Understanding The Carotenoid Biosynthesis Pathway in Orange Actinidia Species
Carotenoids belong to the tetraterpene family and are involved in diverse biological processes in plants. Along with a primary role in photosynthesis, they also provide distinct colours (red, yellow, and orange) to fruit, vegetables, and flowers to attract pollinators and facilitate pollination and seed dispersal. Carotenoids provide novelty to fruits and vegetables and are beneficial to human health primarily as provitamin A, therefore, they are essential for dietary intake. The aim of this thesis is to understand and characterize carotenogenesis in kiwifruit (Actinidia species) and its regulation in the orange Actinidia species. Metabolic analysis of orange Actinidia valvata (OF), yellow A. polygama (YF), and green A. arguta (GF) revealed high accumulation of β-carotene (provitamin A) in orange and yellow fruits, whereas green fruit accumulated xanthophylls (primarily lutein). Transcriptomic analysis between OF and GF revealed upregulation of carotenoid pathway genes (DXS, PSY2, GGPPS, PDS, Z-ISO, and ZDS) in OF fruit during ripening (a contrast from GF). Increased expression of chlorophyll degradation genes (SGR, RCCR, and NYC1) in OF species explains increased chlorophyll turnover in the early ripening stages. Transient overexpression of Actinidia PSY2 (a rate-limiting step in most plant species) and SGRs in Nicotiana tabacum leaves resulted in a significant increase in carotenoids and de-greening, respectively. The rapid de-greening accompanied by β-carotene accumulation can also depend on the transcriptional regulation of the pathway and plastid diversity (for carotenoid sequestration). Key genes that encode pathway enzymes were differentially regulated, therefore, candidate transcription factors based on differential expression analysis were selected and functionally characterized in heterologous plant systems. New candidates that regulate carotenogenesis (MYB1R, DIVARICATA, bHLH154, and bHLH137) and de-greening (bZIP53, bZIP44, and WRKY53) were identified. Sequestration of carotenoids in plastids determines their concentration in diverse tissues. Microscopic analysis revealed distinct differences in plastid type, number, and ultrastructure in the flesh of the three Actinidia species with chloroplast-to-chromoplast development in orange and yellow fruits. Furthermore, over-expression of FIBRILLIN1B, a plastid-lipid associated protein, increased total carotenoid accumulation suggesting the highly expressed FIBRILLIN1B in orange kiwifruit facilitates high carotenoid sequestration. This study provides knowledge on the complex molecular mechanisms regulating carotenogenesis and de-greening in diverse kiwifruit species.
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