Capsanthin-capsorubin Synthase Research Articles

Generating high purity and yield of capsanthin and capsorubin carrot germplasm through synthetic metabolic engineering.

Capsanthin and capsorubin are red κ-xanthophylls exclusively found in a handful of other plant species. Currently, capsanthin and capsorubin are only extracted from red pepper. Here, high purity production of capsanthin and capsorubin has been achieved in carrot taproot by synthetic metabolic engineering strategy. Expression of a capsanthin-capsorubin synthase gene (CaCCS) from pepper resulted in dominant production of capsanthin whereas expression of a LiCCS gene from tiger lily resulted in production of both capsanthin and capsorubin in carrot taproot. The highest content of capsanthin and capsorubin was obtained in LiC-1 carrot taproot hosting the LiCCS gene, 150.09 μg/g DW (dry weight). Co-expression of DcBCH1 with CCS could improve the purity of capsanthin and capsorubin by eliminating the non-target carotenoids (eg. α-carotene and β-carotene). The highest purity of capsanthin and capsorubin was obtained in BLiC-1 carrot taproot hosting DcBCH1+LiCCS genes, 91.10% of total carotenoids. The non-native pigments were esterified partially and stored in the globular chromoplast of carrot taproot. Our results demonstrated the possibility of employing carrot taproot as green factories for high purity production of capsanthin and capsorubin. The capsanthin/capsorubin carrot germplasms were also valuable materials for breeding colorful carrots cultivars.

Development of Gene-based Markers for capsanthin-capsorubin synthase and zeaxanthin epoxidase Genes to Distinguish Fruit Colors in Paprika (Capsicum annuum L.)

Development of Gene-based Markers for capsanthin-capsorubin synthase and zeaxanthin epoxidase Genes to Distinguish Fruit Colors in Paprika (Capsicum annuum L.)

Changes in the Carotenoids of Zamia dressleri Leaves during Development.

It has been observed that the leaves of some Zamia species undergo a kind of "reverse ripening"; that is, they change from their original brown color to green during development. We assumed that this strange color change was due to the change in carotenoid composition, so we followed the changes for several weeks. The detailed carotenoid composition and content at different stages of development of the leaves was determined with HPLC-DAD focusing on the changes in red and yellow carotenoids. The total and relative amounts of red and yellow carotenoids were determined simultaneously from one measurement from a saponified and/or unsaponified extract. At the beginning of development, the concentration of red carotenoids was higher than that of the yellow ones; it decreased drastically until 22 days and continued to decrease slowly until they completely disappeared. The concentration of yellow carotenoids decreased at the beginning as well, but after 22 days it started to increase. The amount of red carotenoids started to decrease when the leaflet stopped growing. Lutein is the main component in old leaflets, which is not a red carotenoid precursor. Red carotenoids can always be found in their esterified form in the leaves. These findings support the hypothesis that red and yellow carotenoid accumulation are independent and probably have different functions in the leaflet. The strange color change was explained based on the compartmentalization of red and yellow carotenoids and on the changing activity of the enzyme capsanthin-capsorubin synthase responsible for the synthesis of red carotenoids capsorubin and capsanthin.

Transcript Analysis Reveals Positive Regulation of CA12g04950 on Carotenoids of Pigment Pepper Fruit under Nitrogen Reduction

