Fine mapping and identification of CaTTG1, a candidate gene that regulates the hypocotyl anthocyanin accumulation in Capsicum annuum

Abstract

Pepper (Capsicum annuum L.) is a typical self-pollinating crop with obvious heterosis in hybrids. Consequently, the use of morphological markers during the pepper seedling stage is crucial for pepper breeding. The color of hypocotyl is widely used as a phenotypic marker in crossing studies of pepper. Pepper accessions generally have purple hypocotyls, which are mainly due to the anthocyanin accumulation in seedlings, and green hypocotyls are rarely observed in pepper. Here we reported the characterization of a green hypocotyl mutant of pepper, Chal, which was identified from a pepper ethyl methanesulfonate (EMS) mutant library. Fine mapping revealed that the causal gene, CaTTG1, belonging to the WD40 repeat family, controlled the green hypocotyl phenotype of the mutant. Virus-induced gene silencing (VIGS) confirmed that CaTTG1 regulated anthocyanin accumulation. RNA-seq data showed that expression of structural genes CaDFR, CaANS, and CaUF3GT in the anthocyanin biosynthetic pathway was significantly decreased in Chal compared to the wild type. Yeast two-hybrid (Y2H) experiments also confirmed that CaTTG1 activated the synthesis of anthocyanin structural genes by forming a MBW complex with CaAN1 and CaGL3. In summary, this study provided a green hypocotyl mutant of pepper, and the Kompetitive Allele Specific PCR (KASP) marker developed based on the mutation site of the underlying gene would be helpful for pepper breeding.

Headnote

ABSTRACT

Pepper (Capsicum annuum L.) is a typical self-pollinating crop with obvious heterosis in hybrids. Consequently, the use of morphological markers during the pepper seedling stage is crucial for pepper breeding. The color of hypocotyl is widely used as a phenotypic marker in crossing studies of pepper. Pepper accessions generally have purple hypocotyls, which are mainly due to the anthocyanin accumulation in seedlings, and green hypocotyls are rarely observed in pepper. Here we reported the characterization of a green hypocotyl mutant of pepper, Chal, which was identified from a pepper ethyl methanesulfonate (EMS) mutant library. Fine mapping revealed that the causal gene, CaTTG1, belonging to the WD40 repeat family, controlled the green hypocotyl phenotype of the mutant. Virus-induced gene silencing (VIGS) confirmed that CaTTG1 regulated anthocyanin accumulation. RNA-seq data showed that expression of structural genes CaDFR, CaANS, and CaUF3GT in the anthocyanin biosynthetic pathway was significantly decreased in Chal compared to the wild type. Yeast two-hybrid (Y2H) experiments also confirmed that CaTTG1 activated the synthesis of anthocyanin structural genes by forming a MBW complex with CaAN1 and CaGL3. In summary, this study provided a green hypocotyl mutant of pepper, and the Kompetitive Allele Specific PCR (KASP) marker developed based on the mutation site of the underlying gene would be helpful for pepper breeding.

Keywords: Pepper; CaTTG1; Anthocyanin; Hypocotyl color; Fine-mapping

1. Introduction

In the commercialization of most field and vegetable crops, F1 hybrids are commonly used due to their consistent performance in various environments. Chili pepper (Capsicum annuum L.), a typical spicy vegetable, is extensively cultivated worldwide and holds a high economic value (Liu et al., 2017; Zou et al., 2022, 2024). Pepper exhibits significant heterosis, and F, hybrids have higher yields, better disease resistance, and superior fruit quality compared to their parents. Heterosis breeding has substantially improved the quality and yield of pepper, and has played a critical role in food security.

Seedling morphological markers are mainly used for breeding new varieties and identifying the seed purity of the F, hybrids. These morphological markers used at the seedling stage can clearly identify the appearance traits, such as hypocotyl color, leaf color, and shape. True F₁ hybrids are widely selected at the early stage using seedling morphological markers in production. Seedling morphological markers play an indispensable role in double-screening parental and hybrid seed purity, strengthening seed quality supervision, reducing production cost, and ensuring variety purity (Du et al, 2020). Markers used for seedling morphology must have distinct characteristics, no lethal defects, no significant disadvantages in hybrids, and good recessive traits without defects in dominant traits. The morphological markers under natural conditions typically exhibit different leaf or hypocotyl colors, such as the sub-red plant marker in upland cotton (Gossypium hirsutum L.) (Cai et al., 2014), purple-red leaf markers in Brassica napus (Rahman et al., 2008), and green hypocotyl markers in tomato (Qiu et al., 2016).

