Study On Chromoplast Carotenoids Accumulation Tuber Root Of Cassava

Carotenoids are plant indepensable secondary metabolites,involved in a series of biological process,such as photosynthesis,antioxidation and hormone biosynthesis.At the same time,Carotenoids are also essential compounds for human health,suppressing the develpment of many chronic diseases and protect heart form cardiovascular disease.Carotenoids biosynthesis is believed to occur and store in chromoplasts which are non-photoynthetic plastids offen present in fruits,flowers and storage root.Therefore the study of mechasim underlying cassava storage root chromoplast struture and different cassava carotenoid content is valuable for both theoretical significant and applied value.In this study,we used four cassava lines to investigate the mechanism leading to the diversity of carotenoid patterns in cassava storage root different peroid.The main results are as follows:1.The expressions of genes and enzymes related with carotenoid metabolic pathway in the storage roots of cassava swelling and mature periods was validated using white cassava SC6068,yellow cassava SC9,purple-leaf and yellow cassava BGM019 and pink cassava Mirasol as research materials.The results show:the key of carotenoid biosynthesis genes(PSY2?LCYB,HYD)and degradation genes(ZEP?CCD1?NCED3)expression changed may cause the different cassava lines carotenoid content.These genes may use as the marker genes to determine yellow cassava and pink cassava varieties.Meanwhile Western blot analyzed the ROS enzymes(GR?APX?Fe-SOD)expression was found during the mature period these enzymes were high expression than swelling period.But these enzymes expression had no different at the same period which suggested the ROS enzymes participated in plastid development.2.The results showed that exogenous ABA and sodium tungstate was incresead the?-carotene content in the tuber root.Both exogenous ABA and sodium tungstate incresead carotenoid biosythase key genes PSY2 and LCYB expression,meanwhile it restrain the degradtion genes ZEP and NCED3 expression.Which can explain the change of ?-carotene content at the level of transcription.The LCYB and ZEP transcription were consistent with translation.Plastid associated gene PAP1 and enzyme FBNla were upregulation which promote the number of plastidglobules.Exogenous ABA and sodium tungstate accelerated the reactive oxygen(ROS)related superoxide dismutase(Fe-SOD and Cu/Zn-SOD)expression which protect the carotenoid stable.These three factor combination incresead the tuber root p-carotene content.3.We reported a comprehensive protemic analysis of chromoplasts purified from cassava storage root using Percoll density gradient centrifugation.The optical mircoscope showed the 40%-50%of Percoll density gradient has many of intact chromplasts.Western blot showed that the expression level of mitochondrial marker Vdacl was the lowest and plastid marker RbcL was highest.34 differentially expression protein spots were detected in SC9 compared with the SC6068(control),of which 17 were up-regulated and 17 were down-regulated.The different spots belong to 4 metabolism processes:carbohydrate and energy metabolism?detoxifying and antioxidant?amino acid metabolism and protein biosynthesis.STRING metabolic network showed that Enolase 2 and elongation factor were the hub proteins,which the core of whole regulatory network.Quantitative analysis by qRT-PCR confirmed theEnolase 2 expression in high carotenoid cassava SC9 was significantly up-regulation than low carotenoid cassava SC6068.Those proteins may be the key points for affecting the carotenoid content between SC9 and SC6068.4.Drought stress affected on ?-carotene accumulation and expression of carotenoid biosynthesis-re-related genes.Mainly including:the drought stress significant affected PSY2?HYD?ZEP expression and LCYB?ZEP enzymes activity.The plastid associated genes(PAP1)and enzyme(FBNla)expression were also great inhabited by soil drought.The cassava storage root endogenous hormone measure by MS/GS showed the ABA and GA3 content was increased but the JA and IAA content was decreased.The NCED3 down-regulation may cause the ABA content decrease and JA content changed was great affected the ROS enzymes expression.Analysis the comparative proteomics was found the ROS related enzymes and carotenoid biosynthesis enzymes consistent with cassava storage root chromoplasts differential proteins.The drought stress cause the carbohydrate and energy metabolism?chaperone?protein biosynthesis?structure protein expression variation.Carbohydrate and energy metabolism protein Enolase and molecular chaperone HSP70 was the core proteins that located on the interaction network.Furthermore the core protein HSP70 down-regulation was consistent with the western blot result.Previously identification the protein Enolase 2 was down-regulation under the drought stress.The soil drought had great influence on hormone?carotenoid biosynthesis-re-related genes and enzymes expression and proteome,which resulting in a significant reduction in the?-carotene content in cassava storage root.