This study investigates the relationship between nitrogen fertilization and pepper fruit color by employing five different nitrogen treatments (N1: 750 kg/hm2, N2: 562.5 kg/hm2, N3: 375 kg/hm2, N4: 187.5, and N0: 0 kg/hm2). Fruits were harvested at 30 (S1: green ripening stage), 45 (S2: color transition stage), and 60 days (S3: red ripening stage) after flowering. Subsequently, pigment content, carotenoid component content, carotenoid enzyme activity, and transcriptome sequence were analyzed, and CA12g04950 function was validated through virus-induced gene silencing (VIGS). The results indicate that a reduction in nitrogen application led to an earlier onset of fruit color breakdown, and increased the contents of total carotenoid, capsanthin, phytoene and PSY (phytoene synthase) activity, LCYB (lycopene β-cyclase) activity and CCS (capsanthin/capsorubin synthase) activity. The analysis of different expression genes indicated that the most differently expressed genes were enriched in the N1 vs. N4 comparison, with 18 genes involved in carotenoid metabolism and 16 genes involved in nitrogen metabolism. Most DE genes were enriched in the pathways of photosynthesis, porphyrin, carotenoid biosynthesis, seleno-compounds, and nitrogen metabolism. There were numerous differential transcription factor families, including ERF, bHLH, MYB, C2H2, and NAC. Pearson correlation analysis revealed a significant positive correlation between CA12g04950 expression and 11 carotenoid genes in the N4 treatment. Subsequent silencing of CA12g04950 using VIGS resulted in delayed color ripening while a significant decrease in total carotenoid content and the expression levels of carotenoid genes. In conclusion, nitrogen reduction led to an increase in carotenoid content in pigment pepper fruits. Furthermore, under nitrogen reduction, CA12g04950 positively influenced the redness of the fruits.

Identification of carotenoids and candidate genes shaping high pigment chili pepper variety

Pepper (Capsicum spp.) is a major vegetable grown and consumed worldwide. Fruit color is an important exterior quality of fresh and processed peppers. The typical red color of pepper fruit is mainly determined by the content of capsanthin and capsorubin. The biosynthetic pathway of capsanthin and capsorubin has been largely clarified, but the regulators of this pathway remain to be investigated. To decipher the regulatory mechanism of high pigment accumulation in mature red pepper (C. annuum) fruit, carotenoid and transcriptome profiling were performed on two red chili peppers with distinct levels of capsanthin and capsorubin. The total carotenoids of high-pigment genotype (GB23) increased by 168 times during fruit ripening, especially carotenoid compounds such as zeaxanthin, β-cryptoxanthin and capsanthin. Moreover, the difference in the transcript level of genes encoding phytoene synthase 1 (PSY1), β-carotene hydroxylase 1 (BCH1), and capsanthin-capsorubin synthase (CCS) was consistent with the difference in carotenoid accumulation between GB23 and GB42 (low-pigment genotype). Further investigation revealed several transcription factors exhibiting similar expression patterns and correlated with capsanthin accumulation. Utilizing weighted gene co-expression network analysis, we identified eight transcription factors, namely bHLH13, Zinc finger protein ZFP3, MADS-RIN, NAC68, PIF3, and WRKY6, as potential regulators of PSY1, BCH1, or CCS genes. Yeast one-hybrid and luciferase reporter assays demonstrated that ZFP3 activates PSY1 and CCS while WRKY6 activates CCS through promoter binding. This comprehensive study sheds light on the regulatory mechanism underlying capsanthin accumulation in chili pepper, offering new insights into this important trait.

Fast and High-Efficiency Synthesis of Capsanthin in Pepper by Transient Expression of Geminivirus.

The color of the chili fruit is an important factor that determines the quality of the chili, as red chilies are more popular among consumers. The accumulation of capsanthin is the main cause of reddening of the chili fruit. Capsanthin is an important metabolite in carotenoid metabolism, and its production level is closely linked to the expression of the genes for capsanthin/capsorubin synthase (CCS) and carotenoid hydroxylase (CrtZ). We reported for the first time that the synthesis of capsanthin in chili was enhanced by using a geminivirus (Bean Yellow Dwarf Virus). By expressing heterologous β-carotenoid hydroxylase (CrtZ) and β-carotenoid ketolase (CrtW) using codon optimization, the transcription level of the CCS gene and endogenous CrtZ was directly increased. This leads to the accumulation of a huge amount of capsanthin in a very short period of time. Our results provide a platform for the rapid enhancement of endogenous CCS activity and capsanthin production using geminivirus in plants.

Putative Daucus carota Capsanthin-Capsorubin Synthase (DcCCS) Possesses Lycopene β-Cyclase Activity, Boosts Carotenoid Levels, and Increases Salt Tolerance in Heterologous Plants.

Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of trans-lycopene. Daucus carota harbors two LCYB genes, of which DcLCYB2 (annotated as CCS-Like) is mostly expressed in mature storage roots, an organ that accumulates high α-carotene and β-carotene content. In this work, we determined that DcLCYB2 of the orange Nantes variety presents plastid localization and encodes for a functional LCYB enzyme determined by means of heterologous complementation in Escherichia coli. Also, ectopic expression of DcLCYB2 in tobacco (Nicotiana tabacum) and kiwi (Actinidia deliciosa) plants increases total carotenoid content showing its functional role in plants. In addition, transgenic tobacco T2 homozygous plants showed better performance under chronic salt treatment, while kiwi transgenic calli also presented a higher survival rate under salt treatments than control calli. Our results allow us to propose DcLCYB2 as a prime candidate to engineer carotenoid biofortified crops as well as crops resilient to saline environments.

Fine mapping and identification of candidate genes for fruit color in pepper (Capsicum chinense)

The quality of pepper fruits is determined by numerous characteristics such as, flavor, color, and nutritional value. Through little is known about it, fruit color impacts breeding preferences in Capsicum chinense in a substantial way. In the present study, two C. chinense germplasms, HNUCC16 (dark green immature and yellow ripe fruit) and HNUCC22 (light green immature and red ripe fruit) were used to produce an F2 population. Based on genotyping-by-sequencing, we constructed a high-density genetic map of the F2 population with 1,347 bin markers and covered 1,206.37 cM in twelve pepper linkage groups. The trait segregation analysis of the F2 population show that the color of immature and mature fruits is controlled by a single locu . Bin markers 19 (chromosome 1) and 849 (Chromosome 6) were closely associated with the locus that regulates the color of immature and ripe fruits, respectively. The transformation of the two bin markers into dCAPS markers indicate that inactive shikimate kinase-like 2 (BC332_00870) may be a strong candidate gene for regulating immature fruit color, and the premature termination mutation of capsanthin–capsorubin synthase (BC332_17237) gene may possibly cause the yellow color in HNUCC16 pepper fruits. Overall, the findings of this study offer fundamental knowledge concerning the development of color and offer a new resource for pepper fruit color genetic improvement and molecular breeding.

Carotenoid analysis and functional characterization of lycopene cyclases in Zinnia elegans L.

Carotenoids, a group of essential pigments for petal coloration in many species, could be generally categorized into linear and cyclic groups. The cyclization of the linear substrate lycopene at both end groups by β- and ε-cyclases (LCYBs and LCYEs, respectively) is a key branching point of the biosynthesis of cyclic carotenoids in flowers. Common zinnia (Zinnia elegans L.), an Asteraceae family plant that has been widely used in landscape greening, is best known for its brilliant flower colors. However, the mechanism of its petal coloration is not well studied, especially with respect to carotenoid pigmentation. In this study, total carotenoid content and carotenoid compositions of petals were analyzed in two common zinnia cultivars, 'Dreamland Red’ (DRE) and 'Dreamland Yellow’ (DY). Despite the lighter color of DY petals, the total carotenoid content in DY flowers was significantly higher than that in DRE. Metabolomic analysis revealed that the major carotenoids of both cultivars are cyclic carotenoids, with β-carotene being the most abundant. In addition, one LCYB, LCYE, and capsanthin/capsorubin synthase (CCS) gene were identified from Z. elegans by homology-based search in transcriptome. After cloning and sequencing, full-length open reading frame of each gene is exactly the same between two cultivars. Bacterial pigment complementation experiments revealed that ZeLCYB and ZeCCS can cyclize lycopene at both ends to produce β-carotene. Unlike most LCYEs characterized from higher plants, ZeLCYE produced predominantly ε-carotene in E. coli system. However, ZeLCYE is likely to have a lower affinity for lycopene or lower activity compared with ZeLCYB. Gene expression analysis showed that DY has higher expression levels of ZeLCYB and ZeCCS than DRE at specific developmental stages, consistent with its higher total carotenoid content. This work would lay a solid foundation for future studies on carotenoid accumulation in Z. elegans.

