| In eukaryotic cells,endocytosis transports the components of plasma membrane into the cell through endosomes formed by plasma membrane invagination.Once inside the cell,the cargo in the endocytoic endosomes can undergo modification,degradation or subcellular relocalization,which are very important for maintaining cell function and communication.Multivesicular bodies(MVBs)are late endosomes in the endocytotic pathway known for their intralumenal vesicles,whose cargo can be degraded upon fusion with lysosomes or vacuoles.The formation of intralumenal vesicles in MVBs is catalyzed by the ESCRT(Endosomal Sorting Complexes Required for Transport)protein complex,which depends on SKD1(Suppressor of K~+transport Growth Defect1)ATPase to provide the required energy.As an activator of SKD1 ATPase,LIP5(LYST-Interacting Protein 5)is a regulatory factor for MVB biogenesis.Molecular genetics studies have shown that LIP5 has no significant effect on the growth and development of Arabidopsis under normal conditions.Under stress conditions,on the other hand,the protein stability of LIP5 is enhanced through phosphorylation,leading to increase in cellular vesicle trafficking to pomote plant disease resistance,salt and heat tolerance.In order to further understand the biological function and action mechanism of LIP5,this study has analyzed the effect of Arabidopsis LIP5 gene mutation on the accumulation and tolerance of heavy metal Cadmium(Cd)and explored the molecular pathway by which LIP5 regulates plant salt tolerance through isolation of salt tolerant suppressor of lip5 mutant(slip).This study has also generated rice oslip5 mutants by CRISPR/cas9 gene editing technology,and found the mutants to have important phenotypes of severe growth defects.The main results of this study include:1?Analysis of phenotypes of Arabidopsis lip5 mutants in Cd accumulation and toleranceAt 50?M Cd,the levels of Cd in all parts of Arabidopsis lip5 mutant plants were significantly higher than that of wild-type plants(Col-0),indicating that LIP5mutation increased the uptake of Cd in roots and the transport to the aboveground parts.The growth of cotyledons and roots of Arabidopsis lip5 mutants at both germination and post-germination stages was inhibited more severely than that of wild type Col-0 when grown at 50?M and 100?M Cd.These results indicate that LIP5 mutation increases the sensitivity to Cd stress.2?Isolation and analysis of genetic salt tolerant suppressors of lip5 mutantScreens were conducted from chemically mutagenized salt sensitive lip5 mutants from Arabidopsis and 5 independent salt tolerant suppressor of lip5(slip)were isolated.Genetic analysis showed that these suppressors were caused by single dominant or recessive mutations.Crosses between salt tolerant slip5-4 and salt sensitive lip5 resulted in an F2 population in which the ratio of salt tolerant to salt sensitive progeny was close to 3:1,indicating that the salt tolerant suppressor was caused by a dominant gene mutation.Through bulk segregant analysis of the F2generation coupled with whole genome sequencing,the chromosomal region of the slip5-4 suppressor gene has been tentatively located and candidate genes identified.3?Generation of rice lip5 mutants using CRISPR/cas9 technologyThe CRISPR/Cas9 gene editing vector for rice LIP5 gene was constructed,and13 independent transgenic rice lines were obtained.From these transgenic lines,four types of LIP5 gene mutations were identified by DNA sequencing,including three knock-out mutations that cause translation frameshifting of the LIP5 gene.It was found that the homozygous lip5 knockout mutants germinated and grew normally during early seedling stages.However,the mutants grew at a reduced rate at 2-3weeks post germination and started to develop lesions in the leaves.With increased age,the growth of the mutants further declined and lesions expanded,leading to ultimate death of the mutant plants.Therefore,unlike Arabidopsis LIP5 gene,rice LIP5 gene also plays an important role in plant growth under normal conditions. |
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