR experiment. The elongation factor 1 alpha (EF-1α) and actin (ACT) genes of durian were selected as reference genes for the normalization of RT-qPCR data according to our in-house transcriptome data of durian fruit from different cultivars, which confirmed the invariant expression levels of EF-1α and ACT under different experimental conditions [6]. Relative P450 expression levels were measured using the 2^−ΔΔCT method [28] according to the average Ct values of the two reference genes [29]. The normalized Ct values of each gene are presented in S3–S5 Tables.

Statistical analysis

Gene expression analysis for ethephon and 1-MCP treatments was performed based on five biological replicates each. Gene expression analyses for developmental and ripening stages were performed with three biological replicates each. Normalized expression values (–ΔCT) were transformed to log(2^−ΔCT) or log(2^−ΔΔCT). The log data were tested for normality, and a T-test, Tukey HDS, and Pearson correlation test were performed using IBM SPSS Statistics version 22 (p = 0.05).

Results and discussion

Identification, classification, and conserved motifs of durian P450s

P450 genes have been identified from the durian genome [3]. As a result, a total of 355 putative durian P450 genes with complete open reading frames was identified. All durian P450s were classified and named based on the protein sequence homology [18]; families share ≥ 40% identity, and subfamilies share ≥ 55% identity when aligned with the officially classified P450s that are publicly provided on the Cytochrome P450 Homepage [17]. Accordingly, durian P450 genes were distributed in 10 groups consisting of 56 families, as listed in S2 Table. This number (355) is comparable to that in cocoa (336), apple (328), and grapevine (315), higher than that in A. thaliana (245), banana (233), watermelon (233), cucumber (229), and strawberry (209), and lower than that in soybean (715), tomato (456), cotton (449), and rice (412). We constructed phylogenetic trees of DzP450s based on the fact that plant P450s are of two types, A-type and non-A-type [9]. As shown in Fig 1, A-type (52.4%, 186/355) consisted of the CYP71 clan and the non-A type (47.6%, 169/355) consisted of the remaining families.

Fig 1 — (A) A-type is represented by clan 71. (B) Non-A type is represented by the other families. The numbers on each clan indicate the family of cytochrome P450. The name of durian P450s is indicated by the protein accession number followed by the predicted (sub) family. The tree was constructed by the maximum likelihood method with 100 replicates. The colors on the trees indicate the bootstrap values (percentages) after 100 replicates. The scale bars in the circular trees represent the number of amino acid substitutions per site.

According to the distribution of P450 families across plant phylogeny, there are 10 clans in plants that are named by their lowest family numbers (CYP71, CYP72, CYP85, CYP86, CYP51, CYP74, CYP97, CYP710, CYP711, and CYP727). In the durian genome, the CYP71 clan was determined to be the largest clan and comprises 52.4% of durian P450 genes. This proportion is similar to that in Arabidopsis (50% A type), soybean (56.08% A type), and apple (56.10% A type). In durian, A-type P450 genes were found to be more divergent than the non-A-type. This is because most A-type genes encode plant-specific enzymes that act in the metabolism of diverse secondary metabolites that enhance plant adaptation. Non-A-type genes are mainly involved in the synthesis of hormones and other compounds related to primary metabolism in plant [30]. Considering the number of angiosperm P450s (see S2 Table), we identified one CYP92, one CYP727, one CYP733, 14 CYP736s, and 21 CYP749s in the durian genome, but these have no orthologs in A. thaliana, a model organism that has been extensively studied for the functions of P450 genes. This suggests an evolutionary path in the durian genome, which differs from that of the Arabidopsis genome. The present analysis and previously published data indicate that CYP92 exists in all higher plants and is involved in brassinosteroid biosynthesis [31]. The functions of CYP727 and CYP733 have not yet been identified. CYP736 is involved in the biosynthesis of biphenyl phytoalexins in apple [32], and CYP749 is required for herbicide tolerance in cotton [33]. To the best of our knowledge, no P450s in durian have been characterized. To further confirm this identification, a total of 355 predicted durian P450 genes was subjected to protein domain and motif analyses. As shown in Fig 2, both A- and non-A types of durian P450 proteins had structures typical of the P450 family [30], including a heme-binding region, PERF motif, K-helix region, and I-helix region. In comparison with those of Arabidopsis [30], the Glu and Arg of the K-helix, the Phe, Gly, and Cys of the heme-binding motif, and the Arg of the PERF motif were found to be respectively conserved in both plants. Meanwhile the residues of the I-helix involved in oxygen binding were slightly different.

Fig 2 — Sequence logos of the consensus motifs were created using MEME online software, containing stacks of letters at each position in the motif. The total height of the stack was the information content of that position in the motif in bits. The height of each letter represents the frequency of amino acids at the corresponding position.

Gene expression analysis reveals putative DzP450s related to fruit ripening

The five stages of durian were divided into two main stages, developmental stages and ripening stages. Developmental stages refer to the pre-harvest to harvest stages (immature 1, immature 2, and mature) and ripening stages refer to harvest to post-harvest stages (mature, mid-ripe, and ripe). The expression levels of DzP450s, analyzed by RNA-seq at five stages of fruit (S1 Fig) were quantified as RPKM data. The expression signals of 44.3% (155/355) of the durian P450 genes were detected. As shown in Fig 3, the expressed P450 genes were grouped into 16 clusters, A to P, according to their expression pattern during the five stages based on ward clustering algorithm. The expression profiles of nine randomly selected DzP450s (from the clusters in which the expression increased during the ripening stages) were validated by RT-qPCR. These selected P450s were analyzed for their relative transcript abundance and are graphically represented in S2 Fig. Pearson correlation values (r) ranged from 0.547 to 0.983, indicating a highly positive correlation between RNA-sequencing data and RT-qPCR analysis. Therefore, our transcriptome data of the Monthong cultivar was highly accurate for examining the expression of other genes.

Fig 3 — The P450s were grouped as groups A–P based on a ward clustering algorithm using MetaboAnalyst 5.0, an open-source R-based program. Data were sum normalized, log-transformed, and auto scaled. The color scale indicates the z-score. Stage abbreviations: IM1, immature1; IM2, immature2; M, mature; MR, mid-ripe; R, ripe.

Considering fruit-specific P450s, gene expression of P450s across four tissues of the Musang king cultivar was determined. Accordingly, CYP71, CYP72, CYP81, CYP83, CYP84, CYP88, CYP89, CYP93, CYP94, CYP96, CYP706, CYP707, CYP714, CYP718, and CYP749 were abundantly expressed in the aril compared to levels in the leaves, stems, and roots (S3A Fig). Taken together with the expression pattern of P450s during the five stages of the Monthong cultivar, CYP93 was decreased, whereas CYP81, CYP83, CYP84, CYP88, CYP89, CYP94, CYP96, CYP706, CYP707, CYP714, CYP718, and CYP749 increased during the five stages. Among the CYP71 genes, the expression of some putative genes decreased, whereas some putative genes were decreased during the five stages (S3B Fig).