Anthocyanin is a natural plant pigment that is synthesized in the cytoplasm and stored in vacuoles, giving fruits, flowers, seeds, and other vegetative tissues of plants different colors (Liu et al., 2018; Wang et al., 2024). Anthocyanins not only play a significant role in plant coloration, but are also involved in plant resistance to biotic and abiotic stresses. Anthocyanin belongs to flavonoid compounds, which consist of two benzene rings and contain a typical flavonoid C6-C3-C6 skeleton (Aza-Gonzalez et al., 2012). Anthocyanin biosynthesis is a branch of the flavonoid biosynthesis pathway, using phenylalanine as the direct synthetic precursor to produce various types of anthocyanins through a series of enzymatic reactions. Firstly, phenylalanine ammonium lyase (PAL) catalyzes phenylalanine to produce cinnamic acid (Chen et al, 2022b), and cinnamic acid 4-hydroxylase (C4H) catalyzes the hydroxylation of cinnamic acid to 4-coumarinic acid. 4-coumaroyl CoA ligase (4CL) acts on 4-coumaric acid to produce 4-coumaroyl CoA (Tang et al., 2020). Then, chalcone is produced from 4-coumarate CoA and malonyl CoA under the action of chalcone synthase (CHS). Chalcone isomerase (CHI) catalyzes the conversion of chalcone to naringenin. Naringenin, under the action of flavanone 3-hydroxylase (F3H), produces dihydroflavonol, including dihydrokaempferol (DHK). The hydroxylation of DHK at different sites forms precursors of other anthocyanins. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) catalyze DHK to produce dihydroquercetin (ОНО) and dihydromyricetin (DHM), respectively (Li et al., 2022). DHK, DHQ, and DHM become colorless anthocyanins under the catalysis of dihydroflavonol-4-reductase (DFR). These colorless anthocyanins are converted into colored anthocyanins under the action of anthocyanin dioxygenase/anthocyanin synthase (LDOX/ANS). Finally, glutathione S-transferase (GST) is responsible for transporting anthocyanins from the cytoplasm to the vacuole for storage (Liu et al., 2020b).

The anthocyanin synthesis pathway is regulated by a complex network of structural genes and multiple transcription factors. These transcription factors can act independently or in combination on the promoters of structural genes, influencing anthocyanin accumulation by either inhibiting or promoting the expression of genes in the pathway. The MBW complex, composed of MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WD40) proteins, has received substantial attention in this regard (Wang et al., 2020). MYB transcription factors, being one of the largest families in plants, play a crucial role in regulating anthocyanin biosynthesis (Masumi, 2022; Sun et al., 2023). For example, LfMYB113 binds to the promoter of LfDFR and activates anthocyanin biosynthesis in Formosan sweet gum (Liquidambar formosana Hance) (Wen and Chu, 2017). R2R3-MYB SIAN2-like gene played a key role in anthocyanin accumulation in tomato fruit (Yan et al., 2020). DcMYB113 could activate the expression of DcbHLH3 and structural genes related to anthocyanin synthesis, indicating that DcMYB113 specifically regulated the transport of anthocyanins in carrot roots (Xu et al., 2020).

The bHLH transcription factor is the second-largest family of transcription factors in plants and is also involved in cyanin (Zhang et al., 2015; Zheng et al., 2021). Large number of studies have shown that C1 or Pll requires the R or B genes of the bHLH family to synergistically regulate the expression of anthocyanin biosynthetic genes. The expression patterns of R1/B1 genes related to anthocyanin synthesis in maize were tissue-specific and directly bounded by the MYB and N-terminal domains of bHLH (Petroni and Tonelli 2011). In pepper, CabHLH1 could directly bind to DFR promoters and activate flavonoid synthesis (Zhang et al., 2022).

