Abstract:
Although many of the enzymes of the carotenoid biosynthesis pathway have been characterised, the regulation of carotenoid levels remains poorly understood. From RNA-sequencing of kiwifruit with different carotenoid profiles, DnaJ20 proteins, which participate in various biological processes in plant cells, were found to be substantially higher in orange (carotenoid-rich) kiwifruit, compared to green (relatively carotenoid-poor) fruit. This implies a potential regulatory role in chlorophyll and carotenoid accumulation in kiwifruit. The role of Dna20J proteins was investigated in this thesis, along with other gene families including 1-deoxy-D-xylulose-5-phosphate synthase (DXS), phytoene synthase (PSY) and Orange (OR), which play crucial roles in the biosynthesis and storage of carotenoid in plants. Candidate genes were functionally characterised using transient expression assays in Nicotiana benthamiana by measuring their effects on chlorophyll and carotenoid contents of infiltrated leaves. Yeast-2-hybrid (Y2H) interaction tests were performed in order to test for interactions between DnaJ20 and other candidate client proteins. Gene expression levels were also assessed using quantitative PCR (qPCR) during induction of fruit colour change. Data obtained from transient expression assays suggests that the over-expression of DnaJ20 facilitates chlorophyll and carotenoid degradation. In agreement, qPCR analysis showed that DnaJ20 transcripts were present at negligible levels during the period of major carotenoid accumulation in kiwifruit. A co-ordinated role of both DXS1 and PSY1 in the production of carotenoid in kiwifruit was also suggested based on transient expression and qPCR data. No direct protein-protein interactions were found between DnaJ20 and the other candidate proteins. This thesis has functionally tested several important kiwifruit proteins involved in fruit colour change and their relationship to DnaJ20. The data presented suggest an important role for DnaJ20 chaperones working with other key enzymes to control chlorophyll and carotenoid accumulation in kiwifruit.