Decoding purple pepper’s dress code

Anthocyanins have long been valued as essential pigments responsible for the colouration of many plants, creating diverse hues ranging from orange, red, purple to blue. Plant anthocyanin levels, determined by both synthesis and degradation, are genetically regulated in a temporal and spatial manner in different tissues at different developmental stages; additionally, it is also influenced by environmental cues. This thesis aims to advance our knowledge regarding genetic and environmental factors that regulate anthocyanin biosynthesis and degradation.

Capsicum spp. (pepper) is an important horticultural crop. It has various fruit colours including purple. However, the fruit is only purple temporarily, at the unripe fruit stage, and the purple starts to disappear at the onset of fruit ripening, which is due to transient anthocyanin accumulation during fruit development. This transient nature can be exploited because it involves both anthocyanin biosynthesis and degradation.

In Chapter 1, I first described the genetic diversity of plant pigmentation in the Solanaceae family, especially in pepper. Then I focused on anthocyanins and reviewed the current knowledge on its biosynthesis and degradation. Findings in the genetic and environmental regulation of anthocyanins were summarised, and knowledge gaps were pointed out. Lastly, I introduced the research content of this thesis and outlined the research approaches to achieve the main objectives.

In Chapter 2, the state of the art with respect to our knowledge regarding anthocyanins in four main Solanaceous vegetables, including the chemical structure, biological function, genetic and environmental regulation of anthocyanins is described. Anthocyanin levels change not only during fruit development but also in response to light and temperature. Fruit anthocyanin discolouration is due to reduced biosynthesis, increased degradation, or a combination of both. The decrease of biosynthesis is controlled by the downregulation of MYB activators and upregulation of MYB repressors. Degradation is likely an active process in Solanaceous fruits. In this respect the lack of knowledge regarding MYB repressors and anthocyanin degradation genes in the main Solanaceous vegetables is a drawback.

To fill the knowledge gap in the study of anthocyanin-related R2R3-MYB repressors in pepper, in Chapter 3, we first carried out a comprehensive genome-wide analysis of the R2R3-MYB family in three pepper species, i.e., C. annuum, C. baccatum and C. chinense. We gained insight into the identification and potential functional prediction of all pepper R2R3-MYBs by comparing gene structure and constructing phylogenetic trees. We proposed candidate R2R3-MYB repressors from all pepper R2R3-MYBs of three species by screening repression motifs. Candidates for anthocyanin-related R2R3-MYB repressors were selected. Among them, the function of CaMYB101 as an anthocyanin repressor was tested by virus-induced gene silencing (VIGS). VIGS led to purple pigmentation (anthocyanin accumulation) in leaves and ovaries whose wildtype phenotype is green.

In Chapter 4, the effect of light intensity and light spectrum on anthocyanin accumulation was demonstrated. Anthocyanin content was hardly affected by light intensity. High blue light fractions preserved anthocyanin accumulation via up-regulating anthocyanin biosynthesis. This is supported by kinetic modelling and elevated expression of anthocyanin biosynthetic genes. High blue light fractions also slowed down several fruit ripening-related processes; anthocyanin production was higher in less ripe fruit. In contrast to biosynthesis, anthocyanin degradation is a light-independent process. But this still needs to be verified in the future.

The results obtained in Chapter 5 demonstrated that the transient anthocyanin accumulation was due to stopped biosynthesis and enhanced degradation upon fruit ripening. The biosynthetic process dominated at the early fruit development stages and two anthocyanin accumulation patterns were observed in purple accessions. The visual purple colouration, anthocyanin accumulation pattern and expression profile of anthocyanin biosynthetic genes coincided with each other. Anthocyanin degradation was a common process in all tested purple accessions. The degradation process seemed to dominate at the onset of fruit ripening, leading to the observation of metabolites that were putatively identified as anthocyanin degradation products. Oxidation and deglycosylation were proposed as two co-existing ways to degrade anthocyanin in pepper fruit. A candidate anthocyanin degradation gene, a pepper monocopper oxidase (CaMCO), was selected based on RNA-seq transcriptomic analysis of fruits from purple accessions at different developmental stages and functionally characterized by VIGS. Transient silencing of CaMCO led to a clear accumulation of anthocyanins in the silenced fruit tissue.

In Chapter 6, the new findings of this thesis were summarised to briefly answer the main research objectives raised in Chapter 1. Additional interpretations were discussed in two main themes: anthocyanin biosynthesis and anthocyanin degradation. For anthocyanin biosynthesis, I first discussed the collaboration, competition and confrontation between genetic regulators of anthocyanin biosynthesis. I put more focus on anthocyanin-related MYB activators and repressors, which regulate anthocyanin biosynthesis from two opposite directions. I believe these two types of regulators interact with and influence each other. However, a clear picture of the exact regulatory network is far from complete. Second, based on the latest knowledge of how different light spectra regulate anthocyanin biosynthesis, I suggest that further understanding of the interactions between photoreceptors would provide clues to fine-tune the light spectrum to achieve maximum anthocyanin production. Third, I hypothesize that the reason for the termination of anthocyanin biosynthesis in ripening fruit might be due to a shortage of substrate (UDP-glucose), since more glucose is transported in the vacuole due to increased activity of the monosaccharide transporter.

For anthocyanin degradation, I first suggest that anthocyanin degradation in pepper fruit is due to oxidation and deglycosylation. Based on current data and literature review, I propose two hypothetical anthocyanin degradation processes. Second, I speculate that anthocyanin degradation might be associated with intracellular redox changes in pepper fruits during ripening. To be specific, as an antioxidant, anthocyanin might be broken down by scavenging free radicals in response to a more oxidative intracellular environment to balance the redox status in response to ripening.

Finally, I present some thoughts for the methodology employed in this thesis and provide future perspectives for breeding, cultivation and postharvest storage.