Comprehensive transcriptome-based characterization of differentially expressed genes involved in carotenoid biosynthesis of different ripening stages of Capsicum

Pepper (Capsicum annuum L.) is one of the widely cultivated vegetable crops in the world. The color formation of pepper fruit is closely related to the carotenoid composition and content. In order to understand the regulatory mechanism of red and orange pepper formation, comprehensive transcriptome and carotenoid content determination were carried out at 25, 40 and 55 days after flower. At the mature stage (55 DAF), the contents of zeaxanthin, β-cryptoxanthin, β-carotene and capsanthin in red chili pepper(R) were significantly higher than those in orange pepper (O), while the content of lutein in orange pepper(O) was higher than that in red pepper(R). Moreover, it was found that many differentially expressed genes (DEGs) were involved in the biosynthesis of carotenoids, phenylpropanoids, flavonoids and metabolism of phenylalanine. A total of 23 DEGs were found to be involved in carotenoid biosynthesis, among which β-carotene hydroxylase 1(CHYB1) and capsanthin-capsorubin synthase(CCS)were significantly different during fruit development. Further analysis showed that there were complete CCS genes in both red and orange pepper fruits. However, the expression level in orange pepper fruits was maintained at a very low level, on the contrary, the red fruits exhibited a remarkable upward trend and extremely high transcript level. Through weighted gene co-expression network analysis (WGCNA), transcription factors F-box protein SKIP23, GATA transcription factor 26, U-box domain-containing protein 52, zinc finger family FYVE/PHD-type, RING/FYVE/PHD-type and CONSTANS-LIKE 9 might be involved in the regulation of CCS gene. This study will lay a base to further analyze the regulation mechanism of carotenoid metabolism pathway in the process of pepper fruit color formation.

A mutation in Zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum).

Phytoene synthase (PSY1), capsanthin-capsorubin synthase (CCS), and pseudo-response regulator 2 (PRR2) are three major genes controlling fruit color in pepper (Capsicum spp.). However, the diversity of fruit color in pepper cannot be completely explained by these three genes. Here, we used an F2 population derived from Capsicum annuum 'SNU-mini Orange' (SO) and C. annuum 'SNU-mini Yellow' (SY), both harboring functional PSY1 and mutated CCS, and observed that yellow color was dominant over orange color. We performed genotyping-by-sequencing and mapped the genetic locus to a 6.8-Mb region on chromosome 2, which we named CaOr. We discovered a splicing mutation in the zeaxanthin epoxidase (ZEP) gene within this region leading to a premature stop codon. HPLC analysis showed that SO contained higher amounts of zeaxanthin and total carotenoids in mature fruits than SY. A color complementation assay using Escherichia coli harboring carotenoid biosynthetic genes showed that the mutant ZEP protein had reduced enzymatic activity. Transmission electron microscopy of plastids revealed that the ZEP mutation affected plastid development with more rod-shaped inner membrane structures in chromoplasts of mature SO fruits. To validate the role of ZEP in fruit color formation, we performed virus-induced gene silencing of ZEP in the yellow-fruit cultivar C. annuum 'Micropep Yellow' (MY). The silencing of ZEP caused significant changes in the ratios of zeaxanthin to its downstream products and increased total carotenoid contents. Thus, we conclude that the ZEP genotype can determine orange or yellow mature fruit color in pepper.

Capsanthin Production in Escherichia coli by Overexpression of Capsanthin/Capsorubin Synthase from Capsicum annuum.

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene β-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

Whole genome resequencing of four Italian sweet pepper landraces provides insights on sequence variation in genes of agronomic value

Sweet pepper (Capsicum annuum L.) is a high value crop and one of the most widely grown vegetables belonging to the Solanaceae family. In addition to commercial varieties and F1 hybrids, a multitude of landraces are grown, whose genetic combination is the result of hundreds of years of random, environmental, and farmer selection. High genetic diversity exists in the landrace gene pool which however has scarcely been studied, thus bounding their cultivation. We re-sequenced four pepper inbred lines, within as many Italian landraces, which representative of as many fruit types: big sized blocky with sunken apex (‘Quadrato’) and protruding apex or heart shaped (‘Cuneo’), elongated (‘Corno’) and smaller sized sub-spherical (‘Tumaticot’). Each genomic sequence was obtained through Illumina platform at coverage ranging from 39 to 44×, and reconstructed at a chromosome scale. About 35.5k genes were predicted in each inbred line, of which 22,017 were shared among them and the reference genome (accession ‘CM334’). Distinctive variations in miRNAs, resistance gene analogues (RGAs) and susceptibility genes (S-genes) were detected. A detailed survey of the SNP/Indels occurring in genes affecting fruit size, shape and quality identified the highest frequencies of variation in regulatory regions. Many structural variations were identified as presence/absence variations (PAVs), notably in resistance gene analogues (RGAs) and in the capsanthin/capsorubin synthase (CCS) gene. The large allelic diversity observed in the four inbred lines suggests their potential use as a pre-breeding resource and represents a one-stop resource for C. annuum genomics and a key tool for dissecting the path from sequence variation to phenotype.

