Understanding the Complex Functional Interplay between Glucosinolates and Cyanogenic Glycosides in Carica papaya

Abstract

Glucosinolates (GSLs) and cyanogenic glycosides (CGs) fulfil functions in plant defence and have been reported to be anticancer agents. Generally, GSL-containing plants do not produce CG, and vice versa, CG-containing plants do not synthesise GSLs. However, the production of both GSL and CG compounds was observed in Carica papaya. Additionally, several studies found both GSL glucotropaeolin and CG prunasin in papaya leaves. The advancement of genome technologies can be explored to elucidate the gene functions and other molecular discoveries in plants that might relate to GSLs and CGs. This review aims to discuss the complex interplay of the rare events whereby these two compounds (GSL and CG) co-occur in a bifurcation pathway in papaya. To our knowledge, this is the first review that highlights novel GSL and CG genes in papaya. Furthermore, species-specific pathways in papaya are also discussed and comprehensively described. The transcription factors involved in regulating GSL and CG biosynthesis pathways are also discussed, accompanied by relevant bioinformatic approaches that can help discover potential regulatory genes that control the production of prunasin and glucotropaeolin in papaya.

1. Introduction

Plants are sessile organisms that produce up to one million metabolites and compounds [1,2]. These metabolites can be used in various industries such as food, agriculture, medicine, and cosmetics [1,2,3]. Furthermore, the secondary (or specialised) metabolites contribute to an effective defence against biotic and abiotic stresses [4]. Terpenes, phenolics, and nitrogen-containing compounds (NCCs) are the three main chemical classes of plant secondary metabolites. Some of the compounds have bitter-tasting properties in nature [5]. Glucosinolates (GSLs) and cyanogenic glycosides (CGs) are examples of bitter-tasting NCCs that are known to participate in essential defence mechanisms in plants [6,7]. The co-occurrence of GSL and CG in a single plant species was initially thought to be due to the presence of contaminant extracts, as the secondary metabolites were perceived as mutually exclusive [8]. However, several articles reported the existence of both CG and GSL in Carica papaya [8,9,10].

GSLs have been found in 16 angiosperm plant species, especially in the Brassicaceae family, including Arabidopsis thaliana. GSLs are responsible for the bitter flavours of Brassica vegetables, including turnip (Brassica rapa ssp. rapa), broccoli (Brassica oleracea var. italica), and cauliflower (Brassica oleracea var. botrytis) [11]. Previous studies have also reported the GSL content in other Cruciferae, Cleomaceae, and Caricaceae species [12,13,14,15,16]. Currently, 130 GSL structures have been identified in GSL-containing plants [17]. GSLs are derived from several amino acids, and depending on their amino acid precursor, GSLs are categorised into three types: aliphatic GSLs from methionine (Met), indolic GSLs from tryptophan (Trp), and benzyl GSLs from phenylalanine (Phe) and tyrosine (Tyr).

These sulfur- and nitrogen-containing compounds and their degradation products are known for their role in plant defence against fungi, bacteria, pests, and insects [12,18]. In the model plant A. thaliana, GSLs can be found in various organs, including seeds, roots, stems, and leaves [12,19]. Different GSL-containing plant organs have different GSL compositions, both quantitatively and qualitatively. Furthermore, the concentrations of GSL in roots are often higher than in shoots [20]. In these GSL-containing plants, a myrosinase enzyme known as β-thioglucosidase in different plant organelles will convert the inactive form of GSL into bioactive GSL hydrolytic products, such as isothiocyanates, nitriles, thiocyanates, and epithionitriles [21,22]. These bioactive compounds are produced upon pest infestation or physical processes, leading to tissue disruption [23].

