Genome-wide identification and expression analysis of the carotenoid metabolic pathway genes in pepper (Capsicum annuum L.)

Abstract

Carotenoids belong to a major family of isoprenoids which play vital roles in plant growth and development. Pepper (Capsicum annuum L.) is not only a vegetable and condiment grown worldwide, but also can be used as a model organism in fruit color research. In this study, 61 genes known to be involved in mevalonate (MVA), plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) and carotenoid metabolism (biosynthesis and catabolism) pathways were identified and characterized based on the genome of pepper plants. In the present study, it was found that 56 of the 61 carotenoid metabolism genes in pepper were unevenly distributed throughout 12 chromosomes. CaGGPPS2, CaGGPPS4a and CaGGPPS4b were generally clustered into one region. This study identified CaHMGR1/CaHMGR3 and CaHMGR1/CaHMGR2 as segregation duplication events. In addition, the pepper carotenoid metabolism genes had high variations in gene structure. The key carotenoid metabolism genes showed different expression patterns during the pepper fruit development and under environmental stress conditions. The results obtained in this research investigation will contribute insight to the evolution and function of carotenoid metabolic pathways in pepper fruit.

Keywords:

• carotenoid metabolic pathways
• comparative analysis
• expression patterns
• fruit development
• abiotic stress

Introduction

Carotenoids are a large group of isoprenoid molecules with over 750 members, which are distributed in plants, algae, fungi and bacteria [1, 2]. Carotenoids participate in various physiological and developmental processes [3], such as light harvesting, and processes related to protecting species against photo-oxidation and during photosynthesis [1, 4–6], photomorphogenesis [7] and non-photochemical quenching [8]. Carotenoids also provide bright colours and flavours which attract insects and animals for seed dispersal [9]. In addition, carotenoids also act as precursors for two types of important phytohormones, abscisic acids (ABA) and strigolactones, which are vital regulators for plant growth and development, as well as biotic/abiotic stress responses [10–12]. Moreover, as part of the composition of various food crops, carotenoids are considered to have essential nutritional benefits for both humans and animals [13, 14].

Carotenoid metabolism pathways have been extensively studied in the majority of the species worldwide. A large number of the genes and enzymes associated with carotenoid biosynthesis and degradation have been identified, cloned and studied in various plants species [1, 15–19]. Carotenoids are derived from two isoprene isomers, the five-carbon precursors isopentenyl diphosphate (IPP) and its double-bond isomer dimethylallyl diphosphate (DMAPP) [20–22]. Two pathways exist for IPP production in plants: the cytosolic-mevalonic acid pathway (MVA) and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway [23, 24], as shown in Supplemental Figure S1. However, carotenoids are generally synthesized from IPP and DMAPP via MEP pathways in plant species.

The first step of carotenoid biosynthesis is the head-to-head condensation of two geranylgeranyldiphosphates to produce carotenoid 15-cis-phytoene, which is catalyzed by phytoene synthase (PSY). This is considered to be the main bottleneck in the carotenoid pathways [1, 20, 25]. Then, uncoloured phytoene is desaturated and isomerized to red-coloured all-trans-lycopene through the actions of two desaturases (phytoenedesaturase (PDS) and ζ-carotene desaturase (ZDS)) and two isomerases (prolycopeneisomerase (CRTISO) and ζ-carotene isomerase (ZISO)) [2]. Phytoene desaturation by PDS and ZDS requires the transfer of two electrons to plastoquinones re-oxidized by a plastid terminal oxidase (PTOX) using O₂ as a terminal acceptor (Supplemental Figure S1) [26]. PTOX is an oxidoreductase linked to carotenoid biosynthesis and chlororespiration in order to prevent photooxidative damage [27].

Subsequently, the cyclization of the lycopene carbon chain ends is a vital step in carotenoid biosynthesis in terms of generating carotenoid diversity [1]. All-trans-lycopene is converted into the formation of δ-carotene and γ-carotene, which are catalyzed by lycopene ε-cyclase (LCYE) and β-cyclase (LCYB), respectively. Then, the orange carotenes (α- and β-carotene) are synthetized by the enzyme LCYB [1]. Thexanthophylls are produced by hydroxylases and epoxidases. There are two different types of hydroxylases: Cytochrome P450 types and CHYB (BCH) types, which hydroxylate the β-ring of cyclic carotenes [1]. Among the former type, the hydroxylases CYP97A and CYP97C hydroxylate the band ε-rings, respectively. The α-carotene is transformed into lutein, and is catalyzed primarily by CYP97-type hydroxylases. Moreover, the lutein can be also converted into lutein epoxides by epoxidases in some plant species [28]. The β-carotene is converted to zeaxanthin and hydroxylated by CHYB. In addition, zeaxanthin is epoxidized to produce antheraxanthin and violaxanthin by zeaxanthinepoxidase (ZEP), and then further to yield neoxanthin via neoxanthin synthase (NXS). Violaxanthin can be converted back to zeaxanthin via violaxanthin de-epoxidase (VDE). In pepper plants, antheraxanthin and violaxanthin are converted by capsanthin–capsorubin synthase (CCS) into capsanthin and capsorubin [29, 30]. Carotenoids are degraded enzymatically by a family of carotenoid cleavage oxygenases (CCOs), which are further divided into carotenoid cleavage dioxygenases (CCDs) and 9-cis-carotenoid dioxygenases (NCEDs) according to the different substrates in the epoxy structure [31–34]. Moreover, these genes have been found to be regulated by WRKY transcription factors in the flower petals of Osmanthus fragrans [35, 36].