CYP71 is the largest P450 clan in plants, containing over half of all known cytochrome P450s identified from the plant kingdom [7, 9, 34]. Similar to a previous genomic study [3], CYP71 was upregulated in durian arils during the ripening process. Most members of this family have been functionally characterized as terpenoid oxidases, including mono-, sesqui-, and diterpenoid-modifying enzymes [35–37]. For CYP81, this family is generally involved in phenolic metabolism, the substrates of which depend on subfamilies. CYP81B and CYP81D subfamilies were upregulated during the ripening stages of the Monthong cultivar. CYP81B has been reported as involved in the in-chain hydroxylation of fatty acids in Helianthus tuberosus [38]. CYP81D can be induced by various exogenous stimuli, such as a number of herbicides [39–42], jasmonic acid [42–44], and salinity [45]. Therefore, CYP81B and CYP81D might be involved in the biosynthesis of defense compounds against biotic and abiotic stresses during the ripening process of durian. CYP84A encodes a C-5 hydroxylase of coniferaldehyde and coniferyl alcohol, leading to syringyl lignin formation [46]. The downregulation of rice CYP84A results in altered lignin that is largely enriched in guaiacyl units, whereas the upregulation of rice CYP84A leads to enrichment in syringyl units [47], suggesting that CYP84A might be a major factor controlling lignin composition in durian fruit. CYP89A has been reported to be involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis [48], suggesting its role in pigment formation in the aril over the course of ripening. CYP93A is the ancestral group distributed in angiosperms and is involved in the biosynthesis of flavones [49]. The increased expression of CYP93A supports the occurrence of flavonoids in durian fruit [50]. CYP96A mediates omega-hydroxylation of fatty acids, which is essential for the synthesis of Arabidopsis cuticles [51], suggesting a role for the synthesis of aril wax during the ripening stage. CYP706 evolved from the CYP71 clan and was reported to be involved in the oxidation of both monoterpenes and sesquiterpenes [52, 53], suggesting a role for durian aril defense during the ripening stage. The biological functions of CYP718 and CYP749 remain unknown. However, CYP718 is a single-copy gene in most plant genomes (see S2 Table), indicating strong purifying selection and a possible role in durian fruit ripening. The other remaining families (CYP72, CYP83, CYP88, CYP94, CYP707, and CYP714) have been reported to be involved in phytohormone metabolism and are mainly discussed in the next section. It should be noted that metabolite information in durian arils is scarce and is necessary for the functional elucidation of DzCYP450s.

Ripening-related cytochrome P450s potentially regulate phytohormones in durian fruit

CYP72, CYP88, and CYP714 are involved in gibberellins biosynthesis

In total, 11 CYP72s were identified in the durian genome, but only XP_022738158, which showed 82.55% identity to cotton CYP72A, was abundantly expressed in the aril of the Musang king cultivar with gradually increasing expression during the ripening stages of the Monthong cultivar (S3 Fig). CYP72A has a gibberellin (GA)-inactivating function via the 13-hydroxylation of GAs and ent-kaurenoic acid (KA), which abundantly occurs in Arabidopsis seeds, indicating that CYP72A might play a role in promoting dormancy in Arabidopsis [54]. Although the expression of DzCYP72A was not changed by 1-MCP and ethephon treatment and was not specific to either fast-ripening or slow-ripening cultivars, it was significantly higher in Phuangmanee cultivar than in the Monthong cultivar during the ripening stages (Fig 4A–4C), suggesting that it is not related to controlling ripening behavior.

Fig 4 — (A) Expression profiles in Monthong fruit treated with exogenous 1-methylcyclopropene (MCP) or ethephon (ETP) at the ripe stage, in relative comparison with untreated fruit (control; C). Five biological replicates were employed for each treatment. Dots and error bars represent the means and standard deviations of log(2^−ΔΔCT), respectively. Tukey’s HSD tests were used for statistical calculations (p = 0.05). The same letter above the error bars indicates no significant difference. (B) Expression profiles during five ripening stages in Phuangmanee cultivar (orange dots) and Monthong cultivar (grey dots). Three biological replicates for each cultivar and stage were employed. Dots and error bars represent the means and standard deviations of log(2^−ΔCT), respectively. Asterisks indicate the significance between two cultivars at the same stage, calculated by a t-test (p = 0.05). The expression of CYP714E at IM1 and IM2 stages of Monthong cultivar were undetectable by RT-qPCR. Abbreviations: IM1, immature 1; IM2, immature 2; M, mature; MR, mid-ripe; R, ripe. (C) Expression profiles in four cultivars of durian at the ripe stage (log scale). Three biological replicates of each cultivar were employed. Dots and error bars represent the means and standard deviations of log(2^−ΔΔCT), respectively. Tukey’s HSD tests were used for statistical calculations (p = 0.05). The same letter above the error bars indicates no significant difference. Cultivar abbreviations: PM, Phuangmanee; CN, Chanee; MT, Monthong; KY, Kanyao.

Among the five CYP88 genes found in the durian genome, XP_022738013 was highly expressed during fruit ripening. This putative gene showed 71.6% identity with cotton CYP88A. This subfamily catalyzes the conversion of KA to GA₁₂, the precursor of all GAs (Fig 5A) [55]. The expression of DzCYP88A was significantly suppressed in the presence of 1-MCP (Fig 4A), suggesting its significant role in the ripening process of durian fruit. Accordingly, the expression of DzCYP88A was significantly higher in Phuangmanee cultivar than in the Monthong cultivar during the ripening stages (Fig 4B) and in the fast-ripening cultivar (Phuangmanee and Chanee) than in the slow-ripening cultivar (Monthong and Kanyao) (Fig 4C). Taken together, DzCYP88A is potentially involved in GA biosynthesis and potentially plays an important role in accelerating the fruit ripening process in durian.

Considering the CYP714 family, five CYP714s were found in the durian genome, among which only XP022743116 was found to be expressed gradually during fruit ripening. This putative P450 showed 86.08% identity with cocoa CYP714E. The CYP714 subfamily generally has inactivating functions by oxidizing GAs on the C and D rings [55]. In contrast to DzCYP88A expression, DzCYP714E was significantly suppressed in the presence of 1-MCP and was unchanged in the presence of ethephon (Fig 4A). In addition, DzCYP714E significantly expressed higher in Phuangmanee than in Monthong at midripe to ripe stages (Fig 4B). However, the expression of DzCYP714E was not different between fast-ripening cultivars and slow-ripening cultivars (Fig 4C).

KA is a key intermediate in the biosynthesis of several terpenoids, including the phytohormone GA (Fig 5A). According to the expression profiles in durian, KA biosynthetic genes (ent-copalyl diphosphate synthase, ent-kaurene synthase, and ent-kaurene oxidase) were downregulated during the ripening process (Fig 5C), suggesting that an increased level of KA accumulated during the ripening process. Subsequently, an appropriate level of KA is catalyzed by DzCYP88A to produce the precursor GA₁₂, which is potentially oxygenated by DzCYP72A and DzCYP714A/E. As previously mentioned, these durian P450s are upregulated during the ripening process. Therefore, we propose two possible functions for oxygenated products. First, 13-hydroxylated GA is an inactivated form of GA that reduces the negative effects of bioactive GAs. This hypothesis is supported by a study showing that GAs play a role in regulating tomato fruit ripening, in which GAs act as negative regulators of ripening regulator genes, ethylene biosynthetic genes, and subsequent ripening processes [56]. Secondly, we proposed that 13-hydroxy GA might directly regulate ethylene-responsive/biosynthetic genes.

CYP83 is possibly involved in auxin oxidation

Four CYP83s were identified in the durian genome. Of these, DzCYP83F (XP_022765972), which exhibited 74.65% identity with cotton CYP83F, showed an increased expression level during fruit ripening. This family has been reported on the Cytochrome P450 Homepage [17] to be lost from several plant genomes, such as rice, tomato, apple, grape, cucumber, strawberry, and watermelon (see S2 Table), suggesting a role in plant specialized metabolism. CYP83 has been reported to regulate the level of indole-3-acetic acid (IAA) by converting indole-3-acetaldoxime to the glucosinolate pathway [57, 58]. A T-DNA insertion in the CYP83B gene leads to plants with a phenotype of auxin overproduction, whereas CYP83B overexpression leads to the loss of apical dominance, typical of an auxin deficit [57, 58]. In addition, in Phaseolus lunatus, CYP83E is involved in the biosynthesis of cyanogenic glucoside via indole aldoxime oxidation [59]. Although the biochemical function of DzCYP83F is still unknown and neither glucosinolates nor cyanogenic glucosides have been found in durian fruit, it potentially utilizes IAA as a substrate to maintain auxin homeostasis during durian fruit ripening.