Multiple studies have shown the therapeutic potential of GSL-derived compounds in various cancer treatments. For example, a bioactive compound, namely Indole-3-carbinol (I3C) produced from indolic GSL, is commercially used as a supplement for recuperating breast cancer patients [24]. Other studies have also provided supporting evidence on the capability of I3C to inhibit tumour invasion, metastasis, and cell-cycle progression in breast [25] and prostate [26] cancer cells. A couple of years later, I3C was shown to induce apoptosis in colorectal [27] and osteosarcoma [28] cell lines. In another example, sulforaphane, a bioactive compound derived from aliphatic GSL, has shown a potential to inhibit the carcinogenic cells of various malignant cancers, such as breast [29], prostate [30], liver, lung [31], and pancreas [32] cancers in rodents. Another type of bioactive GSL is benzyl isothiocyanate (BITC), which is produced from benzyl GSL or glucotropaeolin. This active compound is capable of suppressing cancer progression, including colon [33], breast [34,35,36], and pancreatic [37] tumours. Interestingly, BITC was suggested as a therapeutic agent to treat bacterial infection, potentially inhibiting the toxin production and growth of enterohemorrhagic Escherichia coli [38].

Cyanogenic glycosides (CGs) are another example of NCCs, known as glycosides of α-hydroxynitriles. CGs and GSLs have similar characteristics. Firstly, CGs are also produced from amino acids, including L-valine, L-isoleucine, L-leucine, L-phenylalanine, or L-tyrosine [39]. Secondly, CGs do not cause toxic effects in their intact form but will produce a bioactive compound, such as toxic cyanide (e.g., hydrogen cyanide) upon tissue damage due to the activity of a digestion enzyme [35], resulting in symptoms associated with cyanide poisoning when consumed in high amounts [40]. Ingestion of hydrogen cyanide can lead to intoxication and possible death if consumed at high concentrations. This mechanism protects plants from potential threats (e.g., insects and animals) and ensures plant survival [41]. However, while GSLs are found explicitly in Brassica and other related families, CGs are one of the most extensive classes of secondary plant metabolites found in more than 2600 plant species, with 112 CGs characterised [42]. The general chemical structures of GSL and CG are shown in Figure 1.

The occurrence of CGs is more widespread across the Plantae region than GSLs [43]. Several essential crops contain CGs, such as cassava (Manihot esculenta), sorghum (Sorghum bicolor), and barley (Hordeum vulgare) [41]. To date, dhurrin biosynthesis in sorghum is the most comprehensively studied tyrosine-derivative, also through the cloning and characterisation of the genes being involved [41]. Amygdalin and linamarin are other CGs primarily identified in sorghum and cassava plants [44,45]. Linamarin is a cyanogenic chemical present in the roots and leaves of cassava plants [46]. Thus, eating fresh cassava can produce cyanide poisoning in humans due to the degradation of linamarin by ß-glucosidase enzymes [47].

Amygdalin has been extensively studied for its potential application in human-targeted cancer treatment [48]. However, oral amygdalin treatment causes cyanide poisoning problems, such as decreased consciousness in pancreatic cancer patients [49]. While amygdalin can occasionally trigger cyanide poisoning, in vitro and in vivo evidence also suggests some therapeutic benefits in cancer treatment [50]. Furthermore, such cyanide toxicity can be utilised in innovative cancer-target therapy [51]. Targeted therapy objectively attempts to deliver drugs to particular tumour areas while limiting the side effects on healthy tissues [52].