In recent years, with the development of sequencing technology, many plant genomes have been successfully sequenced, which has provided opportunities to identify members of gene families at whole-genome levels [37–42]. Peppers (Capsicum annuum L.) are currently one of the most economically important vegetable crops in the world. The fruit colour is rich and colourful, ranging from green to ivory and yellow at the green stage, while being brown, orange, red, violet or yellow at the mature stages. Due to the wide ranges of Capsicum fruit colours, peppers have become model organisms of fruit colour research. It has been found that the identification of carotenoid metabolic pathway genes in pepper plants is conducive to improving the understanding of the formation mechanisms of fruit colour.

In pepper plants, carotenoids are not only responsible for the visual appeal of the pepper fruit, but also enhance the nutritional value and health benefits for humans [1, 43, 44]. The aim of this study was to identify and characterize the genes known to be involved in mevalonate (MVA), MEP and carotenoid metabolism (biosynthesis and catabolism) pathways based on the genome of pepper plants, and to explore available data about their differential expression during the pepper fruit development and under biotic and abiotic stress.

Materials and methods

Identification of carotenoid metabolism genes in pepper plants

The identifications of the carotenoid metabolism-related genes were performed via a BLAST-P search in the PGP (pepper genome platform) database (http://passport.pepper.snu.ac.kr/?t=PGENOME) using the amino acid sequences of Arabidopsis thaliana, Vitis vinifera (grape) and Solanum lycopersicum (tomato) as the queries. The gene sequences related to the carotenoid metabolism pathways from Arabidopsis, grape and tomato were acquired from the TAIR database (www.arabidopsis.org); grape genome database (http://genomes.cribi.unipd.it/grape/); and the SNG database (https://solgenomics.net/), respectively. Subsequently, the search processes of the candidate genes in the pepper genome were repeated using BLAST-P. The e-value used was 1e⁻⁵. Then, all the candidate genes were evaluated for further verification using the Pfam database (http://pfam.janelia.org/) in order to accurately classify the genes.

Sequence feature analysis of the pepper carotenoid metabolism genes

The genomic and CDS sequences for each of the carotenoid metabolism genes were extracted from the SNG database. The structures of the genes were predicted using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/chinese.php). Then, the molecular weights and theoretical isoelectric points were predicted using the online tool (http://cn.expasy.org/tools/protparam.html).

Chromosomal locations, duplications and phylogenetic analysis of the pepper carotenoid metabolism genes

The information regarding the chromosomal mapping of each carotenoid metabolism-related gene was obtained from the PGP database, and the maps were drafted using MapDraw3.0 software. The duplication events of the pepper carotenoid metabolism genes across the entire pepper genome were identified by conducting BLASTP searches of the pepper carotenoid metabolism gene sequences against the annotated pepper (CM334) protein sequences. The obtained hits of the carotenoid metabolism genes were filtered using the following threshold: Expectation (E) value ≤10⁻⁴⁰; cumulative identity percentage (CIP) ≥60%; and cumulative alignment length percentage (CALP) ≥70% [45]. The hits which survived were considered as the duplicated events of the pepper carotenoid metabolism genes in the pepper genome. In addition, gene clusters were defined as regions where two neighbouring homologous genes were less than 200 kb apart with fewer than eight non-carotenoid metabolism-encoding genes between the carotenoid metabolism-encoding genes [46, 47]. A tandemly duplicated gene was determined based on the definition introduced by Huang et al. [48], in which tandem-duplicated candidate gene pairs were defined as regions which included two or more adjacent homologous genes within 100 kb.

