| Among most cell compartments,the chloroplast is the largest pool for iron,one hand,it contributes to chloroplast metabolism,on the other hand,it is able to prevent oxidative damage,Moreover,Iron plays a significant role in photosynthetic electron transport chain,Therefore,Iron is critical for function of chloroplast and growth of plants.Besides storing iron,there are specific transporters that could release iron from chloroplast,but mechanism of it is elusive.We have found a transporter AtCBSDUF5 that is located in the inner chloroplast envelope and may release iron,AtCBSDUF5 belongs to Cystathionine-β-synthase(CBS)domain-containning proteins(CDCPs)family,which contains CBSDUF protein including a domain of unknown function(DUF21)and two CBS domains.under iron-deficiency and iron-excessive treatment conditions,the phenotypes of AtCBSDUF5 transgenic lines are significantly different with wild type,indicating that AtCBSDUF5 participates in transport of iron in chloroplast.We study a iron transporter AtCBSDUF5 which may release iron from chloroplast,Experiment methods include histochemical staining,the identification of subcellular localization,function complementation experiment in yeast and biological phenotype analysis in mutant and overexpression lines,indicating that AtCBSDUF5 is significant for growth of Arabidopsis thaliana under iron treatment.The crucial results are collected as following:(1)AtCBSDUF5 encodes 662 amino acids,which contain 14 exons and 13 introns,it may have 4 transmembrane domains predicted on TMHMM.(2)Use q RT-PCR and Pro AtCBSDUF5:GUS histochemistry to observe the AtCBSDUF5 expression pattern.The result turned out that AtCBSDUF5 is expressed in roots,leaves and flowers,and the expression level of the above-ground part is higher than the underground part,the expression level is highest in the flower and lowest in the silique.(3)Clone the gene AtCBSDUF5,connect the C terminus with GFP to construct p XZp008-AtCBSDUF5-GFP vector,the vector is transformed into Arabidopsis thaliana mesophyll cell protoplasts for transient expression using PEG-mediated transformation,according to the result,AtCBSDUF5 is located in the inner chloroplast membrane.(4)Construct p FL61-AtCBSDUF5 vector and transform it into iron-sensitive yeast mutantΔccc1 and yeast mutant Δfet3fe4 by lithium acetate,at the same time,unload p FL61 vector was transformed separately into yeast wild type BY4741 and mutants as the negative controls.It shows that in the case of poisoning,the expression of AtCBSDUF5 makes the mutant Δccc1 more sensitive,indicating that AtCBSDUF5 could transport iron,However,the expression of AtCBSDUF5 cannot compensate for the growth defect of Δfet3fe4.(5)Using T-DNA insertion mutant and overexpression transgenic lines to study the physiological functions of AtCBSDUF5.It is found that under the condition of iron deficiency,the root length of the mutant is longer than that of the wild type,the above-ground part of mutant is more vigor,in contrast,the root length of overexpression lines is shorter than the wild type,the above-ground part of overexpression lines is weaker.Under the treatment,ICP-OES was used to determine the metal ion content of different parts of wild type,mutant and overpression lines.the result shows that in the case of iron deficiency,the amount of iron content is similar in different tissues among wild type,mutant and overexpression lines.The above results indicate that AtCBSDUF5 locates the inner membrane of the cotyledon chloroplast,Under the condition of iron deficiency,the growth of mutant atcbsduf5 is better than wild type,the overexpression lines are more sensitive,according to the results of yeast function complementation experiments and phenotype analysis,it is speculated that AtCBSDUF5 is involved in the release process of iron in the chloroplast,which contributes to maintain iron homeostasis of chloroplast. |
