Yi Wu

Pepper (Capsicum spp.) is one of the most widely cultivated and consumed vegetable crops worldwide. Besides its economic importance, pepper fruit is also an important source of health-related metabolites. These metabolites include flavonoids, carotenoids, chlorophylls, vitamin E and other antioxidant compounds. Many of these metabolites are associated with fruit development, plant stress responses and nutritional quality. In addition, these metabolites contribute to the nutritional value of the human diet and can help to reduce oxidative stress which is associated with prevention of chronic diseases. The accumulation of these metabolites varies widely among pepper accessions, providing opportunities to investigate their genetic regulation and identify candidate genes for improving fruit quality. This thesis aims to improve our understanding of genetic regulation of health-related metabolites in pepper fruits, with a particular focus on flavonoids, anthocyanins and isoprenoid-related non-polar metabolites such as carotenoids, chlorophylls and vitamin E. To achieve this, several complementary approaches were applied including metabolite profiling, QTL mapping, transcriptome analysis, bulked segregant analysis and virus-induced gene silencing. Together, these approaches enabled the identification and functional validation of candidate genes involved in the regulation of target metabolites. Chapter 2 focuses on the genetic regulation of flavonoid accumulation in pepper fruits. A major QTL for flavonoid content was identified on chromosome 5 and a CaMYB12-like transcription factor, homologous to the major known tomato flavonoid regulator SlMYB12, was selected as the candidate gene within this QTL region. Its function was validated by silencing of CaMYB12-like in the high flavonoid line ‘Long Sweet’, which showed significantly reduced expression of flavonoid biosynthetic genes and significantly decreased flavonoid accumulation. In addition, expression of CaMYB12-like locus in a tomato SlMYB12 mutant enhanced the accumulation of flavonoids in the fruit peel. These results demonstrate that CaMYB12-like is a major regulator of flavonoid biosynthesis in pepper fruits and represents a key gene for breeding pepper varieties with improved nutritional quality. Chapter 3 goes further on the analysis of flavonoid regulation by focusing on the R2R3-MYB transcription factor family in Capsicum spp. and anthocyanin degradation. Candidate R2R3-MYB repressors were identified by screening for conserved repression motifs such as the EAR motif. As a result, CaMYB101 was selected for further validation. Virus-induced gene silencing (VIGS) of CaMYB101 resulted in significantly increased tissue specific expression of anthocyanin biosynthetic genes, higher accumulation of anthocyanins and more intense purple pigmentation in both ovaries and leaves, but not fruit, compared to the wild type. These results suggest that CaMYB101 acts as a negative regulator of anthocyanin biosynthesis and is likely part of a more complex regulatory network controlling anthocyanin accumulation in pepper. Chapter 4 shifts the focus from flavonoids to isoprenoid-related non-polar metabolites in pepper fruits including carotenoids, chlorophylls and vitamin E. Several QTLs related to carotenoids, chlorophylls and vitamin E were detected and mainly localized on chromosomes 1, 6 and 10. Notably, multiple QTLs co-localized, suggesting potential shared genetic regulation of different branches of isoprenoid metabolites. Candidate genes were identified within these regions including genes involved in carotenoid, chlorophyll and vitamin E biosynthesis, such as STAY-GREEN (SGR) and CHLI for chlorophyll metabolism, LUT1 and CCS for carotenoid biosynthesis, and GTOMC/γ-TMT/VTE4 for vitamin E biosynthesis. Chapter 5 further investigates the genetic regulation of vitamin E biosynthesis in pepper fruits. A candidate gene, CaGLK2, was identified underlying the QTL at the top of chromosome 10. Silencing of CaGLK2 caused a significant reduction in chlorophyll accumulation and visible changes in fruit color from dark green in non-silencing fruits to light green in silenced fruit tissue. Silencing of CaGLK2 also affected the accumulation of α-tocopherol. However, the effect of CaGLK2 in ripe fruits was not significant compared to unripe fruits, suggesting that CaGLK2 is not the major factor at the ripe-fruit stage. Therefore, bulked segregant analysis (BSA) and RNA-seq analysis revealed the most significant QTL on chromosome 10 with a peak at ~212 Mb and a region between 206 to 213 Mb, associated with chloroplast function and chlorophyll biosynthesis related processes. In Chapter 6, the summary of the major findings in this thesis integrates with a discussion of the genetic regulation of health-related metabolites in pepper fruits from a broader perspective, discussing how different pathways are connected through transcriptional regulation, plastid development and precursor supply during fruit development.