Then, MEGA X software was used to construct the phylogenetic trees with a neighbour-joining (NJ) method [49]. The pairwise deletion gaps, p-distance model and 1,000 bootstrap replications were selected as the parameters for the NJ analysis. The missing sequence data were treated using pairwise deletions of the gaps. The branch lengths were assigned by utilizing the pairwise calculations of the genetic distances. PhyML software (Version 3.0) was adopted to construct the maximum likelihood (ML) trees. In the present study, a Whelan and Goldman amino acid substitution model, 100 nonparametric bootstrap replicates and γ-distribution were selected for the analysis [50]. In addition, the ProtTest software (Version 2.4) was used for the model selection [51].

Expression analysis of the pepper carotenoid metabolism genes

The transcriptome data of the fruit development, along with the multiple pathogen and abiotic responses, were acquired from the scientific data [52, 53]. Then, the expression heatmaps of the pepper carotenoid metabolism genes were built and visualized using TBtools software [54].

Results and discussion

Identification and characterization of 61 genes involved in the pepper carotenoid metabolism pathways

The genes involved in carotenoid metabolism pathways in pepper were identified using a bioinformatics method. The results indicated that at least 61 carotenoid metabolism-related genes could be identified in the PGP database using the amino acid sequences of Arabidopsis, grapevine and tomato as the queries. Each of the carotenoid metabolism genes of the pepper were named based on the enzymatic reactions, similar to those given in the Arabidopsis, grapevine and tomato carotenoid metabolism pathways. Details about each of the carotenoid metabolism (biosynthetic and catabolic) genes are shown in Table 1, including the gene ID, chromosome location, gene length, number of exons, protein length, isoelectric point (pI), molecular weight and the subcellular localization (Table 1).

Table 1. Carotenoid metabolic genes in pepper.