WD40 proteins are composed of 40-60 amino acids and contain 5-8 repetitive domains. The N-terminal contains a glycine-histidine dipeptide structure (Gly-His, G-H), and the C-terminal includes a tryptophan-aspartic acid dipeptide structure (Trp-Asp, W-D) (Ben-Simhon et al., 2011). The WD40 proteins have been identified in various plants and form MBW complexes with MYB and bHLH transcription factors to regulate anthocyanin synthesis (Liu et al., 2016). In pomegranate (Punica granatum L.), the WD40 gene PgTTG1 coordinated with PgAnl and PgAn2 to regulate the expression of anthocyanin synthesis genes, playing an important role in pomegranate fruit development (Ben-Simhon et al., 2011).

However, narrow genetic basis has led to limited pepper yield and quality nowadays. Therefore, artificial mutation breeding, particularly EMS mutation breeding, has recently become one of the most effective ways to create new germplasm resources for peppers (Zhang et al., 2022). Several genes controlling anthocyanin production in pepper have been fine-mapped by EMS mutagenesis combined with the BSA-seq method, such as CaAN3 encoding an R2R3-MYB transcription factor that regulates fruitspecific anthocyanin accumulation (Byun et al., 2022), Ca3GT controlled anthocyanin biosynthesis in mature unripe pepper fruit (Liu et al., 20204).

In pepper, the CaHY5 gene, which regulated anthocyanin biosynthesis, has been identified as a key gene for morphological identification at the seedling stage (Chen et al., 2022a, 2022b). However, few seedlings morphological markers have been reported for use in production practice. In this study, an EMS-induced green hypocotyl mutant, Chal, was identified. Genetic fine-mapping confirmed that the CaTTG1 gene controls hypocotyl color at the seedling stage. Virus-induced gene silencing (VIGS) verified that CaTTG1 is involved in regulating anthocyanin synthesis and yeast two-hybrid (Y2H) analysis indicated that CaTTG1 forms a MBW complex with CaAN1 and CaGL3. These results provided a molecular marker underlying green hypocotyls in seedlings, which could be used to facilitate pepper breeding.

2. Materials and methods

2.1. Plant materials and EMS mutagenesis

C. annuum L. cv Zhangshugang and Nicotiana. benthamiana were used as the WT background. A stable hereditary mutant, Chal, was observed and identified from a pepper mutant library of the inbred line 'Zhangshugang', which was created using 0.6 % EMS. 'Zhangshugang' one-week-old seedlings and tobacco seedlings were grown in growth chambers for full growth cycle at Hunan Agricultural University (Changsha, China) under controlled conditions: a 16 h light (25 °C)/8 h dark (20 °C) photoperiod cycle, 65% relative humidity, and 300 µ · m^-2s^-1 light intensity (Wang et al., 2023). Three-week-old seedlings of the wild type (WT, 'Zhangshugang'), Chal, F, (WT x Chal), and F₂, (F₁ ⊗) were used to observe hypocotyl phenotypes.

2.2. Determination of anthocyanin contents

Hypocotyls of three-week-old 'Zhangshugang' seedlings were used to extract anthocyanins. Hypocotyl tissue (0.2 g) was ground in liquid nitrogen and extracted with a 1% methanolhydrochloric acid solution at 4 °C under dark conditions for 18 h. The powdered samples were centrifuged at 12 000 r - min^-1 for 5 min (D3024, SCILOGEX, America), and the all supernatant was mixed with all acid solution to measure the absorbance values at 530 nm (A₅₃₀) and 657 nm (A₆₅₇) using enzyme calibration (SPARK, Austria). The anthocyanin content was calculated following the method described in Zhang et al. (2022).

2.3. Bulked segregant analysis sequence (BSA)-seq analysis

The WT pepper variety 'Zhangshugang' with purple hypocotyl was used as the female parent, and the F₁ population was obtained by crossing with the mutant Chal (hypocotyl green), and then the F, was selfed to obtain the F, population. The BC, ulation was obtained by backcrossing F, individuals with the Chal mutant. In the F₂ segregated population, 30 purple hypocotyl individuals and 30 green hypocotyl individuals were collected to create two DNA pools, which were then sequenced using the HiSeq 4000 platform (Changsha, China) with the 2 x 150 bp paired-end mode. Raw reads were processed using Trimmomatic (Bolger et al., 2014), and the cleaned reads were mapped to the reference genome 'Zunla' (Qin et al., 2014) using Burrows-Wheeler Aligner (BWA) software v0.7.12 (Li and Durbin, 2009). Single nucleotide polymorphisms (SNPs) and small insertions/ deletions (InDels) were detected using GATK (v4.4.0.0) (DePristo et al., 2011), and ANNOVAR (v2018) was used to annotate the potential functional effects of variants (Wang et al., 2010).