CYP94s involved in the catabolism of jasmonate and its conjugates (JAs)

Twelve CYP94s were identified in the durian genome. Among these, three DzCYP94s were specifically expressed in the aril tissue of the Musang king cultivar and were highly expressed during the ripening stages of the Monthong cultivar (S3 Fig). Two of these, XP_022759757 and XP_022771227, showed 85.27% and 84.27% identity with cocoa CYP94A. However, XP022764670 showed 84% identity with cotton CYP94D. CYP94 has been recognized to be involved in metabolic pathways of fatty acid-derived compounds, and in particular, the catabolism of bioactive JA, an oxidation derivative of polyunsaturated fatty acids. JA and its derivatives are well-known for their role in immune responses, plant growth and development, and secondary metabolism, thus allowing plants to rapidly adapt to changing environmental conditions [60]. Some reports have shown that CYP94A acts as a fatty acid hydroxylase that might be associated with cutin monomer synthesis [61–63]. Interestingly, CYP94B and CYP94C convert jasmonate-isoleucine (JA-Ile) into 12OH-JA-Ile and 12COOH-JA-Ile [64–68]. Enhanced CYP94C expression deactivates and turnovers the content of JA-Ile, conferring stress tolerance [69] and improving plant height in rice [70]. In avocado, JA-Ile was found to be increased abruptly in cold-stored fruit compared to levels in normal-stored fruit [71]. Taken together, the expression of DzCYP94B leads to the inactivation of JA-Ile, possibly resulting in the ripening process in durian. Based on the phylogenetic analysis of the plant CYP94 family, CYP94D was extended from the CYP94B/C clan (S4 Fig), which is well-recognized as a JA-Ile hydroxylase. Although CYP94D has recently been identified as a cholesterol C-26 hydroxylase in phytosteroid diosgenin biosynthesis in Dioscorea zingiberensis [72], the expression of DzCYP94D was induced by exogenous ethephon treatment (Fig 4A), indicating a possible role in regulating durian fruit ripening. The expression of DzCYP94D was significantly higher in the Phuangmanee cultivar than in the Monthong cultivar at mid-ripe and ripe stages (Fig 4B). In addition, it was predominantly expressed in fast-ripening cultivars (Phuangmanee and Chanee) rather than in slow-ripening cultivars (Monthong and Kanyao) (Fig 4C). It is possible that CYP94D is involved in the biosynthesis of a signal molecule that accelerates fruit ripening in durian, which remains to be further elucidated.

CYP707 is involved in the catabolism of abscisic acid (ABA)

Among the 15 CYP707s found in the durian genome, only XP_022755802 was specifically expressed in the aril of the Musang king cultivar and increased throughout the ripening stages of the Monthong cultivar (S3 Fig). This putative gene showed 90.32% identity with cocoa CYP707A. This subfamily has been recognized to catalyze the first committed step in the predominant ABA catabolic pathway (Fig 5B) [73] and appears to be widespread in many crop species such as rice, tomato, soybean, maize, lettuce, and wheat [74]. Constitutive expression of a CYP707A gene in transgenic Arabidopsis results in decreased ABA content in mature dry seeds [75]. In fact, phaseic acid (a catabolite of ABA) is unable to bind to ABA-binding proteins from apple fruit and barley aleurone layers [76, 77]. However, this molecule likely emerged in seed plants as a signaling molecule that fine-tunes plant physiology, environmental adaptation, and development [78].

Upon external 1-MCP administration to the Monthong cultivar, the expression of DzCYP707A was significantly suppressed (Fig 4A). In addition, the expression of this putative gene gradually increased during the ripening process of the Monthong cultivar (Fig 4B). Similar to results of previous reports, CYP707 was upregulated during the ripening process in tomato, citrus, and grape [79], as well as during fruit development in oriental melon [80]. Notably, the expression level of DzCYP707A was significantly higher in Monthong (slow-ripening cultivar) than in Phuangmanee (fast-ripening cultivar) in the late ripening stages (Fig 4B). In addition, DzCYP707A also expressed predominantly in Monthong and Kanyao (slow-ripening cultivars) compared to levels in Phuangmanee and Chanee (fast-ripening cultivars), implying that ABA content is probably higher in fast-ripening cultivars than in slow-ripening cultivars during the late ripening process of durian fruit (Fig 4C). Correspondingly, ABA biosynthetic genes (zeaxanthin epoxidase, 9-cis-epoxycarotenoid dioxygenase, short-chain alcohol dehydrogenase/reductase, and ABA-aldehyde oxidase) were upregulated during the ripening stages of cv. Monthong (Fig 5C). A previous report demonstrated that ABA induces the expression of ethylene-associated genes to trigger the ripening of tomato fruits [81–83]. ABA also determines fruit firmness in tomato and promotes softening, synergistically with ethylene, in banana [82, 84]. Taken together, DzCYP707A might play an important role in fruit ripening and accelerating the ripening process. Therefore, this gene is a potential molecular marker that requires further characterization.

Conclusion

In climacteric fruits, including durian fruit, ethylene is the major regulator controlling fruit ripening and seems to be the most explored phytohormone. After harvest, ethylene continues to increase for a few days before declining, in parallel to the respiration of the whole durian fruit [2]. Evidence suggests that both ethylene and auxin crosstalk with each other, resulting in increased accumulation of IAA during durian ripening [5, 6]. However, the interaction between auxin and GA signaling pathways is essential for the promotion of fruit set and early fruit development stages in fleshy fruits [85]. Therefore, the active GA content might decrease during the ripening process of durian. The active GA also positively regulates the expression of ABA catabolic P450 (CYP707A) [73], which might result in decreased expression of Dz707A and elevated ABA content during the ripening stages of the fast-ripening cultivars. Accordingly, high ABA content can trigger ripening by inducing the expression of ethylene-associated genes [81–83].

Our study revealed that the durian genome has a greater number of P450 clans than various flowering plants with variable numbers of P450 genes in each clan. Phylogenetic analysis provided information about the functional evolution of the P450 gene family in durian. The expression profile of tissue-specific P450s coupled with the expression profile of P450s during the ripening stages provides potential candidates for further study. Based on the expression profile of candidate P450s in the four durian cultivars and upon exogenous treatment with ripening regulators, we suggest that post-harvest ripening of durian fruit might be regulated by auxin, ABA, Ja-Ile, and GAs in crosstalk with ethylene. Considering the rapid climacteric nature of durian ripening, it is important to translate this knowledge to implement techniques along the supply chain to further delay or inhibit ripening. For example, an exogenous regulator of phytohormone-related P450 delays durian ripening. Not only is P450 related to fruit ripening, but this study also provides a solid foundation for the functional characterization of candidate genes with biological significance of economic importance.

Supporting information

S1 Fig. Morphology of durian fruit (Monthong cultivar).

(A) whole fruit. (B) peeled fruit. (C) Arils across five developmental and ripening stages. Stage abbreviations: IM1, immature 1; IM2, immature 2; M, mature; MR, mid-ripe; R, ripe.

(TIF)

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S2 Fig. Correlation between RNA-based expression profile and RT-qPCR validation of selected P450s in Monthong cultivar across five stages.

Protein ID followed by predicted P450 family are indicated above each chart. Blue bars and error bars represent the means and standard deviations of log(2^−ΔCT) based on RT-qPCR. Red lines and error bars represent the means and standard deviations of log-RPKM values. Three biological replicates were used for each stage. Pearson correlation (r) values are shown in green letters (p = 0.05). Stage abbreviations: IM1, immature stage 1; IM2, immature stage 2; M, mature stage; MR, mid-ripe stage; R, ripe stage.

(TIF)

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S3 Fig. RNA-seq based expression profile of durian P450s.

(A) Four tissues of Musangking cultivar. (B) Five ripening stages of Monthong cultivar. To simplify the heatmap, the top 70% ranked by partial least squares discriminant analysis (PLS-DA) and variable importance in projection (VIP) are shown and briefly categorized into two clusters, decreased and increased during the ripening stages. The heatmap was generated by MetaboAnalyst 5.0, an open-source R-based program. Data were sum normalized, log transformed, and auto scaled. Asterisks indicate fruit-specific P450s from the heatmap (a). The color key bars indicate the standard score (Z-score) of each gene expression level. Higher expression for each gene is presented in red; otherwise, blue was used. Stage abbreviations: IM1, immature1; IM2, immature2; M, mature; MR, mid-ripe; R, ripe.

