Transgenic Tobacco Lines Expressing Sense or AntisenseFERROCHELATASE 1RNA Show Modified Ferrochelatase Activity in Roots and Provide Experimental Evidence for Dual Localization of Ferrochelatase 1

Abstract

In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 down-regulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to re-examine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.

Complementation studies of the Arabidopsis fc1 mutant substantiate essential functions of ferrochelatase 1 during embryogenesis and salt stress

“…We are aware that recent reports have cast doubt on the translocation of FC into Arabidopsis mitochondria (Lister, Chew, Rudhe, Lee, & Whelan, ; Woodson et al, ). Nevertheless, we recently demonstrated FC activity in the mitochondria of tobacco plants, supported by immunological evidence for FC (Hey et al, ). In parallel, studies on the recently characterized ATHEMN1 ‐encoded coproporphyrinogen oxidase have shown that this protein is translocated into mitochondria (Pratibha et al, ).…”

“…In the present work, comparison of FC1 and FC2 mRNA levels in wild-type embryos suggests that FC1 is the predominant FC isoform during embryo maturation. The successful complementation of the (Hey et al, 2016). In parallel, studies on the recently characterized ATHEMN1encoded coproporphyrinogen oxidase have shown that this protein is translocated into mitochondria (Pratibha et al, 2017).…”

“…The two isoforms of ferrochelatase, FC1 and FC2, are assumed to act in the synthesis of two different haem pools (Espinas et al, 2016;Hey et al, 2016;Nagai et al, 2007;Scharfenberg et al, 2015;Woodson et al, 2011). Their functional differences were proposed to be due to dissimilarities in the expression of their genes and their protein structure (Chow et al, 1998;Nagai et al, 2007;Woodson et al, 2011).…”

“…Specific up-regulation of FC1 transcripts by stress has been confirmed by coexpression analysis of both genes. The distinct expression profiles of the two FC genes suggest that both isoforms supply different haem pools (Espinas et al, 2016;Hey et al, 2016;Woodson et al, 2011).…”

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics

“…In plants, the heme synthesis pathway is located in the plastids (Tanaka and Tanaka, ), but its last step, i.e. the chelation of iron, was proposed to be also performed in mitochondria (Chow et al ., , ; Hey et al ., ), however this has been disputed (Lister et al ., ; Masuda et al ., ; Woodson et al ., ). Several proteins involved in heme biosynthesis were identified in our analysis (Dataset S3, worksheet #8), albeit at low copy numbers (<80 copies), indicating etioplast contamination of our mitochondria isolations.…”