2.4. Fine-mapping and KASP analysis

The SNP indexes were calculated for each pool and delta SNP indexes were calculated between the two pools. Rlanguage (v4.2.1) was utilized to plot a A(SNP index) distribution plot, and distributions of SNP and delta SNP indexes were drawn along the chromosomes using a sliding window (five SNPs as a window and two SNPs as steps) approach (Voorrips, 2002). Kompetitive Allele Specific PCR (KASP) genotyping analysis was employed for finemapping and narrowing down the genetic distance. Allele-specific primers were designed using the SNP Primer software (www.snpway.com) with sequences 200-bp upstream and downstream of SNPs (Table 51). The 5' ends of the primers were labeled with fluorescent dyes 6-carboxyfluorescein (FAM) and hexachlorofluorescein (HEX) allowing for detection of SNP genotypes by reading the fluorescent signal. The KASP genotyping assay was carried out according to the PARMS SNP Detection Reagent Manual (Gentides Biotech Co., Ltd, Wuhan), and performed on the LightCycle® 96 Real-Time PCR System (Roche, Basel, Switzerland).

2.5. Subcellular localization and phylogenetic analysis

The full-length open reading frame (ORF) of the CaTTG1 gene was amplified via PCR and subsequently cloned into the 35S:eGFP vector to generate two GFP-fusion constructs: 35S:eGFP-CaTTG1-WT and 35S:eGFP-CaTTG1-MU. Each of the constructs, as well as an empty vector control (35S:eGFP), was then introduced into five-weeks-old tobacco leaves via Agrobacterium tumefaciens strain GV3101 (Weidi Biotechnology Co. Ltd, Shanghai, China). Following a three-day incubation period, confocal laser scanning microscopy (Zeiss LSM 510 microscope, Oberkochen, Germany) to visualize the samples. Primers used in PCR amplification are provided in Table S2. AT5G24520 (Arabidopsis, Arabidopsis thaliana), Solyc03g097340 (tomato, Solanum lycopersicum), Bra009770 (Chinese cabbage, Brassica chinensis Linn), Csa_4G097650 (cucumber, Cucumis sativus L.), GLYMA_04G228000 [soybean, Glycine max (Linn.) Merr.], LOC107904228 (cotton, Gossypium spp.), LSAT_1X129320 (lettuce, Lactuca sativa), LOC107800718 (tobacco, Nicotiana. benthamiana), 0s02g0682500 (rice, Oryza sativa L.), PGSC0003DMG400000561 (potato, Solanum tuberosum L.), TraesCS6A02G259400 (wheat, Triticum aestivum LJ), VIT_16s0098g00870 (grape, Vitis vinifera L.), Zm00001d017616 (corn, Zea mays L.), POPTR_012G006100v3 (black cottonwood, Populus trichocarpa), GSMUA_Achr4G29160_001 (banana, Musa paradisiaca L.), and CaTTG1 were together with conducted phylogenetic tree using the neighbor-joining (NJ) method with 1 000 bootstrap replicates, as implemented in the MEGA 7.0 software (Kumar et al., 2018).

2.6. Virus induced gene silencing (VIGS)

A 350-bp fragment of the CaTTG1 gene was amplified via PCR using gene-specific primers (Table 52) and cloned into the pTRV2 vector. The A. tumefaciens strain GV3101carrying the pTRV2 vector, pTRV2-PDS, and pTRV2-CaTTG1 were mixed in equal proportion and adjusted to an ODeoo value of 1.0. The WT 'Zhangshugang' pepper plant with purple hypocotyl, was used as the material for this study. The mixture of bacteria (pTRV1 + pTRV2, pTRV1 + pTRV2-PDS, pTRV1 + pTRV2-CaTTG1) was then infiltrated into the one-week-old pepper seedlings, and three biological replicates per groups. Following infiltration, the seedlings were kept in darkness for one day (25 °C) and then grown under normal conditions [16 h light (25 °C)/8 h dark (20 °C)]. After approximately three weeks, a striking albino phenotype was observed in the pTRV2-PDS group, and the expression of CaTTG1 was quantified using qRT-PCR, and the primers were showed in Table S2.