(TIF)

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S4 Fig. Phylogeny of selected plant cytochrome P450s involved in phytohormone biosynthesis.

(A) CYP72. (B) CYP88. (C) CYPCYP94. (D) CYP707. (E) CYP714. The candidate durian P450s are highlighted in red letters. The tree was constructed by the maximum likelihood method with 100 replicates. The bars in the trees represent protein relationships of the unrooted tree. The list of protein sequences used to construct the tree is presented in S6 Table. Plant abbreviations: At, Arabidopsis thaliana; Cl, Citrullus lanatus; Cs, Cucumis sativus; Dz, Durio zibethinus; Fa, Fragaria ananassa; Gm, Glycine max; Gr, Gossypium raimondii; Ma, Musa acuminata; Md; Malus domestica; Os, Oryza sativa; Sl, Solanum lycopersicum; Tc, Theobroma cacao; Vv, Vitis vinifera.

(TIF)

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S1 Table. Primer list for RT-qPCR.

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Click here for additional data file.^{(79.6KB, pdf)}

S2 Table. Comparison of P450 families in durian and selected flowering plants in the reversed order of angiosperm evolution.

(PDF)

Click here for additional data file.^{(124KB, pdf)}

S3 Table. Normalized Ct values of the genes in durian arils cv. Monthong control (natural ripening), 1-MCP treatment, and ethephon treatment.

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S4 Table. Normalized Ct values of the genes in durian arils (two cultivars of each stage).

The abbreviations N/D indicate no detectable Ct.

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S5 Table. Normalized Ct values of the genes in durian arils (four cultivars at ripe stage).

(PDF)

Click here for additional data file.^{(80.9KB, pdf)}

S6 Table. List of proteins used for construction of phylogeny of selected plant cytochrome P450s involved in phytohormone biosynthesis.

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Click here for additional data file.^{(29.4KB, pdf)}

Acknowledgments

The authors are thankful to Kittiya Tantisuwanichkul, and Pinnapat Pinsorn for assistance in sample collection.

Data Availability

The nucleotide sequences reported in this article have been submitted to Genbank under accession numbers [PRJNA683229 and PRJNA732556].

Funding Statement

This research was funded by Chulalongkorn University (grant number GRU6407023008-1) to S.S. S.S. and H.S. acknowledge financial support from Franco-Thai Mobility Programme/PHC SIAM 2021-2022 international exchange program N°46969PF. N.S. is supported by the Second Century Fund (C2F), Chulalongkorn University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0260665.r001

Decision Letter 0

Frances Sussmilch

15 Sep 2021

PONE-D-21-24817

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

PLOS ONE

Dear Dr. Sirikantaramas,

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.

Please address all points raised by the three reviewers. In addition, please also address the following:

For expression analyses, be aware that fold-change values are not normally distributed and thus error bars should represent the upper and lower bounds of the confidence interval, rather than standard deviation/error, and statistical analyses should not assume normal distribution (or be performed on log fold change values which are normally distributed). If fold change is retained, explain clearly in the legend what comparison the fold change represents.
Include (or cite another paper that includes) the primers for the reference genes and explain how both were used (e.g. was the geometric mean of both housekeeping genes used, or were these used alone as individual reference genes in different experiments?).
There is displacement of some letters in gene names in S5.

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #2: No

Reviewer #3: Yes

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5. 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: In the current work, Suntichaikamolkul et al. present a deep mining of the Durian genome and transcriptome libraries, leading to the identification of P450s that are potentially involved in Durian fruit ripening. They provide insight regarding the probable activity of the different P450 clans.

Major comments:

References for the bioinformatics tools used in this analysis (SATR, tblastn, MEME, htseq clustalW etc) should be included in the materials and methods secion.

Fig 1.: The legend lacks necessary information, including whether or not the tree is rooted, which external group was used, etc. Also, the leaves could be colored to be easier to read.

Fig 2.: More detail in the legend would help to explain the figure. Is it showing the degree of conservation of the Durian P450 for each group? How did you compute the degree of conservation? Furthermore, it would be interesting to redo the same figure with Arabidopsis P450 to be able to compare the motif conservation between Durian and Arabidopsis P450s.

The heat-map in Supplementary Fig. S4 should be included in the main body of work. However, it also needs more detail—how did you define these 5 clusters and which method did you use to produce them?

In regard to Line 237 “The expression profiles of nine randomly selected DzP450s were validated by RT-qPC:” Why did you not choose at least one or two genes within the clusters that you defined in Fig. S4? The C cluste is over-represented in your figure, and we do not know if the profiles for the other cluster are consistent?

Fig. S5: The legend needs to be clearer. You included the “ratio of RPKM-normalized read counts.” Is this an RPKM or a ratio of RPKM, and, if it is a ratio, what did you use as the reference?

Sup. Fig. S6: It is difficult to understand why the RPKM is between -1 and 1 because the legend is not clear.

Line 344 "CYP83 is involved in auxin oxidation:" This part only includes published information from other species and no Durian experiments, so you cannot make a claim regarding Durian CYP83 involvement in auxin oxidation. Furthermore, the authors stated that CYP83 has been lost in several plants, so you cannot truly know what the role of this gene is in Durian, as it could have a totally new activity.

Fig. 4C: Is this a fold change or an RPKM? The column title and the color scale legend are not in agreement. Also, the blue and red labels in Fig. 4A and 4B need more explanation, along with 4C.

In the section "Durian CYPome reveals phytohormone-crosstalk in the regulation of fruit ripening," there are no results from your analysis. Therefore, this should be included in the conclusion or another part.

Line 347 “This family has been reported to be lost from several plant genomes, such as rice, tomato, apple, grape, cucumber, strawberry, and watermelon:” if it has been reported you need to include the references.

Fig. S7: You need to include the method that was used. Can you add a table with the protein ID used in these different phylogeny tree? Is the tree rooted, and, if yes, with which species? How many branches are on the tree? How did you compute these bootstraps?

Reviewer #2: In this manuscript, the authors provide valuable data regarding the molecular mechanisms putatively regulating the ripening process of durian fruit ripening that may serve as a starting point for further studies involving functional characterization of the highlighted genes presented here. A better understanding of these mechanisms could lead to different biotechnological applications, positively impacting post-harvest shelf life and other organoleptic desirable traits.

Most of my comments focus on helping to make the text clearer and more consistent. However, the authors need to pay close attention to the statements made, particularly in the results and discussion section. In general, I suggest that authors change to a more moderate writing style, especially when speculating, which is not accompanied by additional data that clearly supports the speculations.

General comments:

-When the function of a CYP is cited, be careful to always mention in what species this function was studied.

-The expression “ripening stages” used in lines 251, 361, 428 and others is very ambiguous and seems to be used as synonym of ripe developmental stage and late developmental stages in some context, while in the legends is used to refer together to the five ripening stages analyzed.

-According to PLOS Data policy, all the single measurements used for Figure 3 must be available in a table.

Introduction:

- Line 44: Please change “Fruit qualities such as flavor, odor, and color” by “Fruit organoleptic properties such as flavor, aroma, and color.”

- Lines 64-66: Please avoid the repetitive use of “therefore.”

- Line 78:

- Lines 82-83: I would suggest replacing the expression “ripening repressor” and “ripening activator” with the relation of both compounds with ethylene since these expressions sound too broad.

- Lines 86-89: I suggest rewriting this sentence since it is unclear where or when this P450 burst appears and how it is related to phytohormone crosstalk.

Materials and methods:

- Line 92: Please mention which cultivars correspond to slow- and quick-postharvest in one sentence and not in different sections throughout the text.

- Line 95: immature1

- Line 96: harvest stage (mature?)

- Line 98: Please mention briefly the firmness values used for defining each developmental stage.