Name	Gene ID	Chromosome location	Chromosome start	Chromosome end	Gene length (bp)	Number of exon	Protein size (aa)	MW (kDa)	PI	Subcellular location
MVA pathway
CaAACT1	CA03g02190	chr 3	4398902	4401780	2,879	13	433	45320.03	7.14	Peroxisomal
CaAACT2a	CA05g01450	chr 5	2665992	2670695	4,704	12	397	41369.45	8.42	Cytoplasmic
CaAACT2b	CA07g08230	chr 7	119622047	119626500	4,454	13	404	41449.71	6.47	Cytoplasmic
CaHMGR4	CA00g65350	chr un	413811	417523	1,827	4	608	65087.1	6.99	Endoplasmic reticulum
CaHMGR2	CA02g05800	chr 2	77053707	77056728	3,022	4	604	64921.47	5.62	Endoplasmic reticulum
CaHMGR3	CA04g23430	chr 4	222126529	222129810	3,282	4	604	64965.48	5.62	Endoplasmic reticulum
CaHMGR1	CA04g04480	chr 4	13645658	13650152	4,495	5	590	64036.6	5.82	Endoplasmic reticulum
CaHMGS1	CA01g03550	chr 01	5581682	5585805	4,124	12	462	51497.83	6.2	Mitochondrial
CaHMGS2	CA01g33440	chr 01	269488590	269492635	4,046	12	462	50848.9	6.05	Mitochondrial
CaHMGS3	CA12g13960	chr 12	181294289	181299847	5,559	12	462	50913.07	6.18	Mitochondrial
CaMDC	CA12g06650	chr 12	23761553	23768185	6,633	10	432	47859.64	6.18	–
CaMVK	CA00g43930	chr un	218795	224085	1188	5	395	41789.16	5.48	Cytoplasmic
CapMVK	CA01g07990	chr 1	19249685	19255908	6,224	10	510	55146.15	6	–
CaFPS	CA03g13390	chr 3	121063786	121067795	4,010	12	342	39514.38	5.23	Cytoplasmic
MEP pathway
CaDXS1	CA01g25140	chr 1	204585304	204581904	3401	9	700	75618.48	6.58	Chloroplast
CaDXS2	CA00g82780	chrUn	137967	142585	2052	9	683	73407.2	6.34	Chloroplast
CaDXS3	CA08g03180	Chr 8	50179584	50197894	18311	9	662	71822.59	5.82	Chloroplast
CaDXR	CA03g28450	chr 3	241307160	241311876	4717	11	442	48217.62	5.92	Chloroplast
CaMCT	CA08g13720	chr 8	134357213	134362049	4837	11	312	34528.81	5.76	Chloroplast
CaCMK	CA01g13350	chr 1	61235016	61241190	6175	11	355	38972.08	5.53	Chloroplast
CaMDS	CA01g02020	chr 1	3028039	3030510	2472	3	169	18150.17	7.69	Chloroplast
CaHDS	CA11g19050	chr 11	255913327	255919141	5815	19	740	81914.69	5.91	Chloroplast
CaHDR	CA08g18250	chr 8	142954066	142957728	3663	10	380	42906.83	5.09	Chloroplast
CaIDI	CA05g20080	chr 5	232438807	232441462	2656	5	235	27199.11	5.02	Peroxisomal
Carotenoid biosynthetic pathway
CaGGPPS2	CA02g23040	chr 2	158339859	158340944	1,086	1	361	39164.1	5.97	Mitochondrial
CaGGPPS4a	CA02g23050	chr 2	158341901	158343019	1,119	1	372	40715.15	8.69	Endoplasmic reticulum
CaGGPPS4b	CA02g23060	chr 2	158343856	158344971	1116	1	371	40465.84	6.61	Endoplasmic reticulum
CaGGPPS3	CA07g16510	chr 7	221593119	221597622	4,504	6	404	44343.82	5.72	Endoplasmic reticulum
CaGGPPS4c	CA09g01580	chr 9	3265550	3267401	1,852	2	330	36103.21	5.27	Chloroplast
CaGGPPS1	CA12g02760	chr 12	5960090	5961187	1,098	1	365	39805.87	6.56	Chloroplast
CaPSY1	CA04g04080	chr 4	12363130	12365973	2844	6	419	47066.05	9.01	Chloroplast
CaPSY2	CA02g20350	chr 2	154228939	154231923	2985	6	432	48435.43	9.13	Chloroplast
CaPSY3	CA01g12040	chr 1	47542238	47545834	3597	7	358	41548.97	8.44	Chloroplast
CaPDS	CA03g36860	chr 3	257446761	257453556	6796	14	582	65128	6.55	Chloroplast
CaZISO	CA12g20770	chr 12	232059061	232064708	5648	4	372	41894.69	7.9	Chloroplast
CaZDS	CA08g10750	chr 8	129462430	129471900	9471	14	588	64722.5	8.66	Chloroplast
CaCrtISO	CA10g17880	chr 10	224644564	224651489	6926	13	608	66526.43	7.2	–
CaPTOX	CA12g00880	chr 12	2286224	2290071	3848	9	325	37599.45	5.7	–
CaLCYB1	CA05g00080	chr 5	73170	74666	1497	1	498	55626.23	6.34	Chloroplast
CaLCYB2	CA10g20140	chr 10	229907029	229908699	1671	1	556	62318.83	7.51	Chloroplast
CaLCYE	CA00g74190	chrUn	232560	239209	1563	11	520	58247.38	7.06	Chloroplast
CaCYP97C	CA10g15950	chr 10	218131383	218139557	8175	8	560	62913.89	6.04	Chloroplast
CaCYP97A	CA06g02310	chr 6	5538387	5547886	9500	10	394	44725.34	5.41	Chloroplast
CaZEP	CA02g10990	chr 2	132164818	132169619	4802	16	661	72427.63	6.72	Chloroplast
CaVDE	CA12g11380	chr 12	116597311	116600077	2767	5	478	54309.96	6.21	Chloroplast
CaCHYB1	CA06g05750	chr 6	41880804	41882886	2083	7	308	34656.01	8.84	Chloroplast
CaCHYB2	CA03g25820	chr 3	235592338	235594362	2025	7	315	35317.91	8.52	Chloroplast
CaNXS1	CA02g05530	chr 2	72467806	72469387	1,582	5	234	26157.54	9.81	–
CaNXS2	CA02g23460	chr 2	159344067	159346199	2,133	5	234	26049.59	9.51	–
CaCCS	CA06g22860	chr 6	227702469	227703965	1497	1	498	56757.8	8.85	Chloroplast
Carotenoid catabolism pathway
CaCCD1	CA01g20280	chr 1	161737255	161752673	15419	12	584	65776.8	6.41	Cytoplasmic
CaCCD4a	CA01g28360	chr 1	227519917	227523853	3937	4	475	52870.7	6.86	Chloroplast
CaCCD4b	CA01g08910	chr 1	25816545	25821013	4469	2	603	66037.2	6.34	Chloroplast
CaCCD7	CA00g32540	chrUn	612768	618958	1941	7	646	72718.7	6.27	Chloroplast
CaCCD8	CA11g14280	chr 11	232297321	232299320	2000	2	366	41298.5	8.82	Chloroplast
CaCCD-like	CA08g04710	chr 8	85944859	85947997	3139	12	576	65389.5	5.74	–
CaCCD-like2	CA11g20400	chr 11	258320345	258328606	8262	12	575	65850.1	5.4	–
CaCCD-like3	CA01g34880	chr 1	272688150	272693275	5126	12	597	68585.2	5.86	–
CaNCED	CA07g16140	chr 7	219989766	219990719	954	1	317	34957.2	9.39	Cytoplasmic
CaNCED2	CA08g03620	chr 8	60919934	60921739	1806	1	601	un	un	Cytoplasmic
CaNCED3	CA05g17080	chr 5	224637029	224638768	1740	1	579	64672.1	8.25	Chloroplast

The subcellular location of pepper carotenoid metabolic genes by ProtComp 9.0. ‘–’ was any other location without chloroplast, mitochondrion, and secretory pathway in cell.