Candidate Gene Analysis Reveals That the Fruit Color Locus C1 Corresponds to PRR2 in Pepper (Capsicum frutescens).

The diverse fruit colors of peppers (Capsicum spp.) are due to variations in carotenoid composition and content. Mature fruit color in peppers is regulated by three independent loci, C1, C2, and Y. C2 and Y encode phytoene synthase (PSY1) and capsanthin-capsorubin synthase (CCS), respectively; however, the identity of the C1 gene has been unknown. With the aim of identifying C1, we analyzed two pepper accessions with different fruit colors: Capsicum frutescens AC08-045 and AC08-201, whose fruits are light yellow and white, respectively. Ultra-performance liquid chromatography showed that the total carotenoid content was six times higher in AC08-045 than in AC08-201 fruits, with similar composition of main carotenoids and slight difference in minor components. These results suggest that a genetic factor in AC08-201 may down-regulate overall carotenoid biosynthesis. Analyses of candidate genes related to carotenoid biosynthesis and plastid abundance revealed that both accessions carry non-functional alleles of CCS, golden2-like transcription factor (GLK2), and PSY1. However, a nonsense mutation (C2571T) in PRR2, a homolog of Arabidopsis pseudo response regulator2-like (APRR2), was present in only AC08-201. In a population derived from a cross between AC08-045 and AC08-201, a SNP marker based on the nonsense mutation co-segregated fully with fruit color, implying that the mutation in PRR2 may cause the white color of AC08-201 fruits. Transmission electron microscopy (TEM) of AC08-201 fruit pericarp also showed less developed granum structure in chloroplast and smaller plastoglobule in chromoplast compared to those of AC08-045. Virus-induced gene silencing (VIGS) of PRR2 significantly reduced carotenoid accumulation in Capsicum annuum ‘Micropep Yellow’, which carries non-functional mutations in both PSY1 and CCS. Furthermore, sequence analysis of PSY1, CCS, and PRR2 in other white pepper accessions of C. annuum and Capsicum chinense showed that they commonly have non-functional alleles in PSY1, CCS, and PRR2. Thus, our data demonstrate that the fruit color locus C1 in Capsicum spp. corresponds to the gene PRR2.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

Genetic mapping of the c1 locus by GBS-based BSA-seq revealed Pseudo-Response Regulator 2 as a candidate gene controlling pepper fruit color.

The Pseudo-Response Regulator 2 gene was identified in the c1 locus, representing a genetic factor regulating fruit color in pepper using GBS-based BSA-seq. The loci c1, c2, and y have been widely reported as genetic determinants of various ripe fruit colors in pepper. However, c1, which may impact reduced pigmentation in red, orange, and yellow fruits, is not well understood. Two cultivars showing peach or orange fruit in Capsicum chinense 'Habanero' were found to have c2 mutation and were hypothesized to segregate c1 locus in the F2 population. Habanero peach (HP) showed a reduced level of chlorophylls, carotenoids and total soluble solids in immature and ripe fruits. A microscopic examination of the fruit pericarps revealed smaller plastids and less stacked thylakoid grana in HP. The expression of many genes related to chlorophyll and carotenoid biosynthetic pathways were reduced in HP. To identify the genomic region of the c1 locus, bulked segregant analysis combined with genotyping-by-sequencing was employed on an F2 population derived from a cross between Habanero orange and HP. One SNP at chromosome 1 was strongly associated with the peach fruit color. Pepper Pseudo-Response Regulator 2 (PRR2) was located close to the SNP and cosegregated with the peach fruit color. A 41bp deletion at the third exon-intron junction region of CcPRR2 in HP resulted in a premature termination codon. A nonsense mutation of CaPRR2 was found in C. annuum 'IT158782' which had white ripe fruit coupled with null mutations of capsanthin-capsorubin synthase (y) and phytoene synthase 1 (c2). These results will be useful for the genetic improvement in fruit color and nutritional quality in pepper.