-It sounds a bit confusing that three biological replicates were collected in the “Plant material” section. However, in the following section, it is mentioned that five biological replicates were employed. I suggest clarifying that these three biological replicates were stored at -80 °C for qRT-PCR analysis.

- Regarding ethephon and 1-MCP treatments: it is unclear the experimental design. Five replicates were used for each treatment (15 fruits in total?). It is also not mentioned the developmental stage of treated fruits.

Results:

- Please indicate in the legend of figure 3, if the data presented correspond to the mean, and if the error bars correspond to standard deviation or standard error. Besides, indicate the number of biological replicates employed.

- I do not understand the idea of Figure 3C. Could you please clarify what you expect to show/demonstrate with this figure, or explain why the difference of expression level at the R stage between PM/CN and MT/KY is biologically relevant for elucidating the putative role CYPs during the ripening of durian fruit? I think that the pattern of expression of each CYP during the ripening process is more relevant than the total level of expression at the ripe stage. For example, “gene A” has a very high expression level at the ripe stage, but this level remained invariable during the whole ripening process. On the other hand, “gene B” has a lower level of expression compared to “gene A” at the ripe stage; however, the expression of “gene B” has gradually and significantly increased/decreased throughout the ripening process. Could you support that “gene A” is more relevant for the ripening process than a “gene B” based just on their expression level at the ripe developmental stage?

- Also, regarding figure 3, please homogenize “a” as the lowest value and “b” or “c” as the highest. Please, present the data in the order that it is mentioned in the text.

- I have a concern regarding the statistical analysis. Tukey HSD assumes a normal distribution. Was it tested?

-Figure 3B, section CYP88A: Is there no difference between IM1, IM2, M, and MR? or between IM1, IM2, and R in the CYP714E section?

-Lines 311-312: With the provided data, it is impossible to demonstrate that DzCYP88A is involved in GA biosynthesis and plays an essential role in accelerating the fruit ripening process in durian. For this, at least a functional characterization of DzCYP88A must be performed, including substrate tolerance assays and up or downregulation assays by agroinfiltration. Moreover, is there any evidence of the role of GA in durian fruit ripening??

-Line 337: It seems that only CYP714E is upregulated.

-I am not an expert in durian, but as a climacteric fruit, the ripening of durian must be characterized by a burst of ethylene. How would you explain that CYP714E is upregulated during the ripening process but is negatively regulated by the addition of ethephon?

-Line 343: Is there any evidence to support this sentence?

-Line 374: Is there any evidence to support that JA-Ile is a negative regulator of durian ripening?

-Lines 414-46: I am confused by these sentences. First, you present ABA as a positive regulator of ripening in tomato and banana. However, in these two sentences, you say that CYP707A (involved in ABA inactivation) might play a role in accelerating the ripening process.

-Line 425: Is there metabolomic data available to support the affirmation that GA content decreases during durian ripening?

-Line 426: positively regulates.

-Lines 425-429: These assumptions contradict those stated previously in lines 304-312. First, you presented CYP88A as a key gene for GA biosynthesis, induced by ethephon and highly expressed at the ripe developmental stage (stage characterized by high levels of ethylene), speculating with a role of GA in the ripening process. Now, your crosstalk GA-ABA model is based on a decrease of GA content during the ripening.

Reviewer #3: Authors have identified 355 genes related to the cytochrome P450 gene family in Durio zibethinus and were further distributed in 10 groups consisting of 56 families. Additionally, they have also characterized the motifs and phylogenetic analysis. They have further performed the expression analysis and have validated some selected genes using the real-time PCR. The findings of current study represent an important step towards comprehending the molecular regulation of CYPs related to durian fruit ripening. However, the main problem was in the introduction and result and discussion parts. I wrote some my suggestions about this sections;

1. Introduction part; Authors should explain the significance of this study at the end of Introduction part.

2. Materials and methods part; Authors should explain Statistical Analysis in the Materials and methods part.

3. Lines 169-170, 174,…… When providing location details for a vendor, both city and country are mentioned (city, country). In case of US-based vendors, city, state suffice.

4. For the gene family expansion and evolution of novel functions, gene duplication and divergence are essential steps in the plant genome. Authors should add these researches related to Gene Duplication and Syntenic Analysis in revised manuscript.

5. Authors should carefully recheck the manuscript for scientific styles. There are some grammar mistakes; a detailed revision for English is necessary.

6. Authors should consider adding one short paragraph with a conclusion.

7. In the result and discussion section, the authors should add or discuss the following research papers:DOI：10.1007/s13258-013-0170-9 (Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila); DOI: 10.3389/fgene.2020.00044 (The Cytochrome P450 Monooxygenase Inventory of Grapevine (Vitis vinifera L.): Genome-Wide Identification, Evolutionary Characterization and Expression Analysis); DOI：10.1186/s12864-017-4425-8 (Global identification, structural analysis and expression characterization of cytochrome P450 monooxygenase superfamily in rice)

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Reviewer #1: Yes: Axel Poulet

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2021 Nov 30;16(11):e0260665. doi: 10.1371/journal.pone.0260665.r002

Author response to Decision Letter 0

23 Oct 2021

We have attached the response table already.

Table of response to the editor’s comments

Editor’s comments and Authors’ responses

1. For expression analyses, be aware that fold-change values are not normally distributed and thus error bars should represent the upper and lower bounds of the confidence interval, rather than standard deviation/error, and statistical analyses should not assume normal distribution (or be performed on log fold change values which are normally distributed). If fold change is retained, explain clearly in the legend what comparison the fold change represents.

Response - Following the Editor’s comment, the expression analyses have been transformed to log scale and tested for the normal distribution (Kolmogorov-Smirnov and Shapiro-Wilk methods, p=0.05). We have re-calculated the statistical significances and found some slight changes (compared to the previous data set) then we have updated in the main text. In addition, we have been addressed statistical information in the figure legends.

2. Include (or cite another paper that includes) the primers for the reference genes and explain how both were used (e.g. was the geometric mean of both housekeeping genes used, or were these used alone as individual reference genes in different experiments?).

Response - Following the Editor’s comment, the reference papers for housekeeping genes and calculation method have been added in the materials and methods section (line 195) and Table S2.

3. There is displacement of some letters in gene names in S5.

Response - Following the Editor’s comment, the figure S5 has been improved and renamed as S4 Fig.

Table of response to the reviewer’s comments

Reviewer’s comments Authors’ responses

Reviewer #1

1. References for the bioinformatics tools used in this analysis (SATR, tblastn, MEME, htseq clustalW etc) should be included in the materials and methods secion.

Response - Following the reviewer’s comment, we added the references for MEME (line 149), clustalW (line 153), STAR (line 168), HTseq (line 169), and RPKM normalization (line 171).

2. Fig 1.: The legend lacks necessary information, including whether or not the tree is rooted, which external group was used, etc. Also, the leaves could be colored to be easier to read.

Response - Following the reviewer’s comment, we added the sentence “The bars in the trees represent protein relationships of the unrooted tree” in the legend of Fig 1 (line 223). All sequences from durian genome were blasted to the P450 data of a model plant (Arabidopsis thaliana) and malvaceae plants (cotton and cocoa) that share the same family with durian. According to the criteria of the P450 Nomenclature Committee (David Nelson: dnelson@uthsc.edu), we only selected durian sequences that share ≥ 40% identity and consist of major domains of the P450 family. We then unofficially named all putative durian P450s based on the Nelson’s criteria that family share ≥ 40% identity and subfamilies share ≥ 55% identity. Therefore, external P450s may not necessarily be included in these phylogenic trees because durian P450s were generated by alignment with P450s of model plants and we just need to study their distribution of each P450 family in comparison with other plants. In addition, we use the color on the branches to represent the bootstrap percentage. Adding color to the leaves might make it too colorful and make it difficult for the readers. Therefore, we would like to retain the original figure. Please consider our justification.

