Preliminary Studies Of The Chloroplast Proteins PRDA1 And Frataxin

Chloroplast is the specific organelle in photosynthetic organisms. It isthe main place of photosynthesisand many other biosynthetic processes. It exists in all plants and other photosynthetic organisms. Chloroplast development and theregulation of plastid gene expressionare stringent and complex, but themolecular mechanismis not clear.These processesrequirevarious proteins.However, many such proteins have not been identified yet. In the previous wok of our laboratory, by screening an Arabidopsis RNAi library we identified a chloroplast protein named MRL7(Mesophyll-cell RNAi Library line 7)that affects chloroplast development.Later,we identified another protein named PRDA1 (PEP-Related Development Arrested 1) that is related to MRL7 and may also affect chloroplast development.Frataxin was first found in the mitochondria of mammals and proved to interact with ferrochelatase as well as many Fe-S cluster proteins. It is encoded by a nuclear gene.In humans, silencingof the Frataxingene or deficient systhesis of Frataxin protein resulted in an autosomal recessive disease named Friedreich ataxia (FRDA). Plant also containsthe Frataxinhomolog. In Arabidopsis, knock-out of theFrataxinhomologousgene (AtFH)causes embryonic lethal, but the mechanism is not clear. Even the subcellular location of the plant Frataxin homologis not clear.Itwas hypothesized to be also a mitochondria protein.However, at least one ferrochelatasegene product has been proved to locate in chloroplast, implyingthat the Frataxin homolog in plant may locate in chloroplast or in both chloroplast and mitochondria. In this study, preliminary analyses for the Arabidopsis PRDA1 and Frataxin homolog were performed and the followingresults were obtained:1. PRDA1 presents in all photosynthetic organisms, from lower to higher plants.Its transcription can be detected in all tissues examined with a higher amount in young tissues and lower amount in mature tissues.Theexpression is induced by light.2. Protein subcellular localization experiments showed that the fluorescenceof PRDA1:YFP fusion protein can overlap well with the auto fluorescence of chloroplasts and that of MRL7:CFP thatwas known to locate in the chloroplast nucleoid. Bimolecular fluorescence complementation experiments showed thatPRDA1 caninteractwith MRL7, indicating that PRDAlisinvolved in chloroplast developmentregulationtogether with MRL7.3. Detection of the plastid RNA editing sites of the PRDA1 Arabidopsis mutantshowed that PRDA1is not involved in the chloroplast RNA editing.4.Protein subcellular localization experimentsshowed that the green fluorescence of AtFH:GFP fusion protein can overlap well with the red chlorophyll autofluorescence. Co-localization experiments of AtFH:YFP and MRL7-L:CFPshowed that AtFH co-localized with the known chloroplast protein MRL7-L. These results indicate that AtFH is specifically targeted to chloroplast.