Copper excess promotes propagation and induces proteomic change in root cultures of Hyoscyamus albus L.

Hyoscyamus albus L. seedlings respond positively to copper (Cu) excess. In the present study, to understand how roots cope with Cu excess, propagation and proteome composition in the presence of Cu were examined using a root culture system. When H. albus roots were cultured in a medium without Cu, root growth deteriorated. However, in the presence of Cu, root growth increased in a concentration-dependent manner, and vigorous lateral root development was observed at 200 µM Cu. Cu accumulation in the roots increased with the Cu supply. Subcellular fractionation revealed that the highest amount of Cu was present in the cell wall-containing fraction, followed by the soluble fraction. However, the highest specific incorporation of Cu, in terms of fresh weight, was in the mitochondria-rich fraction. High Cu levels enhanced respiration activity. Comparative proteomic analysis revealed that proteins involved in carbohydrate metabolism, de novo protein synthesis, cell division, and ATP synthesis increased in abundance, whereas the proteasome decreased. These results indicate that Cu promotes propagation of H. albus roots through the activation of the energy supply and anabolism. Newly propagated root tissues and newly generated proteins that bind to Cu may provide space and reservoirs for deposition of additional Cu.

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

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/3,4-dihydroxy-2-butanone 4-phosphate synthase; 6,7-dimethyl-8-ribityllumazine synthase; 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.

Iron availability alters ascorbate-induced stress metabolism in Glehnia littoralis root cultures.

Our previous study indicated that formation of furanocoumarin phytoalexins could be induced in Glehnia littoralis root cultures by treatment with 10-40 mM ascorbic acid (AsA). This furanocoumarin production is much less evident when G. littoralis roots are treated with AsA under iron-deficient conditions. Instead, two large unknown peaks appeared in the HPLC chromatogram, whose chemical structures were elucidated by spectroscopic methods as being 6, ß-dihydroxyphenethyl ferulate (DF) and 6-hydroxyphenethyl ferulate (HF), respectively. Their maximal level of induction was observed at 20 mM AsA, and the production of DF always exceeded that of HF. This is the first report of these compounds in G. littoralis and of the modulation of the phytoalexin biosynthetic pathway in G. littoralis by iron deficiency.

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

Hyoscyamus 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 chloride (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 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.

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

Hyoscyamus 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 the root tips into the culture medium and the productivity depended on the number and size of root tips among the clones. A decline of pH was observed before riboflavin production and root development. By studying effects of proton-pump inhibitors, medium acidification with external organic acid, and riboflavin addition upon pH change and riboflavin productivity, we indicate that riboflavin efflux is not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs as a response to an internal requirement in H. albus hairy roots under iron deficiency.