3. Fig 2.: More detail in the legend would help to explain the figure. Is it showing the degree of conservation of the Durian P450 for each group? How did you compute the degree of conservation? Furthermore, it would be interesting to redo the same figure with Arabidopsis P450 to be able to compare the motif conservation between Durian and Arabidopsis P450s.

Response - Following the reviewer’s comment, we have added more detail in the legend of Fig 2 (line 249-253), indicating the program used to compute the degree of conservation. Generally, Arabidopsis is a model for plant P450 study, and its conserved motifs have been well studied (Bak et al, 2011., in Arabidopsis book). Therefore, it may be redundant to recreate conserved motifs of Arabidopsis. However, regarding the reviewer’s comment, we have mentioned the comparison of conserved motifs between durian and Arabidopsis in line 245-248.

4. The heat-map in Supplementary Fig. S4 should be included in the main body of work. However, it also needs more detail—how did you define these 5 clusters and which method did you use to produce them?

Response - Following the reviewer’s comment, we have moved Fig. S4 to the main body as Fig 3. We have regrouped the P450s in this Fig 3 (from 5 to 16 groups) based on Ward’s clustering algorithm which was computed by MetaboAnalyst 5.0. More details have been added in the legend of Fig 3.

5. In regard to Line 237 “The expression profiles of nine randomly selected DzP450s were validated by RT-qPC:” Why did you not choose at least one or two genes within the clusters that you defined in Fig. S4? The C cluster is over-represented in your figure, and we do not know if the profiles for the other cluster are consistent?

Response - This is because we like to focus on ripening -related P450s of durian. As a result, most of the randomly selected P450s came from groups that tended to highly express during the ripening stages. We have added the phrase "..from the clusters in which the expression increased during the ripening stages.. " to the sentence (line 263-264).

In addition, our previous publications on durian transcription factors and other flavor-related genes demonstrated the consistent of RT-qPCR and transcriptomes of which three libraries were also used in this study (Khaksar and Sirikantaramas, 2020; Sangpong et al., 2021). We hope you accept our justification.

6. Fig. S5: The legend needs to be clearer. You included the “ratio of RPKM-normalized read counts.” Is this an RPKM or a ratio of RPKM, and, if it is a ratio, what did you use as the reference?

Response - We have changed to log (2−ΔCT) and log(RPKM) and added information in the legend of Fig S5 (now S4 Fig).

7. Sup. Fig. S6: It is difficult to understand why the RPKM is between -1 and 1 because the legend is not clear.

Response - The color key bars indicate normalized standard score (Z-score) of each gene expression level. The high-to-low expression level for each gene at various stages was presented in red-to-blue, respectively. We have added more details in the legend of Fig S6 (now S5 Fig).

8. Line 344 "CYP83 is involved in auxin oxidation:" This part only includes published information from other species and no Durian experiments, so you cannot make a claim regarding Durian CYP83 involvement in auxin oxidation. Furthermore, the authors stated that CYP83 has been lost in several plants, so you cannot truly know what the role of this gene is in Durian, as it could have a totally new activity.

Response - Following the reviewer’s comment, we have changed the subtitle to be “CYP83 is possibly involved in auxin oxidation”.

9. Fig. 4C: Is this a fold change or an RPKM? The column title and the color scale legend are not in agreement. Also, the blue and red labels in Fig. 4A and 4B need more explanation, along with 4C.

Response - The colors indicate the expression level of each gene across five stages (higher in red, lower in blue). To simplify the biosynthetic pathways of gibberellin and abscisic acid, we highlight cytochrome P450s in red letters and other enzymes in blue letters. These explanations have been added in the fig legend (now Fig 5).

10. In the section "Durian CYPome reveals phytohormone-crosstalk in the regulation of fruit ripening," there are no results from your analysis. Therefore, this should be included in the conclusion or another part.

Response - Following the reviewer’s comment, the section "Durian CYPome reveals phytohormone-crosstalk in the regulation of fruit ripening" has been moved to the conclusion part.

11. Line 347 “This family has been reported to be lost from several plant genomes, such as rice, tomato, apple, grape, cucumber, strawberry, and watermelon:” if it has been reported you need to include the references.

Response - Following the reviewer’s comment, the reference has been added (line 394).

12. Fig. S7: You need to include the method that was used. Can you add a table with the protein ID used in these different phylogeny tree? Is the tree rooted, and, if yes, with which species? How many branches are on the tree? How did you compute these bootstraps?

Response - Phylogenetic trees were constructed using maximum likelihood as a statistical method in MEGAX software with 100 of bootstrap replication as already mentioned in the method section. Therefore, these were unrooted trees show the protein relationships between plant species. Following the reviewer’s comment, we have been added the method detail in the legend and attached the supplementary table of protein ID for construction of these trees (see S8 Table).

Reviewer #2

1. When the function of a CYP is cited, be careful to always mention in what species this function was studied.

Response - Following the reviewer’s comment, the manuscript has been carefully rechecked and corrected.

2. The expression “ripening stages” used in lines 251, 361, 428 and others is very ambiguous and seems to be used as synonym of ripe developmental stage and late developmental stages in some context, while in the legends is used to refer together to the five ripening stages analyzed.

Response - Following the reviewer’s comment, the definition of developmental stage and ripening stage has been addressed at the first paragraph of gene expression analysis (line 256-259), rechecked, and corrected. We have rechecked to avoid using ambiguous words of stages.

3. According to PLOS Data policy, all the single measurements used for Figure 3 must be available in a table.

Response - Following the reviewer’s comment, all the single measurements used for Figure 3 is available in S7 table.

4. Line 44: Please change “Fruit qualities such as flavor, odor, and color” by “Fruit organoleptic properties such as flavor, aroma, and color.”

Response - Following the reviewer’s comment, we have been changed to be “Fruit organoleptic properties such as flavor, aroma, and color.” (line 52)

5. Lines 64-66: Please avoid the repetitive use of “therefore.”

Response - Following the reviewer’s comment, we have deleted the repetitive therefore in line 72-74.

6. Lines 82-83: I would suggest replacing the expression “ripening repressor” and “ripening activator” with the relation of both compounds with ethylene since these expressions sound too broad.

Response - Following the reviewer’s comment, the expression “ripening repressor” and “ripening activator” have been replaced by “ethylene inhibitor” and “ethylene releaser”, respectively (line 90).

7. Lines 86-89: I suggest rewriting this sentence since it is unclear where or when this P450 burst appears and how it is related to phytohormone crosstalk.

Response - Following the reviewer’s comment, the revised sentence has been addressed in line 94-97.

8. Line 92: Please mention which cultivars correspond to slow- and quick-postharvest in one sentence and not in different sections throughout the text.

Response - Following the reviewer’s comment, cultivars correspond to slow- and quick-postharvest have been addressed in one sentence (line 100 -103).

9. Line 95: immature1

Response - Following the reviewer’s comment, the word “immature1” has been corrected (line 105).

10. Line 96: harvest stage (mature?)

Response - Following the reviewer’s comment, the word “mature” has been addressed (line 106).

11. Line 98: Please mention briefly the firmness values used for defining each developmental stage.

Response - Following the reviewer’s comment, the firmness values used for defining each stage have been addressed.

12. It sounds a bit confusing that three biological replicates were collected in the “Plant material” section. However, in the following section, it is mentioned that five biological replicates were employed. I suggest clarifying that these three biological replicates were stored at -80 °C for qRT-PCR analysis.

Response - Following the reviewer’s comment, the phrase “…stored at −80 °C for transcriptome and RT-qPCR analysis” has been added in the plant material section (line 119).

13. Regarding ethephon and 1-MCP treatments: it is unclear the experimental design. Five replicates were used for each treatment (15 fruits in total?). It is also not mentioned the developmental stage of treated fruits.

Response - Five replicates at mature stage were used for each treatment (15 fruits in total). After 5 days, we collected the treated samples which were then ripe stage. Following the reviewer’s comment, we have been rewriting the section Ethephon and 1-MCP treatments for greater understanding.