Identifying genes involved in the GSL and CG biosynthesis pathways could provide further insights for the benefit of various applications, including genetic engineering, to manipulate compounds for relevant industry purposes, such as medical and agricultural uses [41]. For instance, CG compounds could be introduced as biopesticides to control diseases caused by the invasion of pests [8]. In addition, lowering the CG content in cassava would improve the food safety level for human consumption [53]. Similarly, molecular studies in GSL biosynthesis aimed to increase the synthesis of beneficial GSLs such as glucoraphanin [13,54] and reduce other GSLs that contribute to poor taste in crucifers [55]. Additionally, identifying genes would aid future research for cancer treatment [41]. A previous study found two essential enzymes, cytochrome P450 and UDP-glucosyl transferase, that have become vital factors to synthesise both compounds [41,56]. Additionally, some regulators and genes associated with the cytochrome P450 (CYP) enzymes have also been identified [56,57,58]. Furthermore, the CYP79 enzymes have been described in catalysing the rate-limiting steps in CG and GSL biosynthesis [58]. Hence, in this review, we highlight the co-occurrence of GSL and CG in papaya. We then construct the biosynthesis pathways of GSL and CG in papaya using bioinformatics to explore the upstream intermediates involved in their synthesis. Such information is valuable for basic plant science and genetic crop improvement and beneficial for food and agricultural industries and for medicine and cosmetics.

2. The Identification of Glucosinolate (GSL) and Cyanogenic Glycoside (CG) in Papaya

Carica papaya, generally known as papaya, is a significant tropical fruit consumed worldwide [59]. The essential nutrients produced by papaya facilitate the initial papaya development to secure several aspects, including the growth of various parts of the plant, including its foliage, trunks, and roots, leading to higher papaya productivity [60,61]. This tropical plant can be used as an alternative medicine since the leaves, fruits, stems, seeds, and roots could be used for an alternative medical treatment of various disorders, including cancers, ulcers, and gastritis [62]. Furthermore, secondary metabolites that provide essential nutrients for human health are abundant in papaya. Previous metabolomics studies have discovered carotenoids and tocopherols in the papaya seeds with antioxidant activity [63,64]. Papaya is a diploid plant, and the reported genome size is 372 Mb [65,66]. While GSL and CG biosynthesis pathways have been studied in cassava, sorghum, bitter almonds, and Brassicaceae vegetables, to the best of our knowledge, the biosynthesis pathways of both GSL and CG in papaya have not been described comprehensively in any accessible publication. However, Harun et al. [7] extensively reviewed a comprehensive inventory of GSL biosynthetic genes in A. thaliana, whereby the homologs in the Brassicaceae can be retrieved from SuCComBase [67]. Therefore, by using the reference genes from known GSL and CG plants, the homologous genes could be identified in papaya using bioinformatic approaches. This information helps to construct the respective GSL and CG pathways in papaya.

Specifically, this crop has been shown to produce prunasin (CG) and benzyl GSL, also known as glucotropaeolin (GSL) [68]. Hence, it is suggested that the Carica species is unique, as these two compounds co-occur, synthesised in a bifurcation pathway [69].

Table 1. Glucotropaeolin and prunasin in Brassica and papaya.

GSL Compound	Structural Formula	Plant Species
Glucotropaeolin		Arabidopsis thaliana [7] Brassica oleracea [70] Carica papaya [10]
Prunasin

shows the chemical structure of glucotropaeolin and prunasin, which can also be found in A. thaliana and Brassica oleracea. However, the concentration level of this compound in different plant species can be variable. A study conducted in Brassica oleracea found a significant increment of glucotropaeolin concentrations in the organic plants compared to the conventional breeding approach [70]. For instance, the glucotropaeolin content in A. thaliana was only reported in transgenic Arabidopsis rosette leaves expressing CYP79A2 under the control of the CaMV35S promoter. In contrast, the rosette leaves of wild-type plants did not contain detectable amounts of this type of GSL [71].

According to chromatograms, the initial report on the identification of CG and GSL was conducted on the dried papaya leaves, in which the CG was deficient [72]. A couple of years later, Bennet et al. [10] measured cyanide concentration as a proxy to estimate CG in the papaya plant. They reported variations in the distribution pattern of cyanide in papaya organs, in which the concentrations were highest in the young leaves and the tap root, with declining concentration as the leaves age. Similarly, the highest concentration of glucotropaeolin was recorded in the youngest papaya leaves. A similar compound was observed in other plant parts, such as the roots, leaf stalks, and stem internodes. [10]. In another study, a degradation product of glucotropaeolin, benzyl isothiocyanate (BITC), was identified in papaya. BITC was first found in the seeds of papaya [73] and later in its pulp [74,75]. BITC is a bioactive compound with medicinal and pharmacological properties [8]. Furthermore, several studies reported the capability of BITC to suppress mammalian carcinogenic cells [76].