2.7. RNA-seg analysis

Total RNA from three-weeks-old WT "Zhangshugang and mutant Chal hypocotyls was extracted using TransZol kit (TransGen Biotech, Inc., Beijing, China). RNA-seq libraries were constructed and sequenced on an Illumina HiSeq™ X-Ten platform using the paired-end mode (BGI, Shenzhen, China), with three biological replicates for each sample (Table 55). Fastgc (v0.11.9) (Brown, et al., 2017) and Timmmatic (v0.36) (Bolger et al, 2014) were used to filter out low-quality reads and adaptor sequences. The cleaned reads were then mapped to the pepper reference genome 'Zunla' using Salmon (v1.2.0) (Patro et al, 2017), and gene/transcript levels were quantified using transcripts per million (TPM). Differentially expressed genes (DEGs) were identified using DESeq2 (v3.16) (Love et al., 2014), with a threshold of |log,FoldChange| > 1 and a false discovery rate (FDR) < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using the R package clusterProfiler (v3.12) (Yu etal, 2012), with an adjusted P-value < 0.05.

2.8. Real time fluorescence quantitative PCR (qRT-PCR)

cDNA of three-day-old WT and Chal hypocotyl was reversetranscribed using the HiScript® IIQ RT SuperMix (+gDNAwiper) kit (Vazyme Biotech Co. Ltd., Piscataway, NJ, United States). qRT-PCR was performed on a LightCycle® 96 Real-Time PCR System (Roche, Basel, Switzerland) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co. Ltd. Piscataway, NJ, United States), and primers for the genes are listed in Table 52. Relative expression levels of structure genes in anthocyanin biosynthesis were calculated using formula 2-ΔΔCt (Tang et al, 2021), and the actin gene CaREV31 (Capana049002411) was used as the reference gene.

2.9. Yeast two-hybrid (Y2H) assay

The full-length coding sequences (CDSs) of CaAN1 and CaGL3 were inserted into the bait vector pGADT7 (Takara Bio USA, Catalog Numbers 630442) and CaTTG1 was inserted into the prey vector pGBKT7 (Takara Bio USA, Catalog Numbers 630443). Positive control (pGADT7-T + pGBKT7-53) and negative control (pGADT7-T + pGBKT7-lam) from the Matchmaker® Gold Yeast Two-Hybrid System (Takara Bio USA, Catalog Number 630489) were used. The plasmids were co-transformed (pGADT7-T + pGBKT7-53, pGADT7-T + pGBKT7-lam, pGADT7-CaAN1 + pGBKT7-CaTTG1, pGADT7-CaGL3 + pGBKT7-CaTTG1, pGADT7-CaAN1 + pGBKT7, pGADT7-CaGL3 + pGBKT7, pGADT7 + pGBKT7-CaTTG1) into the yeast strain AH109 (Weidi Biotechnology Co., Ltd., Shanghai, China), and plated on SD/-Trp/-Leu medium for 30 "C three days. Protein interactions were screened on selective medium SD/-Trp/-Leu/-His/-Ade according to the Matchmaker® Gold Yeast Two-Hybrid System User Manual. The primers are listed in Table S2.

3. Results

3.1. Phenotypic characterization of the mutant Chal

A stable inherited green hypocotyl mutant Chal was obtained by screening an EMS mutant library constructed from the inbred line 'Zhangshugang'. The WT 'Zhangshugang' had ple whereas the mutant Chal had green hypocotyls at the seedling stage. Additionally, the WT had purple anthers, whereas the Chal anthers were yellow after flowering were observed with no significant differences between 'Zhangshugang' and Chal (Fig. 1, A). The anthocyanin content in hypocotyls of seedlings and anthers of WT was significantly higher than that of Chal, with hypocotyls and anthers of Chal containing small amount of anthocyanins (< 50 µg - g^-1 FW) (Fig. 1, B and C).

3.2. Fine-mapping of a candidate gene CaTTG1

To analyze the genetic factor underlying the mutant Cha1, the WT "Zhangshugang' was crossed with the mutant Chal to construct populations F₁, F₂ and BC₁ Phenotypic investigation and chi-square (&khgr^;2) test showed that the ratio of the purple hypocotyl to green hypocotyl of the BC, population obtained by backcrossing F1 with Chal was close to 1:1 (χ²%0.05 = 0.28 < χ² = 3.84, P > 0.05). The F₂ population contained 295 purple hypocotyl seedlings and 94 green hypocotyl seedlings, with a ratio close to 3 : 1, indicating a monogenic trait (χ² = 0.21 < χ²0.05 = 3.84, P > 0.05). Phenotypic investigation of all populations revealed that the anthers of plants with green stems were yellow, whereas those with purple stems were purple, indicating that the two traits are linked and controlled by a single recessive gene (Table 1).

