Mutation Associated with Orange Fruit Color Increases Concentrations of β-Carotene in a Sweet Pepper Variety (Capsicum annuum L.)

Abstract

Pepper is the second most important vegetable crop in Bulgarian agriculture and has become the subject of extensive breeding programs that frequently employ induced mutagenesis. The success of breeding programs can be enhanced by the efficient and integral application of different biochemical and molecular methods to characterize specific mutant alleles. On the other hand, identifying new cost-effective methods is important under a limited-resources environment. In this paper we compare the levels of five health-related carotenoid compounds of fruits (α-carotene, β-carotene, lutein, β-cryptoxanthin, zeaxanthin) between a mutant variety Oranzheva kapia (possessing high ß-carotene concentration) and a corresponding initial pepper variety Pazardzhishka kapia 794. Both varieties are intended for fresh consumption. Pepper is a major natural source of β-carotene. It was observed that fruit at both commercial and botanical maturity from mutant variety had greater α-carotene and β-carotene concentrations to the initial variety (7.49 and 1.94 times higher, respectively) meaning that the mutant was superior in fruit quality to the initial genotype. Two hydroxylase enzymes, converting α- and β-carotene to lutein and zeaxanthin, respectively, are known to exist in pepper and are encoded by two genes on chromosomes 3 and 6-CrtZ_chr03 and CrtZ_chr06. The molecular characterization of the mutant variety through locus-specific Polymerase chain reaction amplification, gene cloning and sequencing as well as expression was performed. Our results suggest that the increased ß-carotene accumulation in the mutant variety Oranzheva kapia results from a biosynthetic pathway breakdown due to deletion of CrtZ_chr03 gene.

Keywords: carotenoids; high performance liquid chromatography; molecular characterization; mutagenesis; sweet pepper; β-carotene hydroxylase.

Figure 1

Botanical maturity fruit morphology of the two pepper varieties—(A) a red fruit characteristic for the variety Pazardzhishka kapia 794 (I) and an orange fruit of Oranzheva kapia (M), (B)—orange-fruited variety Oranzheva kapia (M) (20 fruits were collected and numbered for the HPLC analysis).

Figure 2

Overview of sample preparation. (a–c) Fruit was cut in three pieces; (d) middle part was used for analysis; (e) middle part was covered by nitrogen, lyophilized and enclosed in an aluminum container.

Figure 3

Schematic diagram of analyzed carotenoid substances in mutant and initial varieties in ripe fruits. Partial schematic representation of the carotenoid biosynthetic grid. Green bars—corresponding carotenoid levels in mutant variety, red bars—corresponding carotenoid levels in initial variety. Names of analyzed carotenoids in black color.

Figure 4

Amplification of ten loci within the CrtZ_ch03 gene. Lanes 2, 4, 6, 8, 10, 12, 14, 16, 19 and 21 are from Pazardzhishka kapia 794 (PK). Lanes 3, 5, 7, 9, 11, 13, 15, 17, 20 and 22 are from the variety Oranzheva kapia (OK) mutant line. Lanes 1 and 18–100 bp are from the DNA Ladder.

Figure 5

Amplification patterns of the loci, selected for sequencing. Lanes 2, 3, 4, 7 and 10 with primers positioned in CrtZ_ch03. FW1_H2 1/b-CRT 8 R and expected length—2072 bp positioned in CrtZ_ch03 gene. Lanes 5, 8 and 13 with primers FW1_H1 1/RE1_H1 1 and expected length—1212 bp positioned in CrtZ_ch06. Lanes 6, 9 and 12 with primers FW1_H1 1/RE2_H1 1 and expected length—1863 bp positioned in CrtZ_ch06. Lanes 2, 3, 4, 5 and 6—Oranzheva kapia. Lanes 7, 8 and 9—Pazardzhishka kapia 794, Lanes 10, 11, 12 and 13 in variety Kurtovska kapia 1. Lane 1–100 bp DNA Ladder.

Figure 6

RT-PCR analysis of CrtZ_ch06 with primers BCH F/BCH R (lane 2 and 3) and CrtZ_ch03 with primers CrtZ-D–F/CrtZ–D–R (lanes 4 and 5). Pazardzhishka kapia 794 (lanes 2 and 4) and Oranzheva kapia (lanes 3 and 5). Lane 6—control amplification of elongation factor EF-1.

Figure 7

Part of the carotenoid biosynthetic pathway related to carotene metabolism including corresponding genes (in green boxes) redrawn according to [42].