Fourteen genes, which were determined to encode enzymes involved in six enzymatic steps, were identified in the MVA pathway, which led to the formation of the IPP and DMAPP. These genes consisted of three AATC genes (CaAACT1, CaAACT2a and CaAACT2b); three HMGS genes (CaHMGS1, CaHMGS2 and CaHMGS3); four HMGR genes (CaHMGR1, CaHMGR2, CaHMGR3 and CaHMGR4); one MDC gene (CaMDC); one MVK gene (CaMVK); one PMVK gene (CapMVK); and one FPS gene (CaFPS). Ten genes which were determined to be involved in eight enzymatic steps were identified in the MEP pathway, which were considered as the main synthesis pathway of the IPP and DMAPP. In addition, the DXS enzyme, which catalyzes the first enzymatic step of the MEP pathway and carotenoid biosynthesis, was encoded by the three genes: CaDXS1, CaDXS2 and CaDXS3. In total, 26 genes involved in carotenoid biosynthesis pathways were identified. Six CaGGPPS genes were identified which were determined to encode enzymes that catalyze the condensation of three IPP and one DMAPP units to generate the GGPP that has the role of a universal precursor for all the plastid isoprenoids. Three CaPSY genes (CaPSY1, CaPSY2 and CaPSY3) were associated with the main bottleneck in the carotenoid pathway, which is the catalysis of the condensation of two GGPP molecules in order to produce 15-cisphytoene were also identified. Two different lycopene cyclases were identified as CaLCYB and CaLCYE, respectively. In addition, two CaLCYB genes (CaLCYB1 and CaLCYB2); two CaCHYB genes (CaCHYB1and CaCHYB2); and two CaNXS genes (CaNXS1 and CaNXS2) were identified, respectively. Eleven members of the carotenoid cleavage oxygenase (CCO) gene family involved in the carotenoid catabolism pathway were identified and subsequently divided into two groups.

Chromosomal distribution and evolution of the pepper carotenoid metabolism genes

As shown in Figure 1, 56 of the 61 identified pepper carotenoid metabolism genes were distributed unevenly throughout the 12 chromosomes, which was similar to the previous research findings regarding rice [55]. Eleven of the genes were located on Chromosome 1, while only one of the carotenoid metabolism genes was mapped in Chromosome 9 (Figure 1). However, based on the data of the current pepper genome, the remaining five genes (CaDXS2, CaMVK, CaLCYE, CaHMGR4 and CaCCD7) were found to be assembled to any scaffold.

Figure 1. Chromosomal locations and duplications of the paralogous carotenoid metabolic genes on the pepper chromosomes. Note: In the figure, the chromosome numbers are shown at the top of each bar; predicted tandem-duplicated genes are indicated by thick blue lines; and gene clusters are denoted by black lines.

Research has suggested that tandem duplications and segmental duplications are probably the main contributors to the evolutionary momentum of the plants [56]. Currently, several genes known to be related to carotenoid metabolism have been identified through either tandem or segmental duplications [2]. In the present study, in order to understand the mechanism underlying the evolution of carotenoid biosynthesis genes in pepper plants more deeply, tandem and segmental duplications were searched. There were found to be three genes (CaGGPPS2, CaGGPPS4a and CaGGPPS4b) clustered into one tandem duplication region on the pepper Chromosome 2. Two pairs of segment duplication events (CaHMGR1/CaHMGR3 pairing and CaHMGR1/CaHMGR2 pairing) were also identified. They indicated that some of the carotenoid biosynthetic genes in the pepper plants have possibly occurred by gene duplication. Moreover, the segment duplication events we determined also provide a reference for understanding the evolutionary relationships and functional predictions of the carotenoid biosynthetic genes in pepper.

Exon-intron organization of the pepper carotenoid metabolism genes

For the purpose of gaining further insight into the pepper carotenoid metabolism genes, a phylogenetic tree was constructed (Supplemental Figure S2) and the exon–intron structures were also analyzed in this study (Figure 2). High variations in the numbers of exons and introns among the carotenoid metabolism genes were observed in the pepper plants in this study, as shown in Figure 2. Ten genes (CaGGPPS1, CaGGPPS2, CaGGPPS4a, CaGGPPS4b, CaNCED, CaNCED2, CaNCED3, CaCCS, CaLCYB1 and CaLCYB2) had no intron in the pepper genome. In addition, among those having introns, the number of introns varied from 1 to 18. However, the CaHDS gene had 19 exons in its coding DNA sequences, which was the highest number of exons contained in this study. The CaDXS3 was the longest carotenoid metabolic gene with 18 kb of genomic sequence. The great variations in the structure of carotenoid metabolism genes indicated that the pepper genome might have undergone significant changes during evolution, as suggested earlier about the genome of grapevine [2].

