Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus

Abstract

Authorship note: AH and YM have equal contribution. 3 SummaryHyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions.In order to examine which elements of the mitochondrial electron transport chain (mtETC) might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity.Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H. albus hairy roots, the alternative NAD(P)H dehydrogenases, complex III and complex IV, but not the alternative oxidase, are actively involved both in respiration and in riboflavin secretion.4

Increased de novo riboflavin synthesis and hydrolysis of FMN are involved in riboflavin secretion from Hyoscyamus albus hairy roots under iron deficiency

“…1b); it was not detected in roots cultured under Fe-replete conditions (Fig. 1a) [13,17]. In this study, therefore, we examined whether or not this secretion was associated with an up-regulation of the riboflavin biosynthetic pathway.…”

“…In our previous paper we showed, for the first time to the best of our knowledge, that of several mtETC-specific inhibitors studied, the ADX inhibitor, dicoumarol, the complex III inhibitor, antimycin and the complex IV inhibitor, KCN, but not other inhibitors, each decreased riboflavin secretion under Fe deficiency [13]. However, it was unclear how respiratory inhibitors might reduce riboflavin secretion, although such inhibitors are known to affect respiratory proteins.…”

“…Previous studies with 7-day-old H. albus roots had shown, by assay of TTC-reducing activity, that respiration was enhanced by Fe deficiency [13], even though aerobic respiration by the mitochondrial electron transport chain (mtETC) requires the participation of many Fe ions. Alongside these studies, plant-mtETC-specific inhibitor-feeding experiments in vivo were performed and, in addition, mitochondrial Fe contents were determined in both Fe-replete and Fe-deficient roots.…”

“…In a previous paper, we demonstrated that both riboflavin secretion and increased respiration occur in response to Fe deficiency in Hyoscyamus albus hairy roots [13]. We focused on aerobic respiration and the involvement of the mitochondrial electron transport chains (mtETC).…”

“…The results we obtained from feeding experiments with respiratory component-specific inhibitors suggested that, in H. albus roots under Fe deficiency, proton pumping is associated mainly with electron flow from ADX to complex III and complex IV; neither complexes I and II, which contain flavins as cofactors, nor AOX, which has no ability to pump protons, appear to be fully functional. We hypothesized that flavins not required for incorporation into fully-functional complexes I and II might be transported outside the mitochondria, reaching the apoplasts and the rhizosphere [13].…”