| Iron(Fe)plays an important role in the growth and development of higher plants.Deficiency of Fe can cause damage to the structure and function of plant cells,lead to the destruction of peroxidase,catalase and other enzyme structure or function,and result in the dysfunction or inhibition on related enzyme system as well as the metabolism.In addition,absence of excess excitation energy from photosynthesis of Fe-deficient leaves and the accumulation of non-coupled electron currents in respiration electron transport chains can generate a large amount of reactive oxygen species(ROS).Therefore,the oxidative damage of ROS to membrane lipid,protein and DNA biomacromolecule is an important way of plant damage under iron stress.Lon protease is a class of highly conserved ATP-dependent proteases that are widely found in organisms.As a member of the protease family,Lon protease play an important role in degrading damage or misfolded proteins.The researches on Arabidopsis thaliana showed that Lonl protease was localized in chloroplast and mitochondria,which is involved in the organogenesis and morphogenesis of seedlings.It has the function of degrading oxidized protein and maintaining the normal metabolism of cells.Although Lonl protease has been known to regulate the protein level of the organelle to be located and play an important role against intracellular oxidative damage,its function in regulating the accumulation of ROS and exacerbating the oxidative damage is still unclear.Besides,the mechanism of action of oxidative stress caused by stress has not been reported yet.In the present study,the cDNA sequence of VcLonl was amplified from Vaccinium spp.(GenBank accession number:MF972079).Based on the analysis of temporal and spatial expression pattern of VcLon1 in blueberry,we obtained the VcLon1 overexpression tobacco seedlings through genetic transformation technique and silenced the expression of the homologue NbLon1 using RNAi method.Afterwards,the transgenic seedlings were used to analyze the funtions of Lon1 protease involved in Fe metabolism in tobacco.The results showed that Lon1 protease could degrade carbonyl compounds in organelles such as chloroplasts and maintain the activity of related antioxidant enzymes in chloroplasts,thus maintaining the physiological metabolism in plants and increasing their tolerance to Fe deficiency.Specific results are as follows:The ORF of VcLon1 was 2982 bp,which encoding 993 amino acids.The theoretical isoelectric point was 5.44 and the molecular weight was 109.5 kDa.The protein sequence encoded by VcLon1 contains a typical AAA + domain and belongs to the AAA+ superfamily.The localization analysis showed that the coding protein was expressed in chloroplast and mitochondria,and had close relationship with Lon1 protease in grape and apple,arabidopsis also has a high homology(amino acid similarity 75%).After 30 days of Fe deficit treatment,blueberry seedlings were subjected to morphological and oxidative analysis.Fe deficit seedlings exhibited the typical Fe deficit symptoms such as chlorosis in newly-bomed leaves.Meanwhile,Fe deficiency significantly increased the H2O2 in leaves,and the content of carbonylated protein in blueberry leaves increased by 65.01%compared with the control under the condition of Fe deficiency.It indicated that the iron deficiency stress had broken the balance between reactive oxygen species production and removal of ROS in blueberry leaves,thus the leaves are oxidatively damaged.The qPCR analysis revealed that VcLon1 was expressed ubiquitously throughout the blueberry plant,with relatively lower expression levels in older tissues,but iron stress significantly increased its transcription level in blueberry.The results showed that under the stress conditions VcLon1 up-regulated expression in response to the accumulation of oxidative damage,indicating its biological function greatly involved in iron deficiency tolerance in blueberry.To further examine the role of VcLon1 in iron deficiency tolerance,we generated transgenic tobacco lines that constitutively overexpress VcLon1.Phenotypically,the T2 transgenic lines(1-1?1-6?1-7)appeared similar to the WT counterparts when grown under non-stress conditions.However,under the Fe deficiency stress,the growth of VcLonl overexpression plants was stronger than the wild type.Comparatively,Fe deficiency induced chlorosis was observed in WT,and the content of carbonylated protein increased significantly which accomplied with the chloroplast slightly swollen and glabrous layer structure.And the chlorophyll content also decreased significantly along with the increase of Fe deficit severity.However,VcLon1 over-expressed lines still maintained normal chloroplast structure and contained 87.8%chlorophyll content under 1/4 Fe-treated condition.In contrast,the chlorophyll content of WT plants had dropped to 66.9%of the control.Meanwhile,after 1/4 Fe treatment for 30 d,the content of MDA in the VcLon1 over-expressed lines was only 1.8 times of that in control,which was 36.5%lower than WT plants in 1/8 Fe treatment.In addition,the over-expressed lines also reduced the accumulation of H2O2 and increased the activities of antioxidant enzymes such as SOD and APX.The results showed that VcLon1 protease can effectively control the quality of the important proteins in the organelle and regulate the activity,thereby significantly reducing the distribution of H2O2 in leaves and decreasing the lipid peroxidation.So that the content of carbonyl group in the leaves was maintained at a relatively low level,thus keep the normal metabolism of plants and improve their Fe deficit tolerance.The results of overexpression analysis was further comproved by generating transgenic plant lines in which the endogenous expression of the NbLon1 gene,which encodes a protein with a high degree of sequence similarity to VcLon1,was reduced by RNAi gene silencing.Although,NbLon1-silenced seedlings(141-G?141-H?141-I)displayed no significant differences in growth parameters between WT and NbLon1-silenced plants under standard growth conditions.Under Fe deficiency stress,the growth of NbLon1-silenced seedlings were weaker than wild type and exhibited the typical Fe deficiency induced chlorosis in leaves.The chloroplast structure was more severely damaged than that of WT,with more swollen chloroplasts and blurred grana lamella observed.The number of damaged chloroplasts were more than 50%,and the chlorophyll content also showed a downward trend.At the same time,the MDA and carbonyl protein content in NbLon1-silenced seedlings leaves also increased significantly than in WT plants.In addition,the level of DAB staining in the leaves of NbLon1-silenced seedlings was observed an increase and the activities of APX and SOD was decreased exposed to Fe deficit tolerance.NbLon]-silenced tobacco seedings showed suceptibilty to abiotic stress such as Fe deficiency and significantly higher accumulation of ROS and oxidation products with damaged structure and function of chloroplast.It is indicated that Lon1 protease may play a pivotal role in maintaining the activity or content of antioxidant enzyme in chloroplast and controlling the quality of protein such as carbonyl protein in cells. |
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