Figure 2. Structural analysis of the carotenoid metabolic genes: (A) NJ phylogenetic tree of carotenoid metabolic proteins; (B) Exon-intron structure of carotenoid metabolic genes. Note: In the figure, yellow indicates the exons; black indicates the introns; and blue indicates the UTR.

Expression patterns of the carotenoid metabolism genes during the development of the pepper fruit

In the present study, in order to gain insight into the functions of the pepper carotenogenic genes during fruit development, the expression levels of 61 genes in two pepper organs during fruit development were analyzed based on published transcriptome data [52] and heatmaps were built from the results (Figures 3A and 3B). These pepper carotenogenic genes exhibited significant differential expression patterns in the placenta (PL) and pericarp (PR) tissue during the development stage of the pepper fruit. In the MVA pathway, 10 genes were found to be up regulated in the placenta tissue during pepper fruit development (Figure 3A). It was also observed that the CaAATC1 expression exhibited large changes throughout the pepper developmental stages. CaHMGR2 was found to be similar in expression to CaAATC1, with the exception of the mature green stage. In addition, CaHMGS4 also displayed an expression similar to CaAATC1, with the exception of the 16 DPA and the mature green stage. This study also revealed that CaAATC2a was expressed higher step-by-step following the completion of the fruit development stage. Furthermore, it was determined that CaAATC2b, CaHMGR1, CaHMGS2, CaMDC and CapMVK displayed the highest expression levels at 16 DPA, 25 DPA, 43 DPA, 16 DPA and 48 DPA, respectively. CaMVK was expressed at higher levels at 25 DPA and during the mature green stage. Three of the genes were expressed at higher levels during the fruit development stage in the pericarp tissue. CaAATC1 and CaAATC2a displayed similar expressions at higher levels, with the exception of the 16 DPA and 25 DPA stages. CaHMGR1 was found to be expressed at the highest level at 25 DPA. The remainder of the genes in both the placenta and pericarp tissue during the pepper developmental stages exhibited no or only slight changes. In the MEP pathway, the pepper carotenogenic genes (with the exception of CaDXS3) in the placenta and pericarp tissue were found to be expressed at low to medium level changes during all of the pepper developmental stages. However, CaDXS3 was observed to be expressed at higher levels at the 43 DPA and 48 DPA stages in placenta and pericarp tissue, respectively. These findings indicated that CaDXS3 may play a predominant role in the carotenoid biosynthesis of pepper fruit.

Figure 3. Expression heatmaps of the pepper carotenoid metabolic genes in placenta tissue (A) and pericarp tissue (B) during the fruit development stage based on the RNA-Seq data [52]. Sample names were assigned as follows: placenta (PL); pericarp (PR); Stage 1, 6 DPA (1); Stage 2, 16 DPA (2); Stage 3, 25 DPA (3); mature green, 36 DPA (MG); breaker, 38 DPA (B); breaker plus 5, 43 DPA (B + 5); and breaker plus 10, 48 DPA (B + 10). Note: In the figure, the colour scale represents the relative expression levels based on log2-scaledfold changes of the RPKM during the later stages compared with those in Stage 1; Red indicates high level fold changes and green represents low level fold changes.

In the carotenoids biosynthesis pathways, the majority of the carotenoid biosynthesis genes will be expressed at high levels during the different fruit development stages. In this study, it was observed that CaGGPS4b was expressed at higher levels at the 38 DPA, 43 DPA and 48 DPA stages in placenta tissue, but had either no or only slight changes in expression in the pericarp tissue throughout the pepper developmental stages. In addition, the transcriptome data showed that CaGGPS3 displayed no or only slight expression changes in the pericarp tissue throughout the pepper developmental stages, with the exception of the 48 DPA. Three CaPSY genes were found to exhibit different expression patterns during the pepper fruit development. For example, CaPSY1 was highly expressed at 38 DPA, as well as during the later development stages both in the placenta and pericarp tissue. CaPSY2 and CaPSY3 displayed only slight changes (Figures 3A and 3B) in their expression levels during all of the fruit development stages, which suggested that the CaPSY2 and CaPSY3 may not be majorly associated with pepper fruit carotenogenesis. Previous research findings have reported that PSY2 and PSY3 are mainly expressed in the leaves and roots [2]. Higher expressions of CaPTOX were found in the pericarp tissue throughout the pepper developmental stages, and CaCHYB2 was found to be up-regulated earlier in the pericarp than the placenta tissue. CaCCS is known to catalyze the synthesis of capsanthin and capsorubin, which are the main pigments and pungent substances. It was also determined that CaCCS expressed higher levels in the placenta and pericarp during the mature green fruit stage, as well as during the later development stages. It is speculated that the high expression levels of CaCCS may also help to synthesize capsanthin and capsorubin during the fruit development stages.

