KNOCKOUT OF FRATAXIN GENE CAUSES EMBRYO LETHALITY IN ARABIDOPSIS

Friedreich ataxia (FRDA) is an autosomal recessive neurological disease affecting up to 1:30.000-50.000 individuals in the Caucasian population. This form of ataxia, a neurological dysfunction which severely impairs motor coordination, also causes cardiomyopathy and diabetes, with consequent reduction of life expectancy. FRDA is caused by a deficiency in frataxin, a mitochondrial targeted protein. Frataxin deficiency dramatically affects mitochondrial metabolism in all different model systems studied so far (e.g. mice, Saccharomyces cerevisiae, Caenorhabiditis elegans). Different roles proposed for frataxin include involvement in the process of iron homeostasis and storage, stimulation of oxidative phosphorylation, as well as iron-chaperone activity for heme synthesis or iron-sulphur cluster assembly and repair. It has also been recently proposed that frataxin could have a more general role as a regulator of Fe metabolism and trafficking in mitochondria. Recently, a frataxin homolog has been identified in the model plant Arabidopsis: this plant frataxin, named ATFH, shares 65% identity with human sequence and it is a functional isoform able to restore normal rate of yeast respiration and growth in Saccharomices cerevisiae mutant strain yfh1, which is deficient in frataxin. We applied the in situ hybridisation tecnique on flowers prior to fertilization, as well as on embryos at different Days After Pollination (DAP) by using, as labelled probe, a RNA sequence spanning the last exon of AtFH and part of the 3’UTR. We show that AtFH is expressed in ovule primordia as well as in stamen prior to fertilization. Also, AtFH transcript accumulates in embryos at different stages up to mature seeds where it accumulates in the epidermis as well as in the root meristem of fully developed embryos. To investigate AtFH role during development, we analysed two independent Arabidopsis insertional mutant lines, S-594203 and S-622008 (Salk collection, http://www.Arabidopsis.org), with T-DNA inserted in the coding region of AtFH gene. Screening of hundreds of progeny plants of the two heterozygous mutant lines failed to identify plants homozygous for such T-DNA insertions. In particular, screening of the progeny of Salk-594203 heterozygous mutant (indicated from now on as AtFH/atfh) reveals that not only the genotypic class atfh/atfh is missing, but also that the segregation ratio of the expected remaining genotypic classes AtFH/AtFH:AtFH/atfh is 1:2. The heterozygous AtFH/atfh plants show no obvious phenotype, and indeed they are phenotypically indistinguishable from wt plants by naked eye. However microscopy inspection of siliques produced from AtFH/atfh plants, at different DAP, reveals that indeed around ¼ of the seeds in the siliques of AtFH/atfh plants are dramatically reduced in size and will not reach maturity, leaving a “hole” in the mature silique. We also sampled seeds from immature siliques of AtFH/atfh plants, with either wt or defective phenotype and analysed them by Nomarski optics; results show that embryos in seeds of regular size also show normal development, whereas embryos in defective seeds arrest at globular stage and cannot proceed further into development. All these evidences suggest that such defective embryos truly correspond to the missing genotypic class atfh/atfh. Our results show that plant frataxin has a key role in planta: lack of frataxin indeed severely affects development of Arabidopsis embryos which arrest at globular stage. Interestingly, mice embryos lacking frataxin die early in uterus suggesting that physiological role of plant frataxin may be similar to the mammalian counterpart. Also, we show that reatment with 100 or 500 M Fe-citrate, able to induce the accumulation of the Arabidopsis AtFer1 ferritin isoform in liquid cell cultures, does not affect frataxin transcript levels indicating that frataxin could act, in plants, not as a mitochondrial ferritin-like protein. However the relationship between plant frataxin and mitochondrial iron metabolism, and in particular the relationship between frataxin and ferritin necessitate further elucidation and are currently under investigation.