To further identify the causal gene of Chal, 30 purple hypocotyl plants and 30 green hypocotyl plants from the F, population were selected to generate two DNA mixing pools. After BSA-seq, reads were mapped to the pepper reference 'Zunla' genome to identify SNP loci (Fig. S1). A total of 2 222 polymorphic SNPs were obtained after filtering, and an obvious peak (33.69-43.16 Mb) was found on chromosome 3. The A (SNP index) values (differences of allele frequencies of SNPs between the two mixed pools) of this region was significantly higher than other regions of the whole genome. A series of KASP molecular markers were developed to further narrow the candidate 9.4-Mb (33.69-43.16 Mb) interval, and phenotypes and genotypes of all individuals in the F₂ populations were used for this analysis.

Based on the characteristics of EMS mutagenesis, the physical location of the candidate interval was further reduced to about 1.0 Mb, located between SNP35186736 and SNP36284351. Only one SNP36141610 marker was co-segregated with the phenotype of F₂ individuals, resulting in a non-synonymous mutation between green and purple hypocotyls (Fig. 2, A; Table S3), a G/A mutation in the CDS of the Capana039001813 gene, 845 base pairs (bp) from the start. The candidate gene Capana03g001813, also known as CaTTG1, encodes a WD40 repeat protein and is orthologous to the Arabidopsis TRANSPARENT TESTA GLABRA 1 gene that was involved in the regulation of anthocyanin biosynthesis (Walker et al., 2019). CaTTG1 was a single-exon gene located on chromosome 3 (36140766-36141794), with the CDS of 1 029 bp in length (Fig. 2, B, Table S4). The CaTTG1 protein contained two typical WD domains (G-beta repeats) in the 172-203 and 258-293 regions. The G/A mutation in the CDS of Capana039001813 resulted in a mutation of cysteine (Cys, C) at position 281 to tyrosine (Tyr, Y) in the encoded protein sequence (Fig. 2, C).

3.3. Basic features of CaTTG1

According to the qRT-PCR study, the expression of CaTTG1 in WT was much higher in stems, fruits, and leaves than in flowers and roots. Furthermore, the expression of CaTTG1 in stems, fruits, flowers, and leaves of WT was significantly higher than that of Chal (Fig. 3, A). Phylogenetic analysis was used to compare CaTTG1 protein sequences with those from Arabidopsis, Chinese cabbage, soybean, cucumber, cotton, lettuce, rice, wheat, tobacco, tomato, and potato, etc. Proteins with high similarity to the CaTTG1 protein included SITTG1 (Solyc03g097340) in tomato (Fig. 3, B). The subcellular localization of CaTTG1 was determined using the 35S:eGFP reporter transiently transformed into tobacco leaves. The green fluorescent protein (GFP) signal of CaTTG1-WT was detected in the nuclear and cytoplasmic membranes, with the mCherry signal of the nuclear marker, indicating that CaTTG1-WT is located in both the nuclear and cytoplasmic membranes. However, the GFP signal of CaTTG1-MU was only detected in the cytoplasmic membrane, indicating that the mutation of CaTTG1 caused it to be no longer located in the nucleus but only in the cytoplasmic membrane (Fig. 3, C).

3.4. Silencing CaTTG1 results in reduced anthocyanin т hypocotyls

In order to confirm the role of CaTTG1 in anthocyanin biosynthesis, VIGS was used to silence CaTTG1 in WT 'Zhangshugang' pepper plants with purple hypocotyls. Four weeks after the pepper was injected with Agrobacterium, the new leaves of the positive control plant pTRV2-CaPDS began to bleach. Purple pigmentation in stems and hypocotyls of pTRV2-CaTTG1 injected plants was significantly reduced, whereas purple was widely enriched in internodes and hypocotyls in the control pTRV2 plants (Fig. 4, A). The expression of CaTTG1 and anthocyanin pathway genes in the hypocotyls of silenced and control plants was analyzed using qRT-PCR, which revealed that CaTTG1 was expressed at a significantly lower level in hypocotyls of silenced plants than in the control (Fig. 4, B). Meanwhile, the anthocyanin content in hypocotyls of silenced plants was significantly lower than that of the control (Fig. 4, C). The expression levels of the upstream genes in the anthocyanin pathway (CHS, CHI, F3H, and F3'5'H) were not significantly different between the silenced and control plants. However, the expression levels of downstream genes (DFR, ANS, and UF3GT) were significantly lower in silenced plants than those in control plants (Fig. 4, D).