14. Please indicate in the legend of figure 3, if the data presented correspond to the mean, and if the error bars correspond to standard deviation or standard error. Besides, indicate the number of biological replicates employed.

Response - Following the reviewer’s comment, the definition of bars, error bars, and the number of biological replicates have been addressed in the legend of Fig 3 (now Fig 4).

15. I do not understand the idea of Figure 3C. Could you please clarify what you expect to show/demonstrate with this figure, or explain why the difference of expression level at the R stage between PM/CN and MT/KY is biologically relevant for elucidating the putative role CYPs during the ripening of durian fruit? I think that the pattern of expression of each CYP during the ripening process is more relevant than the total level of expression at the ripe stage. For example, “gene A” has a very high expression level at the ripe stage, but this level remained invariable during the whole ripening process. On the other hand, “gene B” has a lower level of expression compared to “gene A” at the ripe stage; however, the expression of “gene B” has gradually and significantly increased/decreased throughout the ripening process. Could you support that “gene A” is more relevant for the ripening process than a “gene B” based just on their expression level at the ripe developmental stage?

Response - We found that the expression of gene A increased throughout the ripening stages in both fast ripening cultivar (Phuangmanee) and slow ripening cultivar (Monthong). It was also seen that in midripe and ripe stages, the expression of gene A was significantly higher in fast ripening cultivar (Phuangmanee) compared to slow ripening cultivar (Monthong). This leads to doubt that “Is this gene A highly expressed only for fast-ripening cultivars?” High expression of gene A, especially in fast-ripening cultivars, could lead to the discovery of a molecular key to accelerate ripening in durian fruit. Therefore, we selected another fast-ripening cultivar (Chanee) and another slow-ripening cultivar (Kanyao). We then compared the significance of gene A expression at ripe stage between fast ripening cultivars (Phuangmanee and Chanee) and slow ripening cultivars (Monthong and Kanyao) as represented in Fig 4C.

16. Also, regarding figure 3, please homogenize “a” as the lowest value and “b” or “c” as the highest. Please, present the data in the order that it is mentioned in the text.

Response - Following the reviewer’s comment, these letters have been rearranged in Fig 3 (now Fig 4).

17. I have a concern regarding the statistical analysis. Tukey HSD assumes a normal distribution. Was it tested?

Response - Following the reviewer’s comment, we have transformed the data to log scale and tested for normal distribution (Kolmogorov-Smirnov and Shapiro-Wilk method, p = 0.05) before the statistical analysis.

18. Figure 3B, section CYP88A: Is there no difference between IM1, IM2, M, and MR? or between IM1, IM2, and R in the CYP714E section?

Response - For Fig 3B (now Fig 4B), we have employed t-test (p = 0.05) to calculate the significance between the two cultivars at each ripening stage. The significant differences are indicated by asterisks above each stage.

19. Lines 311-312: With the provided data, it is impossible to demonstrate that DzCYP88A is involved in GA biosynthesis and plays an essential role in accelerating the fruit ripening process in durian. For this, at least a functional characterization of DzCYP88A must be performed, including substrate tolerance assays and up or downregulation assays by agroinfiltration. Moreover, is there any evidence of the role of GA in durian fruit ripening??

Response - Cytochrome P450s with the same subfamily (>55% identity) generally share the same or similar functions. CYP88A encodes ent-kaurenoic acid oxidase and produces GA12 which is the key intermediate for gibberellin biosynthesis. In 1992, Mamat and Wahab detected the presence of endogenous GA4 during the development of durian fruit by GC-MS. The application of GA4 to the pedicel of durian fruits at week 6 after anthesis prevented fruit drop, enhanced fruit development, increased fruit size and weight. Although there is no evidence on how GAs are related to durian ripening, GAs have been found to decrease before ethylene production in climacteric fruit such as tomato. In addition, exogenous GAs resulted in slower fruit ripening due to reduced ethylene content and downregulated ethylene synthesis genes. However, we are functional characterizing the CYP72, CYP88, and CYP714 in model plant and planning to elucidate GA profiles of each stage of durian arils by metabolomic approach. We hope to publish this GA work in the next article. Following the reviewer’s comment, the sentence “DzCYP88A is involved in GA biosynthesis and plays an essential role in accelerating the fruit ripening process in durian” has been adjusted to “DzCYP88A is potentially involved in GA biosynthesis and potentially plays an essential role in accelerating the fruit ripening process in durian” (line 352).

20. Line 337: It seems that only CYP714E is upregulated.

Response - CYP72, CYP88, and CYP714 are upregulated during the ripening stages (see Fig 4B).

21. I am not an expert in durian, but as a climacteric fruit, the ripening of durian must be characterized by a burst of ethylene. How would you explain that CYP714E is upregulated during the ripening process but is negatively regulated by the addition of ethephon? Response - We would like to thank the reviewer for raising this question. We rechecked the information carefully and found an error. In fact, the expression of CYP714E was unchanged when treated with ethephon but was significantly down regulated by 1-MCP. We have edited the fig4 and the content in the main text.

22. Line 343: Is there any evidence to support this sentence?

Response - This is our hypothesis based on the fact that the most active forms are dihydroxylated gibberellins such as GA3. Following the reviewer’s comment, we have added the word “proposed” into the sentence (line 388).

23. Line 374: Is there any evidence to support that JA-Ile is a negative regulator of durian ripening?

Response - Although there is no evidence in durian, it has been reported that JA-Ile level was significant higher in 4 °C-stored Avocado than Avocado stored room temperature and JA-Ile biosynthetic gene is induced by auxin, suggesting a role for ripening-inhibition (Vincent et al., 2020).

Regarding the reviewer’s comment, we have adjusted the sentence to "the expression of DzCYP94B leads to the inactivation of JA-Ile, possibly resulting in the ripening process in durian" (line 420-421).

24. Lines 414-46: I am confused by these sentences. First, you present ABA as a positive regulator of ripening in tomato and banana. However, in these two sentences, you say that CYP707A (involved in ABA inactivation) might play a role in accelerating the ripening process.

Response - ABA induces the expression of ethylene associated genes to trigger the ripening of tomato fruits. When there is sufficient ethylene level for the ripening process, then the level of ABA should be reduced by the increased expression of the CYP707A. As shown in Fig 4, Monthong has a higher expression of CYP707A (low ABA, low ethylene) at MR and R stages compared to Phuangmanee, resulting in slower ripening of Monthong.

25. Line 425: Is there metabolomic data available to support the affirmation that GA content decreases during durian ripening?

Response - We are planning to elucidate GA profiles of each stage of durian fruit by metabolomic approach and planning to publish this GA work in the next article. Although there is no evidence in durian, studies in other climacteric fruits and associated gene expression in durian suggest that GA content is reduced during the ripening process of durian. Following the reviewer’s comment, the sentence “the active GA content may decrease during ripening process of durian” has been replaced (line 470-471).

26. Line 426: positively regulates.

Response - Following the reviewer’s comment, the word “positively” has been added in the sentence (line 471).

27. Lines 425-429: These assumptions contradict those stated previously in lines 304-312. First, you presented CYP88A as a key gene for GA biosynthesis, induced by ethephon and highly expressed at the ripe developmental stage (stage characterized by high levels of ethylene), speculating with a role of GA in the ripening process. Now, your crosstalk GA-ABA model is based on a decrease of GA content during the ripening.

Response - There are at least three P450s involved in the biosynthesis of gibberellins. The first P450 is CYP88, which is involved in the biosynthesis of the intermediate GA12 (well-recognized as inactive form), the key precursor producing more than a hundred of gibberellins. The other two P450s, CYP72 and CYP714, are normally involved in the conversion of active GAs to inactive GAs, resulting in a decrease in active GA level. Regarding the reviewer’s comment, the word “the active GA” has been added in the sentence (line 471).

Reviewer #3

1. Introduction part; Authors should explain the significance of this study at the end of Introduction part.

Response - Following the reviewer’s comment, the significance of study has been added at the end of introduction part (line 94-97).