Phenylalanine, a bitter amino acid [71], is the crucial precursor for papaya CG and benzyl GSL biosynthesis pathways. A recent study compared the correlation between phenylalanine, GSL, BITC, and CG and the bitter taste at different temperatures in papaya. The bitterness intensity is maximum in unripe fruit and gradually decreases as it matures. Furthermore, the bitterness intensity in cool-season fruits is more significant than in warm-season fruits. In their study, Jioe et al. [9] corroborated the previous findings where phenylalanine served as CG and GSL precursor. Based on their calculated correlation values, they also suggested that GSL was not the only component that generated a bitter taste in immature papaya fruits [9].

3. The Construction of Glucosinolate (GSL) Biosynthesis Pathway in Papaya

The GSL biosynthesis pathway has been elucidated by identifying various biological factors involved, such as regulators [77,78,79,80,81,82], enzymes [83,84,85], and protein transporters [86,87,88,89] that also seem to participate in the cross-talk with other essential metabolic processes such as phenylpropanoids, sulfur, and nitrogen in A. thaliana [90,91]. A comprehensive set of 113 known GSL genes were identified from the literature and using public pathway databases, encoding for transcriptional regulators, enzymes, and protein transporters [7]. Generally, GSL biosynthesis comprises several groups of genes initiated by the transcription factors that regulate the production of various secondary metabolites and GSL derivatives. For instance, six MYB genes originating from the R2R3-MYB transcription factor family have been described as regulating the production of GSL in A. thaliana. MYB34, MYB51, and MYB122 control the production of indolic GSLs, whereas MYB28, MYB29, and MYB76 control the production of aliphatic GSLs [92,93]. However, the transcription factor that controls benzyl GSL biosynthesis in A. thaliana is still unclear since the production of glucotropaeolin can only be detected in the engineered lines of Arabidopsis [71]. The ultimate step in GSL biosynthesis is the core structure synthesis, in which most of the biosynthetic genes involved in the indolic and benzyl GSLs are similar. In indolic GSL biosynthesis, CYP79B3 (tryptophan N-monooxygenase 2) catalyses the derivation of tryptophan, whereas CYP79A2 (phenylalanine N-monooxygenase) prepares the phenylalanine substrate in benzyl GSL (glucotropaeolin) biosynthesis [71].

Then, CYP83B1 (CYP83B1 monooxygenase) converts both tryptophan-derived and phenylalanine-derived acetaldoximes into aci-nitro compounds [94]. The remaining steps of GSL core structure formation involve several GSL biosynthetic enzymes that accommodate all GSL precursors regardless of their associated side chains. In this step, the S-alkylthiohydroximates are converted to thiohydroximic acids in a reaction catalysed by SUR1 (C–S lyase) [95]. In the glucosylation process, UGT74B1 was suggested to metabolise thiohydroximates based on the enzyme’s in vitro and in vivo analysis [96]. The final step in the GSL core structure synthesis is the sulfation process of the desulfoglucosinolates to form intact glucosinolates involving the cytosolic sulfotransferase group (ST5a), such as SOT16, SOT17, and SOT18. The biochemical characterisation of sulfotransferases suggests the role of SOT16 to metabolise phenylalanine- and tryptophan-derived desulfoglucosinolates, such as glucobrassicin (3-indolylmethyl GSL) [97,98].

To identify GSL genes and reconstruct the biosynthesis pathway in papaya, we used the available information on the known gene-encoded enzymes in A. thaliana phenylalanine-derived GSL core structure biosynthesis, as it is an important precursor for pathways leading to both benzyl GSL and CG. The gene list was set as queries to identify the homologous genes in the papaya genome.