3.5. Gene expression related to anthocyanin pathway in 'Zhangshugang' and Chal

To further understand the transcriptional changes of anthocyanin biosynthesis genes, transcriptome sequencing (RNA-seq) was performed on hypocotyls of WT and Chal mutant plants. A total of 1794 DEGs were identified between Chal and WT, of which 791 were down-regulated (44%) and 1 003 (56%) up-regulated in Chal (Fig. S2, B). GO analysis showed that DEGs were enriched with various GO terms in the three categories: biological process, molecular function, and cellular component. In the molecular function category, DEGs were mainly enriched for heterocyclic compound binding, protein binding, organic cyclic compound binding, and transferase activity. In the biological process category, DEGs were mainly enriched in organic substance metabolic process, primary metabolic process, and cellular metabolic process. In the cell component category, DEGs were mainly enriched in intracellular, intracellular part, and intracellular organelle (Fig. 5, A). KEGG enrichment analysis suggested that the DEGs were mainly enriched in metabolic pathways, biosynthesis of secondary metabolite, MAPK signaling pathways-plant, flavonoid biosynthesis, brassinosteroid biosynthesis, phenylpropanoid biosynthesis, plant-pathogen interaction, and other pathways (Fig. S2, C). RNA-seq analysis showed that the expression levels of upstream genes in the anthocyanin biosynthesis pathway (CHS, and CHI) were significantly higher in WT than those in the mutant hypocotyls. The expression levels of middle genes (F3H, and F3'5'H) showed no significant differences, while the expression levels of downstream genes (DFR, ANS, and UF3GT) were also significantly higher in the WT hypocotyls than those in the mutant hypocotyls (Fig. 5, B-D).

3.6. CaTTG1 formed a MYB-bHLH-WD40 (MBW) complex to regulate anthocyanin biosynthesis

Since CaTTG1 encodes the WD40 protein, and WD40 proteins can bind to R2R3-MYB and bHLH proteins to form a MBW complex, thereby regulating anthocyanin synthesis. Therefore, we tested whether CaTTG1 protein could bind to the bHLH protein GLABRA3 (CaGL3, Capana01g000256) and R2R3-MYB protein CaAN1 (Capana10g001433) using the Y2H assay. The results showed that the yeast cells transfected with positive control (PGADT7-T and pGBKT7-53), pGBKT7-CaTTG1 and pGADT7-CaAN1, pGBKT7-CaTTG1 and pGADT7-CaGL3 could survive in both SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade selective media. However, negative control pGADT7-T and pGBKT7-lam, pGADT7-CaAN1 and pGBKT7, pGADT7-CaGL3 and pGBKT7, pGADT7 and pGBKT7-CaTTG1 only survived in SD/-Leu/-Trp but could not survive in SD/-Leu/-Trp/-His/-Ade selective media (Fig. 5, E).

4. Discussion

The chili pepper (C. annuum L.) originated in the tropics of South America, and belongs to the Solanaceae family with high economic values (Wang et al, 2022). Study have shown that CaHY5 controls anthocyanin synthesis in pepper hypocotyls and regulated anthocyanin accumulation by directly binding to the promoters of genes involved in the anthocyanin pathway (Chen et al, 2022a, 2022b). In this study, a mutant Chal with an obvious green hypocotyl was identified. However, inconsistent with the phenotype of the e1898 mutant controlled by CaHY5, mutant Chal was most clearly observed after the 2-3 true leaves expanded, with a significantly different hypocotyl color from the WT (Chen et al., 2022a, 2022b) (Fig. 1).