In the carotenoid catabolism pathway, four genes in the placenta tissue and three genes in the pericarp tissue displayed higher expression levels during the fruit developmental stages. CaCCD1 was highly expressed at 25 DPA, as well as during the later stages of fruit development in the placenta tissue. However, there were only slight changes in the expression levels observed in the pericarp tissue. CaCCD4b was highly expressed at 16 DPA, 25 DPA and during the mature green stage in the pericarp, with no changes observed in the placenta tissue. In addition, CaCCD8 was highly expressed at 16 DPA, 25 DPA and during the mature green stage in the placenta tissue, with no changes observed in the pericarp tissue. There were higher expression levels of CaNCED during the mature green stage and the later stages in the placenta, as well as higher expression levels at 16 DPA, 38 DPA, 43 DPA and 48 DPA, respectively, in the pericarp tissue. CaNCED2 displayed higher expression levels during the mature green stage and the later stages in the placenta, with higher expression levels observed at 16 DPA and the later stages in the pericarp tissue.

Expression patterns of the pepper carotenogenic-related genes following pathogen infection

This study investigated the functions of pepper carotenogenic genes in response to pathogens by analyzing available data [52]. The results can be seen in the heatmaps displayed in Figures 4A–C. A greater number of up-regulated genes following the infection with the P. infestans and TMV P0 strains were involved in the MVA pathways. In addition, the CaAACT2b gene was observed to display higher expression levels at 6 h, 12 h and 2 days following the onset of P. infestans infection. CaHMGR4 and CaHMGS1 displayed similar expression patterns of higher expression levels at 6 h, 12 h, 1 day, 2 days and 3.5 days after the onset of P. infestans infection. CaHMGR2 and CaHMGS2 had identical expression patterns, in which higher expression levels were evident following the onset of the P. infestans infection. CaMDC and CaFPS were expressed at higher levels at 12 h, 1 day, 2 days and 3.5 days after the onset of the P. infestans infection. In addition, CaMVK was expressed at higher levels at 6 h and 12 h after the onset of the P. infestans infection. Higher expression levels of CaAACT2b, CaHMGR2, CaHMGS1, CaMDC, CaMVK and CaFPS were observed 2 and 3 days after the onset of the TMV P0 strain infection. Also, CaHMGR4 was more highly expressed at 2 days after the onset of the TMV P0 strain infection. Higher expression levels of CaHMGS2 were observed at 1 day, 2 days and 3 days following the onset of the TMV P0 strain infection.

Figure 4. Expression heatmaps of the pepper carotenoid metabolic genes following pathogen infections based on RNA-Sequence data [52]. Infection by P. infestans (A), pepper mottle virus (PepMov) (B), or tobacco mosaic virus (TMV) P0 strain (C). Note: In the figure, the colour scale represents the relative expression levels based on the log2-scaled fold changes of the RPKM injections at each time point compared with the CK; Red indicates high level fold changes and green represents low level fold changes.

Only one gene (CaIDI) which had been involved in the MEP pathways displayed higher levels of expression following both the P. infestans and TMV P0 strain infections. In addition, two genes (CaGGPS1 and CaCCS) and three genes (CaGGPS1, CaNXS1 and CaCCS) involved in the carotenoid biosynthetic pathways were determined to display higher expression levels following the P. infestans and TMV P0 strain infections, respectively. Furthermore, one gene (CaCCD-like) and two genes (CaCCD8 and CaCCD-like) involved in the carotenoid catabolism pathways were respectively expressed at higher levels following the P. infestans and TMV P0 strain infection. However, only a few carotenogenic-related genes had displayed strong responses following the onset of pepper mottle virus (PepMov) infection. It was observed that genes CaHMGS2, CaGGPS1, CaCCS, CaCCD-like and CaNCED were all up-regulated following the onset of pepper mottle virus (PepMov) infection in the current study.