Table 2. Novel benzyl GSL biosynthetic genes identified in papaya using BLASTp.

Query Gene	Description	Gene	E-Value	Per. Identity	Accession
CYP79A2	tyrosine N-monooxygenase-like	CYP79A1 *	0.00	55.78	XP_021889957.1
	tyrosine N-monooxygenase 2-like	CYP79B3	0.00	57.99	XP_021887085.1
	tryptophan N-monooxygenase 1-like	CYP79B3	0.00	54.13	XP_021887088.1
CYP833B1	cytochrome P450 83B1	CYP83B1	0.00	69.78	XP_021902795.1
	cytochrome P450 71B35-like	CYP71B35	4.00 × 10−135	43.87	XP_021901245.1
	LOW-QUALITY PROTEIN: cytochrome P450 71B35-like	CYP71B35	1.00 × 10−133	43.94	XP_021901202.1
SUR1	S-alkyl-thiohydroximate lyase SUR1	SUR1	0.00	64.09	XP_021900833.1
	tyrosine aminotransferase-like	TAT	0.00	62.79	XP_021900789.1
	probable aminotransferase TAT2	At5g53970	0.00	52.94	XP_021911536.1
UGT74B1	UDP-glycosyltransferase 74B1-like	UGT74B1	1.00 × 10−179	56.89	XP_021908832.1
	UDP-glycosyltransferase 74B1-like	UGT74B1	7.00 × 10−162	53.26	XP_021908582.1
	UDP-glycosyltransferase 74E2-like	UGT74E2	2.00 × 10−137	48.28	XP_021894659.1
ST5a	LOW-QUALITY PROTEIN: cytosolic sulfotransferase 16-like	SOT16	1.00 × 10−148	66.56	XP_021909632.1
	LOW-QUALITY PROTEIN: cytosolic sulfotransferase 15-like	SOT15	7.00 × 10−76	41.99	XP_021912467.1

* CYP79A2 should be the GSL biosynthetic gene encoding enzyme; however, the gene cannot be found in papaya.

shows the identified homologous benzyl GSL genes using protein sequences searched for with the BLASTp program via the NCBI database (accessed on 16 February 2022).

Based on Table 2, the homologous GSL genes identified in papaya showed more than 40% sequence identity with an e-value ranging from 7.00 × 10⁻⁷⁶ to 0.00 with the query sequence of GSL genes from Arabidopsis. The identified novel GSL biosynthetic genes were then used to construct the glucotropaeolin pathway generated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp/) (accessed on 18 February 2022). The primary biosynthetic genes encoding enzymes in GSL core structure synthesis are shown in Figure 2. However, CYP79A1 was identified in the BLASTp analysis instead of CYP79A2, which catalyses the phenylalanine substrate in the production of glucotropaeolin [71].

To elucidate the potential of regulatory mechanisms involved in GSL formation in papaya, bioinformatic analysis of MYB transcription factor genes was conducted with reference to the six known MYB genes from A. thaliana. In addition, the BLASTp (accessed on 22 February 2022) analysis of Arabidopsis against the genome sequences of papaya and its closely related species showed the existence of several MYB orthologous genes in papaya.

To determine the evolutionary relationship among MYB genes in A. thaliana, C. papaya, and other closely related species, a phylogenetic tree was constructed for the 26 selected MYB genes in MEGA version 11.0 [99] using the Maximum-Likelihood method with 1000 bootstrap replicates (accessed on 22 February 2022). In addition, the conserved motifs of MYB genes from A. thaliana, B. rapa, B. oleracea, and C. papaya were also analysed using Multiple Expectation Maximisation for Motif Elicitation (MEME) version 5.4 (https://meme-suite.org/meme/tools/meme, accessed on 24 February 2022) [100] according to the following parameters: site distribution was set to zero or one occurrence (zoops), the maximum number of motifs for searching was set to 10, and the motif width was set between 6 and 50.

