Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency

Abstract

SummaryHyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron deficient condition secreted riboflavin from root tips into the culture medium and the productivity depended on the number and size of root tips among the clones, although the addition of sucrose was essential for riboflavin production. A decline of pH was observed before riboflavin production and root development using either a root tip or propagated roots: propagated roots were employed for further work due to their lesser variation.Additions of proton-pump inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD) at 100 and 10 μM and erythrosine B at 100 μM, suppressed the pH decline at 100 and 10 μM accompanied by inhibition of riboflavin secretion and root growth; at 10 μM of erythrosine B, pH decline occurred with a moderate delay, but both growth and riboflavin efflux were inhibited. Neither inhibition of the pH decline nor riboflavin production was observed at 1 μM. To examine the necessity of acidification and riboflavin secretion by the roots themselves, artificial pH reduction of culture medium with organic acids and the addition of exogenous riboflavin with/without pH reduction were performed. When hairy roots were cultured in iron-deficient medium acidified with citric acid (pH 4.0) or malic acid (pH 3.7), pH increased rapidly to around 5 overnight, following which riboflavin production and root growth occurred. Addition of riboflavin did not affect riboflavin secretion by the roots, but acidification with citric acid (pH 4.0) helped achiever greater riboflavin production and earlier pH elevation. These results indicate that riboflavin efflux does not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs by internal requirement in H. albus hairy roots under iron deficiency.

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

“…As described previously [17], riboflavin secretion was detectable in the tips of H. albus roots cultured for more than 7 days in Fe-deficient medium without pre-culture. To confirm further the site of riboflavin secretion, 7-day old root tips, from which riboflavin was not yet visibly detectable, were subjected to confocal microscopy.…”

“…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.…”

“…Hairy roots of H. albus L. (Solanaceae) used in these experiments were established as reported previously [17]. Roots were maintained on Murashige and Skoog basal medium [26] solidified with 0.2% gellan gum.…”

“…The content was routinely determined using a UV-VIS spectrophotometer (Shimazu UV-1600) at 444 nm, as previously reported [13,17]. …”

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

“…Recently, we have also reported that Hyoscyamus albus hairy roots cultured in Fe-deficient medium secrete riboflavin into the medium (Higa et al, 2008). Interestingly, these roots can grow under Fe-deficient condition.…”