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.

Changes in cell size and number and in rhizodermal development contribute to root tip swelling of Hyoscyamus albus roots subjected to iron deficiency.

Root tip swelling is a common phenomenon observed when plant roots are subjected to Fe deficiency. We analysed whether an increase in cell number or an enlargement of cell width was involved in this phenomenon. Root tips of Hyoscyamus albus cultured with or without Fe were stained with fluorescent SYTO14 and analysed by confocal laser-scanning microscopy. Time-course and position-based examination revealed that the inhibition of longitudinal cell elongation and acceleration of transverse cell enlargement under Fe deficiency started from the tips and then extended towards the base during the time-course period. An increase in cell number also occurred behind the tips. In addition, the development of rhizodermal protrusions was observed on the surface of roots subjected to Fe deficiency. These results indicated that changes in cell size and number and in root hair development were all involved in root tip swelling.

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 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.

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls.

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

Ascorbic acid induces furanocoumarin production in organ cultures of Glehnia littoralis.

Exogenously supplied ascorbic acid (AsA) strongly induced furanocoumarin production in leaf and root cultures of GLEHNIA LITTORALIS, but not in cell suspension cultures, after 24 h of treatment. The dose dependency showed that both organ tissues responded well to AsA supplied at concentrations of 10 - 40 mM. For induction of furanocoumarin production, roots required contact with AsA for at least 6 h and productivity markedly increased after 8 h of treatment. This is the first report of the induction of furanocoumarin biosynthesis by AsA alone and of the detection of furanocoumarin biosynthesis in a root culture system.

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.

[Historical study on the introduction and cultivation of medicinal plants in the Edo-era. 1. A paper on the cultivation of 12 medicinal plant species.].

This article describes a two-page response written by a translator in 1721, in which a reply is given regarding an inquiry made on October 5 to a Chinese person related to the cultivation of 12 medicinal plant species imported to Japan from China. One page, dated October 7, describes the cultivation method for seven species and the other page, dated October 10, describes the cultivation method for the other five species. These plants were imported to Japan for the purpose of domestic propagation as important materials for Chinese medicine at the Jyuzengi Medicinal Plant Garden in Nagasaki, and some of them were apparently sent to Koishikawa Medicinal Plant Garden in Edo (Tokyo). This is a historical document concerning when and which plants were imported for cultivation.