The phylogenetic analysis suggested that these MYB genes could be divided into three major clades: indolic GSL, aliphatic GSL, and MYB-like family (Figure 3). Motif identification revealed five conserved motifs (Motifs 1, 2, 3, 4, and 10) present in all MYB genes, highlighting the conservation of MYB motifs in these species (Figure 4). Motif 1 and Motif 2 were both 50 bp in length, containing the DNA-binding domains of Myb proteins and the SANT domain family specifically involved in the transcriptional regulation [101]. The indolic GSL clade contained the evm.model.supercontig 3.239 gene sequence from C. papaya and several MYB orthologs from Brassicaceae, and the group can be represented by Motif 5, which belonged to the Myb-like DNA binding domain (Figure 4). Interestingly, the evm.model.supercontig 3.239, which has been annotated as Myb domain protein 122, also contained Motif 7 (Myb-like DNA-binding domain) specifically found in the aliphatic GSL clade. Based on this finding, papaya is speculated to possibly regulate the aliphatic GSL biosynthesis as well; further investigation is needed.

Meanwhile, the MYB-like family contains MYB orthologs from both Brassica species and papaya but lacks representation of specific (aliphatic or indolic) GSL motifs. This could suggest that the eight MYB genes in the MYB-like family may undergo rapid functional divergence and do not carry any specific function in the GSL biosynthesis.

4. The Construction of Cyanogenic Glycoside (CG) Biosynthesis Pathway in Papaya

In general, the biosynthesis pathway of CG can be described in three processes. In the initial step, an enzyme from the cytochrome P450 family will convert the α-amino acids via N-hydroxylation to an N-hydroxylamino acid that will eventually be converted to aldoxime. Next is the conversion of the aldoxime to cyanohydrin as catalysed by another member of the cytochrome P450 family. Lastly, the cyanohydrin molecules are glycosylated by a soluble enzyme, UDP-glucosyltransferase a [39,41]. The detailed analysis of the biosynthesis pathway indicated that it has evolved from the pre-availability of CGs, compounds being widely distributed among plant kingdoms. The independent evolution of GSL from an ancient CG is suggested to occur through the evolution of ancestral CYP enzymes capable of metabolising reactive oximes [102].

Prunasin, one of the major CGs, is commonly found in stone fruits such as apricot (Prunus armeniaca) [103], peach (Prunus persica) [104], and bitter almond (Prunus dulcis) [105]. CGs such as amygdalin and prunasin contribute to the bitterness of almonds. The prunasin biosynthesis in P. dulcis involves three enzymatic reactions catalysed by three gene-encoded proteins: PdCYP79D16, Pd71AN24, and PdUGT94AF3 [57]. Here, we used the established CG genes from P. dulcis as queries to identify the homologous prunasin biosynthetic genes in papaya using BLASTp (accessed on 25 February 2022) (

Table 3. Novel biosynthetic CG genes identified in papaya using BLASTP.

Query Gene	Description	Gene	E-Value	Per. Identity	Accession
CYP79D16	tryptophan N-monooxygenase 2-like	LOC110806501	0.00	57.7	XP_021887085.1
	tryptophan N-monooxygenase 1-like	LOC110806504	0.00	58.13	XP_021887088.1
	tyrosine N-monooxygenase-like	LOC110808696	0.00	56.82	XP_021889957.1
CYP71AN24	cytochrome P450 71A1-like	LOC110810705	5.00 × 10−162	52.13	XP_021892654.1
	cytochrome P450 71B35-like	LOC110817138	5.00 × 10−136	46.02	XP_021901245.1
	cytochrome P450 71D10-like	LOC110808646	7.00 × 10−132	45.3	XP_021889891.1
UGT94AF3	beta-D-glucosyl crocetin beta-1,6-glucosyltransferase-like	LOC110819221	1.00 × 10−161	55.11	XP_021904037.1
	putative UDP-rhamnose: rhamnosyltransferase 1	LOC110816080	6.00 × 10−76	35.38	XP_021899800.1
	anthocyanidin 3-O-glucosyltransferase-like	LOC110821584	5.00 × 10−64	32.02	XP_021907153.1

CYP79D16, CYP71AN24 and UGT94AF3 should be the CG biosynthetic gene encoding enzymes; however, the gene cannot be found in papaya.

