Etsuro Yoshimura

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

The KUP6 subfamily transporters regulated directly via an abscisic acid signaling complex act as key factors in osmotic adjustment by balancing potassium homeostasis in both cell growth and drought stress responses. Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K+ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K+ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using 86radioactive rubidium ion (86Rb+) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.

OsTZF1, a CCCH-Tandem Zinc Finger Protein, Confers Delayed Senescence and Stress Tolerance in Rice by Regulating Stress-Related Genes

OsTZF1, a CCCH-type zinc finger protein, acts as a negative regulator of leaf senescence in rice under stress conditions and confers abiotic stress tolerance by delaying stress-response phenotypes, possibly through the control of RNA metabolism of stress-responsive genes. OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of β-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with β-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3′ untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.

Micro Total Bioassay System for Ingested Substances: Assessment of Intestinal Absorption, Hepatic Metabolism, and Bioactivity

Oral medicines and food constituents are absorbed in the intestine and metabolized in the liver, after which they exhibit their activity toward a target tissue. Micromodels of human tissues were developed to mimic these processes and bioactivities. By integrating the micromodels, we realized a micro total bioassay system for oral substances; this system comprised a microintestine, microliver, and the target components. The microchip was composed of a slide glass and polydimethylsiloxane (PDMS) sheets with microchannels fabricated by photolithography. Caco-2 cells were cultured in the intestine component, and HepG2 cells, in the liver component. The human breast carcinoma MCF-7 cells were cultured in the target component, and the activities of anticancer agents and estrogen-like substances were successfully assayed. By using this system, the overall properties of the ingested cyclophosphamide, epirubicin, 17-β estradiol, and soy isoflavone, i.e., their intestinal absorption, hepatic metabolism, and bioactivity toward target cells, could be assayed with operative ease. Further, the assay time and cell consumption were reduced compared to those in conventional in vitro bioassay systems.

A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides.

RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the “cupin” superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni2+, Cu2+, Fe3+, Fe2+, Mn2+, and Co2+) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn2+). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the “minimal PKS” gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.

Interference by mineral acids in inductively coupled plasma atomic emission spectrometry

Interferences caused by low concentrations of mineral acids have been investigated in inductively coupled plasma atomic emission spectrometry. There appear to be two mechanisms that lower the emission intensities: the decrease in the excitation temperature and the reduction in the aspiration rate. The former was predominant at lower concentrations of mineral acids (⩽1 M) and the latter became influential at higher acid concentrations (1 M). The excitation temperature was found to decrease in the presence of mineral acids. The decreases in emission intensities were in fair agreement with those estimated from the decreases in excitation temperature.

The role of potassium in the secretion of mugineic acids family phytosiderophores from iron-deficient barley roots

Mugineic acid family phytosiderophores (MAs) are secreted from iron-deficient barley roots with high equimolar correlation of potassium. To determine the form of MAs when it is secreted, we investigated the effect of anion channel blockers and valinomycin on the secretion of MAs. Among the anion channel blockers, anthracene-9-carboxylic acid and phenylglyoxal drastically reduced the amount of secreted MAs, while 4,4-diisothiocyano-2,2- stilbene disulfonate slightly inhibited the MAs secretion. Trifluoromethyl-3-phenylamino-2-nicotinic acid reduced the secreted amount to the half of non-treated. This result suggested that MAs are secreted in the form of anion through an anion channel. The elimination of potassium gradient between the cytoplasm and the cell exterior by treatment with valinomycin reduced the amount of secreted MAs. Analysis of potassium distribution in root by LV-SEM-XMA indicated that potassium in the cortex cells of iron-deficient roots is released with MAs secretion and the amount of potassium in the cortex cells decreases after secretion. These results suggested that MAs are secreted in the form of a monovalent anion via anion channels using the potassium gradient between the cytoplasm and the cell exterior.

