| Vitamin C (VC), the most abundant water-soluble small molecular antioxidant in plants and eukaryotic algae, plays roles in plant development and response to abiotic and biotic stresses. It has been verified that there are several pathways of VC biosynthesis in plants, including L-galactose pathway, Gulose pathway, Uronic acid pathway, and myo-inositol pathway. The L-galactose pathway integrates VC biosynthesis to carbohydrate metabolism, amylase synthesis and protein glycosylation, which makes it to be the major route of VC synthesis in plants. VTC1 encodes one key enzyme GDP-D-Mannose pyrophosphorylase (GMPase). VC-reduced Arabidopsis thaliana mutant vtc1-1 habors a mis-sense mutation, resulting in the enzyme activity reduction by 35% compared with that of wild type without any changes of VTC1 transcripts. And the VC contents in the mutant decrease to 25-30% of that in wild type, consequently increasing the sensitivity to ozone, UV-B, SO2, high light, salt stress and H2O2.Light intensity is the major environmental factor affecting leaf VC contents, meanwhile VC plays a role in preventing plants from light-induced damage. The reactive oxygen species (ROS) produced in photosynthesis, photorespiration and oxidative stresses could be scavenged through VC transformation from reduced to oxidized status. The key enzymes of L-galactose pathway in A. thaliana are light-regulated in transcript level, and expression of VTC1,GPP,GalLDH,VTC2 increase in constant light-acclimated leaves and decrease under constant darkness. However, the mechanism that how light regulates VC synthesis, especially in post-translation level, is still not clear.In this work, we used the yeast two-hybrid system to isolate proteins able to interact with AtVTC1. By the system, we have isolated 3 putative proteins that might interact with AtVTC1, screened from a 3 day-old etiolated Arabidopsis seedling cDNA library. One of the proteins is CSN5B known as a subunit of COP9 signalosome (CSN), which is consist of 8 subunits named CSN1-8. The CSN complex plays a part in promoting an alternative developmental program (skotomorphogenesis to photomorphogenesis) through ubiquitin degradation pathway in response to a changing light environment. By monitoring the derubylation status of SCF E3 ubiquitin ligases, CSN participates in ubiquitin degradation system, CSN5 is relatively independent to the whole complex, the single mutated CSN5 would results in the phenotypes of all CSN mutants, and CSN5 has a crucial modulation in plant development.After identification of the interaction between AtVTC1 and AtCSN5B by way of yeast two hybrid, BiFC, pull-down and CoIP, we then detected plants VC content and tolerance to oxidative stress. The VC content in csn5b mutant, complete lack of the CSN5B transcripts, was 20% more than that in wild type, although seedlings did not show obvious different phenotypes between wild type and csn5b. The transgenic lines overexpressing CSN5B decreased VC content, compared to that of wild type, which resulted in the ROS in OECSN5B increased. And AtVTC1 protein quantities in OEVTC1/OECSN5B were apparently less than that in OEVTC1.The results above indicate that CSN5B plays an important role in modulation of plant VC synthesis and in influence of plant response to oxidative stress in A. thaliana, as a result of the degradation of VTC1 protein by CSN5B.
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