).

Table 3 shows the identified homologous biosynthetic CG genes in papaya that are likely involved in the prunasin biosynthesis. The novel CG genes in papaya share more than 30% sequence identity and e-value ranging from 5.00 × 10⁻⁶⁴ to 0.00 with the reference CG genes identified in P. dulcis. The three identified CG genes were mapped onto the prunasin biosynthesis pathway generated from the KEGG database (Figure 5) (accessed on 25 February 2022).

The basic helix-loop-helix (bHLH) transcription factors are essential in regulating CG biosynthesis. The mutation of bHLH transcription factor gene clusters located at the Sweet kernel (Sk) locus of the P. dulcis disrupted the regulation of CG biosynthesis, thus leading to a reduced CG content in the plant and resulting in the sweet kernel trait.

To elucidate the potential involvement of prunasin biosynthesis in papaya and the possible evolutionary relationship of bHLH genes between Prunus species and papaya, the bioinformatics analysis of bHLH transcription factor genes was conducted in reference to five bHLH genes from P. dulcis [105]. The BLASTp analysis (accessed on 28 February 2022) of P. dulcis bHLH genes against the genome sequences of P. persica and papaya revealed two and seventeen orthologous bHLH genes in papaya and P. persica, respectively. The evm.model.supercontig_141.19 and evm.model.supercontig_1892.1 genes in papaya share 41–50% sequence identity and e-value, ranging from 1.8 × 10⁻¹²⁸ to 3.6 × 10⁻⁸⁰ with bHLH1, bHLH2 and bHLH4 from P. dulcis.

A phylogenetic tree was constructed for the five bHLH sequences from P. dulcis and ten non-redundant orthologous sequences from BLASTp hits in MEGA version 11.0 [99] using the Maximum-Likelihood method with 1000 bootstrap replicates (accessed on 28 February 2022). The protein sequences were previously aligned using ClustalW. Furthermore, the conserved motifs of bHLH genes from P. dulcis, P. persica, and papaya, were also analysed using Multiple Expectation Maximisation for Motif Elicitation (MEME) version 5.4 (accessed on 1 March 2022) according to the above-mentioned parameters (refer to Section 3). In addition, the conserved motifs for these genes were determined using the ScanProsite interface in the PROSITE web server [106].

The phylogenetic analysis suggested that these genes may be divided into three clades: bHLH1-bHLH2 group, bHLH4 group, and MYC-like family (Figure 6). The evm.model.supercontig_141.19 and evm.model.supercontig_1892.1 genes in papaya belong to the Myc-like family cluster with the absence of several motifs dominated by bHLH genes from P. dulcis. These bHLHs probably got lost during the process of evolution.

Motif identification revealed the presence of Myc-type, basic helix-loop-helix (bHLH) domain profile (Prosite ID: PS50888) in all sequences except the bHLH1 from sweet almond variant (bHLH1sweetLauranne), which also reflected the presence of Motif 2 that contained 50 amino acids in MEME motif profiles (Figure 7, coloured in cyan). The absence of the bHLH domain in the bHLH1sweetLauranne gene sequence is in accordance with the point mutation occurring in the gene that prevents the transcription of P450 genes involved in the amygdalin biosynthesis pathway [105]. Thus, the presence of the bHLH domain in evm.model.supercontig_141.19 and evm.model.supercontig_1892.1 from papaya could potentially indicate the presence of a CG biosynthesis pathway. Interestingly, the evm.model.supercontig_141.19 also contained an additional domain, the ACT domain (Prosite ID: PS51671), which has not been detected in other sequences. The domain is predicted to be a regulatory domain for small ligand binding such as amino acids and is often involved in protein dimerisation [107], but the role of the ACT domain in papaya remains elusive.

