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

Abstract

Riboflavin secretion by Hyoscyamus albus hairy roots under Fe deficiency was examined to determine where riboflavin is produced and whether production occurs via an enhancement of riboflavin biosynthesis or a stimulation of flavin mononucleotide (FMN) hydrolysis. Confocal fluorescent microscopy showed that riboflavin was mainly localized in the epidermis and cortex of the root tip and, at the cellular level, in the apoplast. The expressions of three genes involved in the de novo biosynthesis of riboflavin (GTP cyclohydrolase II/3,6, riboflavin synthase) were compared between Fe-starved and Fe-replete roots over a time course of 7 days, using RT-PCR. All three genes were found to be highly expressed over the period 1-7 days in the roots cultured under Fe deficiency. Since riboflavin secretion began to be detected only from 3 days, there was a lag phase observed between the increased transcript accumulations and riboflavin secretion. To determine whether FMN hydrolysis might contribute to the riboflavin secretion in Fe-deficient root cultures, FMN hydrolase activity was determined and was found to be substantially increased after 3 days, when riboflavin secretion became detectable. These results suggested that not only de novo riboflavin synthesis but also the hydrolysis of FMN contributes to riboflavin secretion under conditions of Fe deficiency. Respiration activity was assayed during the time-course, and was also found to be enhanced after 3 days under Fe deficiency, suggesting a possible link with riboflavin secretion. On the other hand, several respiratory inhibitors were found not to affect riboflavin synthase transcript accumulation.Key words: Hyoscyamus albus; hairy roots; iron deficiency; riboflavin secretion; riboflavin biosynthesis; FMN hydrolase; respiration. RibA: GTP cyclohydrolase II · RibB: 3,4-dihydroxy-2-butanone 4-phosphate synthase · RibC: 6,7-dimethyl-8-ribityllumazine synthase · RibD: riboflavin synthase

Identification and Characterization of RibN, a Novel Family of Riboflavin Transporters from Rhizobium leguminosarum and Other Proteobacteria

“…It has been reported that iron deficiency induces riboflavin biosynthesis and secretion in the roots of some plants. This would make riboflavin available as a nutrient or signal molecule for bacteria in the rhizosphere (39)(40)(41). It is tempting to speculate that bacteria harboring a riboflavin transporter, in addition to a complete riboflavin biosynthesis pathway, are able to economize in synthesizing riboflavin when it is available from the environment.…”

Does Abiotic Stress Cause Functional B Vitamin Deficiency in Plants?

“…Similarly, temperature extremes, high light levels, pH excursions, and stress-driven accumulations Table I. of metabolites with which cofactors react all directly accelerate diverse types of spontaneous cofactor damage (Treadwell and Metzler, 1972;Baggott, 2000;Mills et al, 2006;Marbaix et al, 2011). Abiotic stresses can also promote cofactor damage indirectly by altering compartmentation (Akhtar et al, 2010;Mohammadi et al, 2012) by inducing enzymes that break down cofactors (Rapala-Kozik et al, 2008;Higa et al, 2012) and by creating harsh cellular conditions in which enzymes that normally act on other substrates become more promiscuous (Piedrafita et al, 2015) and mistakenly attack cofactors. Figure 1 uses color-coded arrows to show the site and nature of damage reactions that vitamins and cofactors can undergo in physiological conditions, and Table I catalogs the reactions corresponding to the arrows.…”

A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency

“…In the case of Cyp80F1, primer pairs were used, according to the previous report (Li et al, 2006). The expression of riboflavin synthase ( RibD ) was also determined as a representative gene involved in riboflavin biosynthesis, according to our previous report (GenBank accession, AB712370) (Higa et al, 2012). Primers used were as follows: H6H , forward (5′-GGTCTCTTTCAGGTGATCAA-3′) and reverse (5′-CTTCACAGATGTAGTCCAGCA-3′); Cyp80F1 , forward (5′-CACAGTTGAATGGACATTGGTGGAGC-3′) and reverse (5′-GAACAGTAATGGCGCCGGAGGATGC-3′); RibD , forward (5′-GTTGTCGGAATTTAGTGTCGG-3′) and reverse (5′-TCCCAGTCTTGACCTTCACC-3′).…”

“…It is noteworthy that complexes I and II contain a large number of Fe ions, whilst AOX does not contribute to the generation of a proton gradient (Ohnishi, 1998; Taiz and Zeiger, 2002; Vigani et al, 2009). On this basis, we have proposed that riboflavin secretion occurs as a result of the underuse of flavoprotein complexes I and/or II (Higa et al, 2010), although both increased de novo riboflavin synthesis and hydrolysis of FMN could be involved in riboflavin secretion (Higa et al, 2012). On the other hand, it has been proposed that flavins accumulated in the roots may act as electron donors or as cofactors for Fe (III) reductase (López-Millán et al, 2000; Rodríguez-Celma et al, 2011a,b), because the Fe reductase contains FAD as a cofactor (Schagerlöf et al, 2006).…”