Comparative Analysis of Fruit Metabolites and Pungency Candidate Genes Expression between Bhut Jolokia and Other Capsicum Species

Abstract

Bhut jolokia, commonly known as Ghost chili, a native Capsicum species found in North East India was recorded as the naturally occurring hottest chili in the world by the Guinness Book of World Records in 2006. Although few studies have reported variation in pungency content of this particular species, no study till date has reported detailed expression analysis of candidate genes involved in capsaicinoids (pungency) biosynthesis pathway and other fruit metabolites. Therefore, the present study was designed to evaluate the diversity of fruit morphology, fruiting habit, capsaicinoids and other metabolite contents in 136 different genotypes mainly collected from North East India. Significant intra and inter-specific variations for fruit morphological traits, fruiting habits and 65 fruit metabolites were observed in the collected Capsicum germplasm belonging to three Capsicum species i.e., Capsicum chinense (Bhut jolokia, 63 accessions), C. frutescens (17 accessions) and C. annuum (56 accessions). The pungency level, measured in Scoville Heat Unit (SHU) and antioxidant activity measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay showed maximum levels in C. chinense accessions followed by C. frutescens accessions, while C. annuum accessions showed the lowest value for both the traits. The number of different fruit metabolites detected did not vary significantly among the different species but the metabolite such as benzoic acid hydroxyl esters identified in large percentage in majority of C. annuum genotypes was totally absent in the C. chinense genotypes and sparingly present in few genotypes of C. frutescens. Significant correlations were observed between fruit metabolites capsaicin, dihydrocapsaicin, hexadecanoic acid, cyclopentane, α-tocopherol and antioxidant activity. Furthermore, comparative expression analysis (through qRT-PCR) of candidate genes involved in capsaicinoid biosynthesis pathway revealed many fold higher expression of majority of the genes in C. chinense compared to C. frutescens and C. annuum suggesting that the possible reason for extremely high pungency might be due to the higher level of candidate gene(s) expression although nucleotide variation in pungency related genes may also be involved in imparting variations in level of pungency.

Fig 1. Morphological diversity of Capsicum species.

Selected Capsicum germplasm from North East India showing contrasting phenotypes for fruit morphology, color, and fruiting habits. Accessions in 1-3^rd rows are contrasting Bhut jolokia genotypes (C. chinense), 4^th and 5^th row contains C. chinense, C. frutescens and C. annuum accessions.

Fig 2. SHU range of different Capsicum species.

Total capsaicinoids content observed in C. chinense (63 accessions), C. frutescens (17 accessions) and C. annuum (56 accessions) accessions in Scoville Heat Unit (SHU).

Fig 3. Metabolite range of different Capsicum species.

Metabolite range of C. chinense, C. frutescens and C. annuum varieties.

Fig 4. Range of antioxidant activity of different Capsicum species.

Anti-oxidant activity using DPPH assay obtained for C. chinense, C. frutescens and C. annuum varieties and represented in 25mg/ml dilutions.

Fig 5. Principal component analysis of metabolites identified using gas chromatography–mass spectrometry (GC–MS) analysis.

For GC–MS, different genotypes of C. chinense [Acc 1–63 (major accession formed red circle)], C. frutescens [Acc 64–80 (major accession formed blue circle)] and C. annuum [Acc 81–136 (major accession formed brown circle)] were analysed and the correlation variances explained by the PC1 and PC2 components are 51% and 11%, respectively.

Fig 6. Pungency and capsaicinoid biosynthesis gene expression analysis.

(A) Pungency analysis of selected Capsicum genotypes (B) Quantitative real time PCR analysis to analyze the expression of candidate genes involved in capsaicinoid biosynthesis pathway in highly pungent Bhut jolokia (Acc 23 and Acc 50), moderately pungent C. frutescens (Acc 65) and low pungent C. annuum (Acc 93 and Acc 95) accessions. The expression analysis was done in leaf, flowers, and three different stages of fruit developmental i.e. green (20 days after anthesis), Breaker (30–45 days after anthesis) and Mature stage of each genotype. The majority of the important genes involved in the capsaicinoid biosynthesis pathway (Pun 1, AMT, ACS, ACL, KAS and BCAT) were expressed very high in C. chinense accessions followed by C. frutescens. The low pungent C. annuum accessions showed very low expression of these genes. The other genes (PAL, COMT, FatA and C4H) were expressed variably among the three species. ***P<0.001