A Study On The Plastid Terminal Oxidase In Arabidopsis

Photosynthesis and respiration are two very important energy-producing processes in plant cells.It was previously thought that photosynthesis occurs in chloroplasts and respiration occurs in mitochondria.The discovery of the plastid terminal oxidase(PTOX)in plants confirmed that chloroplasts also possess an oxygen-consuming respiratory electron transfer chain.PTOX is located on the chloroplast thylakoid membrane,acting as an effective safety valve to maintain the PQ pool redox balance and protect plants from photodamage.At present,our knowledge of the physiological functions of PTOX is still very limited.In this study,taking advantage of the PTOX-deficient variegation mutant immutans(im)of Arabidopsis,I examined the functional relationship between PTOX and its distantly related mitochondrial homologs AOX(alternative oxidase),and found that chloroplast targeted AOXs,not mitochondria localized AOXs,could compensate for the PTOX function.Surprisingly,I found that the C-terminal His tag could enhance the function of AOX replacing PTOX in chloroplasts.I also analyzed the regulation of PTOX in the early chloroplast development using two mutants with defects in chloroplast development.From a phylogenetic point of view,mitochondrial AOX and chloroplast PTOX are homologous.When AOXs were engineered into chloroplasts by replacing their own mitochondrial transpeptide with a chloroplast specific targeting sequence of RbcS1A,AOX1b,lc,Id,and AOX2 could completely suppressed the variegation phenotype of im,while AOXla could only partially rescued the im defect,indicating that all AOXs could act as PQH2 oxidase and replace the function of PTOX at varying degrees.Overexpressing the full-length coding sequence of AOXs in im,I found that only AOXlb and AOX2 could completely rescue the varigation defect of im,while the the other three AOX(1a,lc,1d)could not.In order to explore the subcellular localization of overexpressed full-length AOXs in plants,we performed in vivo confocal fluorescence analysis and subcellular fractionation assays.In vivo fluorescence confocal analysis showed that AOXlb and AOX2 could partially localize in chloroplasts,while AOX1a,AOX1c and AOX1d exist in mitochondria.However,the subcellular fractionation assays showed that a small amount of AOXla could also be sorted into chloroplasts.In order to further confirm the subcellular localization of AOXla in Arabidopsis,we fused the transpeptide of each AOX with the mature peptide of PTOX and expressed the fused proteins in im,I found that the transpeptide of AOX1a,AOX1b or AOX2 could guide PTOX into the chloroplasts,while the transpeptide of AOX1c or AOX1d could not.These results demonstrated that chloroplast-localized AOXs,rather than mitochondria-localized AOXs,could functionally compensate for the deficiency of PTOX in chloroplasts.The functional relevance of AOXs and PTOX revealed the interaction between mitochondria and chloroplasts and the complex mechanism of dual targeting of protein in plant cells.While studying the functional relationship between AOX and PTOX,I accidentally found that each full length AOX carrying a His tag at the C-terminus was transformed into im,and resulting transgenic plants appeared all green in leaves.The chloroplast isolation and immunoblotting analysis of these transgenic plants carrying His tagged AOXs showed that all AOXs could enter chloroplasts,and the accumulation of AOXs in chloroplasts increased significantly higher than AOXs without the C-terminal His tag.By analysis of the sub-localization of His-tagged AOXlb,it was found that the protein was present on the thylakoid membrane.When fusing the transpeptide of each AOX with the mature peptide of PTOX plus a C-terminal His tag and expressing the fused proteins in im,I found that the localization of the recombinant PTOX was not affected by the His tag.These results suggested that the C-terminal His tag could promote the transport of AOX into chloroplasts.ZDS(zeta-carotene desaturase)is a key enzyme in the desaturation reaction of carotenoid biosynthesis,and shares the PQ pool with PTOX.The weak mutant zds showed a virescent phenotype,its leaves showed slight yellowish spots at the early stage of development.But the appearance of six-week-old mutant are not significantly different from wild-type plants.In order to study the relationship between PTOX-mediated PQ redox balance and carotenoid biosynthesis in the early development of chloroplasts,I crossed im with the zds mutant and obtained the im zds homozygous double mutant.Phenotypic analysis showed that the im zds double mutant showed a very serious albino phenotype.The double mutant was stunt and lethal under strong light,and it was difficult to obtain the mutant seeds under low light.Microscpe analysis of the seed development of heterozygous plants showed that the proportion of green ovules(genotype imim/ZDS)to white ovules(genotype imim/zdszds)in the pod was about 3:1.This indicated that the absence of ZDS is a genetic enhancer of im and aggravates the developmental defects of chloroplasts in im.I speculated that the production of carotenoids in the im zds double mutant may be severely inhibited.Compared with the redox balance of the PQ pool mediated by PTOX and ZDS,the production of enough carotenoids may play a more dominate role during the early stage of the plant growth and development.Araidopsis arc6(accumulation and replication of chloroplast 6)mutant exhibits a phenotype of blocked chloroplast division.Its mature mesophyll cells only contain two enlarged chloroplasts,but the growth,development and appearance of arc6 are not significantly different from wild-type plants.In order to further explore the regulatory mechanism of PTOX involved in early chloroplast development,I crossed im with arc6 and obtained the im arc6 homozygous double mutant.It was found that the im arc6 homozygous double mutant showed a severe albino phenotype with very few green spots on its leaves.This suggested when the cells of im only contain a few chloroplasts,the growth and development of the plant will have serious defects.Thus,I speculated that the early development of chloroplasts mediated by PTOX is strictly regulated by light,and chloroplasts in the im arc6 double mutant may not overlap with each other due to the small number of chloroplasts,so that all chloroplasts are exposed to light stress to produce more severe light damage.Above all,the generation and phenotypic analysis of im zds and im arc6 double mutants provided important basis for the study of PTOX involved in the regulation of early chloroplast development in response to light stress,and open avenues for further research of the functional mechanism of PTOX.