Timing, magnitude, and location of initial soluble aluminum injuries to mungbean roots

Abstract Despite a centurys knowledge that soluble aluminum (Al) is associated with acid soils and poor plant growth, it is still uncertain how Al exerts its deleterious effects. Hypotheses include reactions of Al with components of the cell wall, plasmalemma, or cytoplasm of cells close to the root tip, thereby reducing cell expansion and root growth. Digital micros copy was used to determine the initial injuries of soluble Al to mungbean (Vigna radiata L) roots Roots of young seedlings were marked with activated carbon particles and grown in 1 mM CaCl2 solution at pH 6 for ca. 100 min (control period), and AICl3 solution was added to ensure a final concentration of 50 µM Al (pH 4). Further studies were conducted on the effects of pH 4 with and without 50 iM Al Four distinct, but possibly related, initial detrimental effects of soluble Al were noted First, there was a 56–75% reduction in the root elongation rate, first evident 18–52 min after the addition of Al, root elongation continuing at a decreased rate for ca. 20 h. Decreasing solution pH from 6 to 4 increased the root elongation rate 4-fold after 5 min, which decreased to close to the original rate after 130 min. The addition of Al during the period of rapid growth at pH 4 reduced the root elongation rate by 71% 14 min after the addition of Al The activated carbon marks on the roots showed that, during the control period, the zone of maximum root growth occurred at 2,200–5,100 im from the root tip (i.e the cell elongation zone) It was there that Al first exerted its detrimental effect and low pH increased root elongation Second, soluble Al pre vented the progress of cells from the transition to the elongation phase, resulting in a considerable reduction of root growth over the longer term. The third type of soluble Al injury occurred after exposure for ca. 4 h to 50 µM Al when a kink developed at 2,370 im from the root tip. Fourth, ruptures of the root epidermal and cortical cells at 1,900–2,300 im from the tip occurred ≥4.3 h after exposure to soluble Al The timing and location of Al injuries support the contention that Al initially reduces cell elongation, thus decreasing root growth and causing damage to epidermal and cortical cells.

Genome-wide screening of aluminum tolerance in Saccharomyces cerevisiae

Genome-wide screening has identified 37 Al-tolerance genes in Saccharomyces cerevisiae. These genes can be roughly categorised into three groups on the basis of function, i.e., genes related to vesicle transport processes, signal transduction pathways, and protein mannosylation. The largest group is composed of genes related to vesicle transport processes; severe Al sensitivity was found in yeast strains lacking these genes. The retrograde transport of endosome-derived vesicles back to the Golgi apparatus is an important factor in determining the Al tolerance of the vesicle transport system. The PKC1-MAPK cascade signalling pathway is important in the Al tolerance of signal transduction. The lack of the gene implicated in this process leads to weakened cell wall architecture, rendering the yeast Al-sensitive. Alternatively, Al might attack the cell wall and/or plasma membrane, and, as signalling is prevented in cells devoid of the genes related to signalling processes, the cells may be unable to alleviate the damage. The genes for protein mannosylation are also associated with Al tolerance, demonstrating the importance of cell wall architecture. These genes are involved in cell integrity processes.

Aluminum rapidly inhibits cellulose synthesis in roots of barley and wheat seedlings

Summary The change in polysaccharide synthesis was studied in the roots of barley and wheat seedlings exposed to Al. Rapid inhibition of 14 C-glucose incorporation into the cellulose fraction, which occurred within 15 min of Al exposure, occurred in both barley and wheat root cells. The reduction in cellulose synthesis was more severe in an Al-sensitive wheat cultivar (Scout 66) than in an Al-tolerant wheat cultivar (Atlas 66). From these results, it can be hypothesized that a reduction in cellulose synthesis causes the Al-induced rapid inhibition of root elongation. Coumarin, a potent inhibitor of cellulose synthesis, inhibited root elongation, which supports the hypothesis. Roots exposed to Al also showed a rapid enhancement of radioactivity incorporation into the hemicellulose fraction, which was attributed to the induction of callose formation. Callose was formed simultaneously with the inhibition of cellulose synthesis, in inverse proportion to the decrease in cellulose. Therefore, the cellulose synthesis system can be switched to a callose synthesis system in Al-injured root cells.

Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.