Study on the Profile of Capsanthin-Capsurobin Synthase (Ccs) Gene responsible for Carotenoid Synthesis in Chili Pepper (Capsicum frutescens L.) Mutants G1M6 M2 Generation

The Capsanthin-capsorubin synthase (Ccs) is one of the enzymes that play a role in carotenoid pigment bio-synthesis. Mutations in the Ccs gene cause the formation of yellow or orange fruit colour instead of red. This study aims to analyze the genetic profile of the Ccs gene and its effect on the carotenoid content of the chili pepper mutant G1M6 M2generation. The profile of the Ccs gene was analyzed using Polymerase Chain Reaction (PCR) continued with sequencing methods.The carotenoid content was measured from chili fruit harvested at 35 and 65 days after planting. Comparison of the partial sequence of Ccs gene of the mutants and the Capsicum annuum var. Landrace, showed a substitution of adenine into guanine in the 124 bp nucleotide base sequence and the cytosine nucleotide substitution to thymine in the 556 bp nucleotide base sequence. However there was no difference between the Ccs gene sequence of the chili pepper G1M6/13, G1M6/7, G1M6/8 compared to the control plant (G1K1). The consistency showed by these sequence did not reflect the carotenoid content. Carotenoid content in the three mutant plants varied compared to control plants. The G1M6/8 mutant had higher carotenoid content, while the G1M6/13 mutant had a lower carotenoid content compared to the control plant. Since there was no differences on the partial sequence of the Ccs gene observed, the deviation of carotenoid content between mutant and control plants maybe governed by other part of the gene.

Single-molecule real-time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.).

SummaryThe diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1,PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.

Stepwise pathway engineering to the biosynthesis of zeaxanthin, astaxanthin and capsanthin in rice endosperm

Carotenoid pigments are valuable components of the human diet. A notable example is β-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond β-carotene, we specifically used a Capsicum β-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus β-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart β-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 μg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 μg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 μg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on β-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension.

Comparative transcriptome analyses of a late-maturing mandarin mutant and its original cultivar reveals gene expression profiling associated with citrus fruit maturation.

Characteristics of late maturity in fruit are good agronomic traits for extending the harvest period and marketing time. However, underlying molecular basis of the late-maturing mechanism in fruit is largely unknown. In this study, RNA sequencing (RNA-Seq) technology was used to identify differentially expressed genes (DEGs) related to late-maturing characteristics from a late-maturing mutant ‘Huawan Wuzishatangju’ (HWWZSTJ) (Citrus reticulata Blanco) and its original line ‘Wuzishatangju’ (WZSTJ). A total of approximately 17.0 Gb and 84.2 M paried-end reads were obtained. DEGs were significantly enriched in the pathway of photosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, chlorophyll and abscisic acid (ABA) metabolism. Thirteen candidate transcripts related to chlorophyll metabolism, carotenoid biosynthesis and ABA metabolism were analyzed using real-time quantitative PCR (qPCR) at all fruit maturing stages of HWWZSTJ and WZSTJ. Chlorophyllase (CLH) and divinyl reductase (DVR) from chlorophyll metabolism, phytoene synthase (PSY) and capsanthin/capsorubin synthase (CCS) from carotenoid biosynthesis, and abscisic acid 8′-hydroxylase (AB1) and 9-cis-epoxycarotenoid dioxygenase (NCED1) from ABA metabolism were cloned and analyzed. The expression pattern of NCED1 indicated its role in the late-maturing characteristics of HWWZSTJ. There were 270 consecutive bases missing in HWWZSTJ in comparison with full-length sequences of NCED1 cDNA from WZSTJ. Those results suggested that NCED1 might play an important role in the late maturity of HWWZSTJ. This study provides new information on complex process that results in the late maturity of Citrus fruit at the transcriptional level.