Expression patterns of the carotenoid metabolic genes of pepper subjected to temperature stress

It is well known that abiotic stress (low or high temperature levels and/or a deficient or excessive amount of water, heavy metals and hormone molecules, etc.) will affect plant growth and development [54–69]. In order to investigate the functions of pepper carotenogenic gene responses to temperature stress, the available transcriptome data of pepper plants subjected to cold (10 °C) and heat treatments (40 °C) were acquired [53]. The results are illustrated in the heatmaps displayed in Figures 5A and 5B. CaAACT1, CaHMGS2 and CaPTOX displayed higher expression levels at 24 and 72 h after the cold treatments (Figure 5A). CaAACT2b, CaHMGR2, CaHMGS1, CaFPS, CaIDI and CaCCD8 had higher expression levels at 72 h after the cold treatments. In addition, the expression levels of CaHMGR4, CaGGPS3 and CaLCYB1 were higher at 6 h after the cold treatments. CaDXS1, CaZEP, CaCHYB1 and CaCCS were more highly expressed at 12 h after the cold treatments. CaCCD7 displayed a higher expression level at 24 h after the cold treatments. Meanwhile, CaCHYB2 and CaNCED2 had higher expression levels at 6, 12 and 24 h after the cold treatments. Higher expression levels were observed for CaNCED at 3, 6, 12 and 24 h after the cold treatments. As can be seen in Figure 5B, according to the abiotic stress experiment results, only CaGGPS4a, CaGGPS4b, CaCHYB1, CaCCD7, CaNCED and CaNCED2 displayed higher expression levels following the heat treatments.

Figure 5. Expression heatmaps of the pepper carotenoid metabolic genes under temperature stress based on the RNA-Sequence data [52]. Cold stress at 10 °C (A) and heat stress at 40 °C (B). Note: In the figure, the colour scale represents the relative expression levels based on the log2-scaledfold changes in the RPKM of the injections at each time point compared with the CK; Red indicates high level fold changes and green represents low level fold changes.

Expression patterns of the carotenoid metabolic genes of pepper subjected to osmotic stress

Drought and salinity stress conditions are important abiotic environmental factors and significant plant stressors. Such stress conditions will potentially have negative effects plant development and productivity processes, and result in serious agricultural yield losses [70–81]. In order to examine the expression patterns of pepper carotenoid metabolic gene responses to osmotic stresses, data regarding pepper plants treated with mannitol (400 mmol/L) and NaCl (400 mmol/L) were acquired [53]. The acquired data are detailed in the heatmaps shown in Figure 6A and B, respectively. Figure 6 details the majority of the carotenoid metabolic gene responses to osmotic stress conditions. It can be seen that CaDXS2 was expressed at higher levels at 72 h after the mannitol treatments. CaGGPS1 was expressed at higher levels at 24 and 72 h following the mannitol treatments. The levels of CaCHYB1 and CaCCD-like2 expression were higher at the 12-hour point following the mannitol treatments. CaCCS was expressed at higher levels at 12 and 24 h after the mannitol treatments. CaCCD4a displayed higher levels of expression at 6 h after the mannitol treatments. At the 24-hour point following the mannitol treatments, it could be seen that the CaCCD4b expression levels were also higher. Higher levels of expression were observed for CaCCD7 at 6 and 24 h after the mannitol treatments. However, slight decreases in expression were observed at the 12-hour point following the mannitol treatments. In addition, at the 6-, 24- and 72-hour points following the mannitol treatments, the CaCCD8 expression levels were at higher levels. It was also noted that CaNCED was expressed at higher levels, with the exception of the 72-hour point after the mannitol treatments. CaNCED2 was also generally expressed at higher levels, with the exception of the 3- and 72-hour points following the mannitol treatments. In the present study, it was confirmed that the expression patterns of CaGGPS1, CaCHYB1, CaCCS, CaCCD7 and CaNCED subjected to salinity stress conditions were almost identical to the expression patterns of those subjected to mannitol stress conditions.

Figure 6. Expression heatmaps of the pepper carotenoid metabolic genes under osmotic stress based on the RNA-Seq data [53]. Osmotic stress following 400 mmol/L mannitol treatment (A) and 400 mmol/L NaCl treatment (B). Note: In the figure, the colour scale represents the relative expression levels based on the log²-scaled fold changes in the RPKM of the injections at each time point compared with the CK; Red indicates high level fold changes and green represents low level fold changes.

Conclusions

In this study, we analyzed the carotenoid metabolism genes based on the pepper genome and their expression patterns using available transcriptome data during the pepper fruit development and under biotic and abiotic stress. A total of 61 carotenoid metabolism-related genes were identified and have different expression patterns during the pepper fruit development and under biotic and abiotic stress. The results indicated that the carotenoid metabolism-related genes play important roles in growth and development, and responses to environmental stress. Overall, comparative genome analysis can provide reference for the research of the carotenoid metabolism in pepper and molecular breeding in pepper.

Disclosure statement

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [HJW], upon reasonable request.