2. Materials and methods part; Authors should explain Statistical Analysis in the Materials and methods part.

Response - Following the reviewer’s comment, the section of statistical analysis has been added in the materials and methods part (line 197-203).

3. Lines 169-170, 174,…… When providing location details for a vendor, both city and country are mentioned (city, country). In case of US-based vendors, city, state suffice.

Response - Following the reviewer’s comment, the location details for vendors have been edited.

Response - As we currently only have draft genome of durian, the exact number of chromosomes is still unknown, so the analysis of P450 duplication and syntenic may provide incomplete information. However, genome-wide analysis for P450 gene identification, transcriptome and gene expression studies during the developmental/ripening stages and the exogenous treatments of ripening agents are sufficient to predict gene function and sufficient to select the gene of interest for further functional characterization.

5. Authors should carefully recheck the manuscript for scientific styles. There are some grammar mistakes; a detailed revision for English is necessary.

Response - The manuscript has already been edited by an English language editing service (the certificate has been attached). Moreover, following the reviewer’s comment, the manuscript has been rechecked and corrected.

6. Authors should consider adding one short paragraph with a conclusion.

Response - Following the reviewer’s comment, the conclusion has been revised to be more succinct.

Response - All three research papers are good articles and include gene duplication and syntenic section. As we have mentioned above, we do not yet know the exact number of chromosomes in durian so it may not be suitable for duplication and syntenic analysis at this stage. And the information we have in this study is sufficient to annotate gene function and enable the selection of candidate genes of interest for further functional characterization. Please note that the grape and rice papers were already cited in the introduction part. We did not cite the fungi paper because there are sufficient references for plant P450s in the result and discussion parts and the fungi paper may not be relevant to our study. Please consider our justification.

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PLoS One. doi: 10.1371/journal.pone.0260665.r003

Decision Letter 1

Frances Sussmilch

10 Nov 2021

PONE-D-21-24817R1Genome-wide identification and expression profiling of durian CYPome related to fruit ripeningPLOS ONE

Dear Dr. Sirikantaramas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not yet 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.

Please address the following points prior to resubmission:

Table S7a, S7b, S7c should be separate supp tables (S7, S8, S9).
Fig 1 legend (L223 track changes version) – please reword to clarify what you mean by “bars in the trees”
Fig 4
- Bar charts are not logical with these x axes (esp. as y intercept not at 0) – please change to an appropriate alternative (e.g. scatter charts, box plots, violin plots)
- Please make the x axis labels consistent (can also remove unnecessary repetition) – e.g. “Relative Expression (log FC)” together with an explanation of calculation in the methods would be sufficient.

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: In the current work, Suntichaikamolkul et al present a deep mining of the Durian genome and transcriptome libraries leading to the identification of all P450s that are potentially involved in Durian fruit ripening. They provide insight of the probable activity of the different P450s clan. In this current version all the comments made during the first round of review has been addressed.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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Reviewer #1: Yes: Axel Poulet

Reviewer #2: No

Reviewer #3: No

PLoS One. 2021 Nov 30;16(11):e0260665. doi: 10.1371/journal.pone.0260665.r004

Author response to Decision Letter 1

11 Nov 2021

Editor's comments

1. Table S7a, S7b, S7c should be separate supp tables (S7, S8, S9).

Response: Following the Editor’s comment, the tables S7a-S7c have been changed to S7 Table, S8 Table, and S9 Table, respectively.

2. Fig 1 legend (L223 track changes version) – please reword to clarify what you mean by “bars in the trees”

Response: Following the Editor’s comment, we have adjusted the sentence to " The scale bars in the circular trees represent the number of amino acid substFig 4

3. Bar charts are not logical with these x axes (esp. as y intercept not at 0) – please change to an appropriate alternative (e.g. scatter charts, box plots, violin plots)

Please make the x axis labels consistent (can also remove unnecessary repetition) – e.g. “Relative Expression (log FC)” together with an explanation of calculation in the methods would be sufficient.

itutions per site" instead (line 223).

Response: Following the Editor’s comment, figure 4 has been changed. The labels of the y-axis and the legend of Fig 4 have been rewritten to be more concise.

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PLoS One. doi: 10.1371/journal.pone.0260665.r005

Decision Letter 2

Frances Sussmilch

15 Nov 2021

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

PONE-D-21-24817R2

Dear Dr. Sirikantaramas,

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.

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Frances Sussmilch

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0260665.r006

Acceptance letter

Frances Sussmilch

17 Nov 2021

PONE-D-21-24817R2

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

Dear Dr. Sirikantaramas:

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.

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on behalf of

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PLOS ONE

Associated Data

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

Supplementary Materials

S1 Fig. Morphology of durian fruit (Monthong cultivar).

(A) whole fruit. (B) peeled fruit. (C) Arils across five developmental and ripening stages. Stage abbreviations: IM1, immature 1; IM2, immature 2; M, mature; MR, mid-ripe; R, ripe.

(TIF)

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S2 Fig. Correlation between RNA-based expression profile and RT-qPCR validation of selected P450s in Monthong cultivar across five stages.

(TIF)

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S3 Fig. RNA-seq based expression profile of durian P450s.

(TIF)

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S4 Fig. Phylogeny of selected plant cytochrome P450s involved in phytohormone biosynthesis.

(TIF)

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S1 Table. Primer list for RT-qPCR.

(PDF)

Click here for additional data file.^{(79.6KB, pdf)}

S2 Table. Comparison of P450 families in durian and selected flowering plants in the reversed order of angiosperm evolution.

(PDF)

Click here for additional data file.^{(124KB, pdf)}

S3 Table. Normalized Ct values of the genes in durian arils cv. Monthong control (natural ripening), 1-MCP treatment, and ethephon treatment.

(PDF)

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S4 Table. Normalized Ct values of the genes in durian arils (two cultivars of each stage).

The abbreviations N/D indicate no detectable Ct.

(PDF)

Click here for additional data file.^{(91.3KB, pdf)}

S5 Table. Normalized Ct values of the genes in durian arils (four cultivars at ripe stage).

(PDF)

Click here for additional data file.^{(80.9KB, pdf)}

S6 Table. List of proteins used for construction of phylogeny of selected plant cytochrome P450s involved in phytohormone biosynthesis.

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Data Availability Statement

The nucleotide sequences reported in this article have been submitted to Genbank under accession numbers [PRJNA683229 and PRJNA732556].

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PERMALINK

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

Nithiwat Suntichaikamolkul

Lalida Sangpong

Hubert Schaller

Supaart Sirikantaramas

Roles

Abstract

Introduction

Materials and methods

Plant materials

Ethephon and 1-MCP treatments

Identification and classification of durian P450 genes

Conserved motif analysis

Phylogenetic analysis

Transcriptome analysis of Musang king and Monthong cultivars

RT-qPCR analysis

Statistical analysis

Results and discussion

Identification, classification, and conserved motifs of durian P450s

Fig 1. Phylogenic tree of durian cytochrome P450s.

Fig 2. Comparison of sequence logos for conserved motifs of the predicted P450 proteins, A-type and non-A type, in durian.

Gene expression analysis reveals putative DzP450s related to fruit ripening

Fig 3. RNA-seq based expression profile of durian P450s during the five stages of the Monthong cultivar.

Ripening-related cytochrome P450s potentially regulate phytohormones in durian fruit

CYP72, CYP88, and CYP714 are involved in gibberellins biosynthesis

Fig 4. Relative expression of durian P450s.

Fig 5. Proposed reaction and RNA-seq based expression profiles involved in phytohormone biosynthesis.

CYP83 is possibly involved in auxin oxidation

CYP94s involved in the catabolism of jasmonate and its conjugates (JAs)

CYP707 is involved in the catabolism of abscisic acid (ABA)

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Frances Sussmilch

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Author response to Decision Letter 0

Decision Letter 1

Frances Sussmilch

Roles

Author response to Decision Letter 1

Decision Letter 2

Frances Sussmilch

Roles

Acceptance letter

Frances Sussmilch

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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