Additionally, the MEME motif profiles (Figure 6 and Figure 7) also showed that these sequences exhibited three highly conserved motifs, Motif 1 (37 amino acids, red), Motif 3 (41 amino acids, green), and Motif 10 (15 amino acids, yellow); thus proposing the importance of these sequence fragments for the CG biosynthesis pathway, thus causing their preservation throughout the evolutionary process. Furthermore, further characterisation of Motif 3 in MEME predicted that the motif could represent a nitrilase/cyanide hydratase signature (ProSite ID: PS00921). The sequence signature is commonly found in enzymes associated with the degradation of nitriles and cyanides.

5. Future Perspectives and Concluding Remarks

Although GSL and CG have been extensively described in Brassicaceae and almonds, respectively, the molecular details of these compounds in papaya are still lacking. This review exemplifies the functional interplay of GSL and CG in papaya using the genomics data and bioinformatics approach. In our study, comparative genomics analysis and construction of the GSL and CG biosynthesis pathways have revealed several candidate genes and transcription factors (TFs) potentially involved in the GSL and CG biosynthesis pathways. Results from this study provide new insights into the biological process for the candidate genes and TFs that could be used to enhance the quality and quantity of papaya yields in agriculture and medication selection.

A continuous improvement in omics and computational technologies holds an essential key to unlocking valuable molecular information on GSL and CG in papaya, which could ultimately be used for treating crop diseases. This approach has successfully identified the MYB TFs responsible for GSL production in B. oleracea [108].

The next-generation sequencing (NGS) technology permits the researchers to design large-scale transcriptomics experiments to capture and enumerate the transcripts representing the GSL and CG. In addition, the gene co-expression networks would facilitate identifying potential key genes [109] that contribute to the molecular mechanism of GSL and CG biosynthesis in papaya. Previous efforts have generated the gene co-expression network of CG and performed the qRT-PCR analysis to investigate the regulatory mechanism of hydrogen cyanide (HCN) synthesis, which could provide a molecular basis for breeding new cultivars with low HCN content in common vetch [110].

The dynamic interplay of signalling and metabolic pathways governing GSL and CG in papaya tissues (i.e., peel, flesh, leaf) could also be unravelled via integrative analysis of transcriptomics, proteomics, and metabolomics datasets. For instance, CG compound (i.e., linamarin, lotaustralin) has been identified in flax seed by integrating genomics, transcriptomics, metabolomics and bioinformatics approaches [111]. In another study by Zhang et al. [112], metabolomics, qRT-PCR, and comparative genomics analysis were used to obtain insights into GSL profiles and accumulation patterns in a medicinal plant, Isatis indigotica Fort [112]. The findings laid a foundation to study further the accumulation and regulation of GSLs in medicinal plants. Integrating the multi-omics approach allows a detailed picture of interactions between two interconnected pathways and enables us to build predictive models on how different molecules interact to respond to various stresses, especially how defence mechanisms are triggered and activated.

Furthermore, a central knowledge-based repository is needed to ensure other researchers can use the molecular information on GSL and CG. Data curation of GSL and CG from multiple sources, including current literature, is essential to ensure comprehensive data is provided to serve the community scientifically. However, such a platform requires continuous effort to remain relevant to the community, particularly in terms of regular system maintenance and data updates.

We have discussed limitations and suggestions to conduct integrated research for studying GSL and CG in future work. Various studies have demonstrated using GSL and CG as plant food resources but lack their involvement as a bioresource in plant defence mechanisms. Hence, studying these two essential biosynthesis pathways will enhance GSL and CG’s role, ultimately providing valuable biological resources for plant defence systems.