In the candidate interval, a SNP marker with G/A mutation was co-segregated with the phenotype of F, individuals, resulting in a non-synonymous mutation in the CaTTG1 gene, which encodes a WD40 repeat protein that regulates anthocyanin accumulation in the hypocotyl. Silence of CaTTG1 gene in pepper showed that anthocyanin was significantly reduced in the hypocotyls of WT 'Zhangshugang' compared with control (Fig. 4). Other evidence also supported our finding, such as pTRV2-WD40 resulted in an almost complete loss of anthocyanin in the pericarp of pepper fruit, indicating that it affected anthocyanin synthesis (Aguilar-Barragan and Ochoa-Alejo, 2014). Interestingly, CaTTG1 protein was localized in both nuclear and cytoplasmic membranes in 'Zhangshugang', but the G/A mutation in CaTTG1 of Chal resulted in it not being localized in the nuclear membrane. Some studies showed that mutations affect the subcellular localization of proteins, which further affect the stability of the interaction proteins (Jin et al., 2022). It is reasonable to hypothesize that mutation from cysteine to tyrosine in the CaTTG1 protein might lead to its secretion or transfer, affecting anthocyanin synthesis in pepper hypocotyls. However, the exact reason underlying the change of protein localization caused by the mutation needs further verification, but these results demonstrated that CaTTG1 was the causal gene of Chal.

The MBW complex is the well-known regulatory factors that control the transcription of structural genes involved in the anthocyanin biosynthesis in various plants. The MBW complex AtTTG1-AtTT8-AtPAP1 in Arabidopsis affected anthocyanin accumulation, with the ttgl mutant being unable to synthesize anthocyanin. Both apple MdTTG1 (An et al., 2012) and grape TTG1 (Matus et al., 2010) could complement the ttgl mutant and restore the phenotype of anthocyanin accumulation in leaves and hypocotyls of apple and grape. The PhAN2-PhAN1-PhAN11 complex affected anthocyanin accumulation in petals by regulating CHS and DFR expression in petunia (Spelt et al., 2002). Our results showed that CaTTG1 could form a MBW complex with CaAN1 and CaGL3, thereby regulating anthocyanin biosynthesis.

Morphological marker can be used to select target traits at the seedling stage and to remove non-target traits in a timely manner. Seedling morphological markers effectively avoid the emergence of false hybrids and facilitate early removal of non-target traits to save land, manpower, and material resources. It also play an important role in the breeding of pepper, a crop with obvious heterosis. However, seedling markers are generally achieved through a large number of long-term backcrossing, and due to the existence of reproductive isolation, good marker traits cannot be comprehensively applied (Karim et al., 2021). Therefore, it is imperative to achieve precise molecular marker cloning and use seedling marker traits. Previous studies have revealed that the single-base mutation of the rice GRY79 gene affected chloroplast development in the early seedling stage, and could be used as a leaf color markerto efficiently identify and eliminate false hybrids in commercial hybrid rice production (Wan et al., 2015). In addition, the color of the hypocotyl is also an obvious seedling morphological marker. For example, the sterile gene ms-10 in tomato was closely linked to the anthocyanin deficiency gene SIGSTAA, which controls the green stem trait and plays an active role in tomato hybrid seed production (Zhang et al., 2016). In this study, we also provided a loss-of-function CaTTG1 that leads to green hypocotyls in pepper seedlings, which could be used as a seedling morphological marker. For example, using green stem lines to hybridize with purple stem male parents could screen hybrids through color at the seedling stage to ensure seed purity and reduce labor cost. In addition to having a green hypocotyl phenotype at the seedling stage, the mutant Chal also had easily distinguishable yellow anthers at flowering, in contrast to WT purple anthers (for most pepper accessions). Therefore, in the case of the omission of seedling screening or high recombination exchange rate, this morphological marker could be further distinguished at flowering. This study provided important information that could facilitate the identification of pure hybrid F, seeds in pepper breeding and help to reduce the cost of manual screening.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by grants from the Special Funds for Construction of Innovative Provinces in Hunan Province (Grant No. 2021NK1006), the Science and Technology Innovation Program of Hunan Province (Grant No. 2021JC0007), China Agriculture Research System of MOF and MARA (Grant No. CARS-24-A-15), National Natural Science Foundation of China (Grant No. 32130097), and National Natural Science Foundation of China (Grant No. U19A2028).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, https://doi.org/10.1016/j.hpj.2023. 05.016.

Sidebar

Received 19 February 2023; Accepted 11 May 2023; Available online 22 November 2023

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

Footnote

* Corresponding authors.

E-mail addresses: [email protected]; [email protected]; [email protected]

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