Toru Kuboi

Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu

Summary Citrus unshiu dehydrin (CuCOR19) was characterized, and its cryoprotective activity and response to water stress were studied. Detached leaf of C. unshiu was treated with cold (4 °C), NaCl (50-200 mmol/L) and abscisic acid (ABA, 0.1-10 μmol/L). CuCOR19 mRNA or protein were detected by Northern or Western hybridization, respectively. A small amount of CuCOR19 mRNA was present in leaf when it was detached, and the mRNA level decreased during the control experiment. Cold induced the gene expression remarkably, but treatment with ABA or NaCl did so only slightly. The CuCOR19 protein accumulated only under cold stress, suggesting that cold is a crucial environmental cue for dehydrin production in the leaf of C. unshiu. In order to characterize CuCOR19 protein, we obtained the protein using the expression system of Escherichia coli and subjected it to the cryoprotection assay and circular dichroism spectrum analysis. CuCOR19 protected catalase and lactate dehydrogenase against freezing inactivation, and it was more effective than compatible solutes or BSA. The circular dichroism spectrum for CuCOR19 showed that the major secondary structure of CuCOR19 is a random coil in the solution. It is likely that the random coil structure allows CuCOR19 to protect plant cells from cold stress, since the random coil is considered to bind water or maintain protein structure.

Enhancement of anthocyanin biosynthesis by sugar in radish (Raphanus sativus) hypocotyl

Radish (Raphanus sativus) hypocotyl accumulates anthocyanins as the plant grows. Although anthocyanins were detected in both the peripheral cortex and pericycle, the major site of accumulation was the pericycle. Detached hypocotyls of radish seedlings produced large amounts of anthocyanins using exogenous glucose, fructose, or sucrose, but little anthocyanin using mannose or 3-O-methyl-d-glucose. Treatment with 175 mM sucrose was most effective in inducing anthocyanin biosynthesis in the hypocotyl system. Two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were significantly activated by sucrose, whereas these genes were expressed weakly in the control. These results suggested that sugars transported into hypocotyls promote anthocyanin biosynthesis by inducing expression of the biosynthetic genes in radish.

DNA binding of citrus dehydrin promoted by zinc ion.

Dehydrins are hydrophilic proteins that accumulate during embryogenesis and osmotic stress responses in plants. Here, we report an interaction between citrus dehydrin Citrus unshiu cold-regulated 15 kDa protein (CuCOR15) and DNA. Binding of CuCOR15 to DNA was detected by an electrophoretic mobility shift assay, a filter-binding assay and Southwestern blotting. The binding was stimulated by physiological concentrations of Zn2+, but little stimulation occurred when other divalent cations, such as Mg2+, Ca2+, Mn2+, Ni2+ and Cu2+, were substituted for Zn2+. Ethylenediaminetetraacetic acid cancelled the Zn2+-stimulated binding. A binding curve and competitor experiments suggested that the DNA binding of CuCOR15 exhibited low affinity and non-specificity. Moreover, tRNA competed with the DNA binding. Histidine-rich domains and a polylysine segment-containing domain participated in the DNA binding. These results suggest that CuCOR15 can interact with DNA, and also RNA, in the presence of Zn2+. Dehydrin may protect nucleic acids in plant cells during seed maturation and stress responses.

Exogenously applied isothiocyanates enhance glutathione S-transferase expression in Arabidopsis but act as herbicides at higher concentrations

Isothiocyanates (ITCs) are sulfur-containing compounds that are generated by the glucosinolate-myrosinase system in plants. Although previous greenhouse studies have demonstrated the phytotoxicity of ITCs, their action modes are still unknown. In this study, we report the physiological responses of Arabidopsis thaliana treated with three exogenous ITCs: methyl ITC, allyl ITC, and phenethyl ITC. Administration of a high dose of each ITC inhibited plant growth and induced severe bleaching in the rosette leaves. The bleaching was concomitant with the elevation of electrolyte leakage and the generation of hydrogen peroxide. Although the three ITCs showed bleaching symptoms, phenethyl ITC was the most potent. A low dose of phenethyl ITC, at which the ITC did not promote leaf bleaching, enhanced the accumulation of transcripts of glutathione S-transferases (GSTs) in Arabidopsis. When 16 GST genes were tested, the levels of transcripts corresponding to 5 of the GST genes were enhanced in response to the phenethyl ITC treatment. In particular, the expression of a Tau class gene (AtGSTU19, At1g78380) responded to the phenethyl ITC treatment. Enhancement of the AtGSTU19 gene expression also occurred in the treatment of both allyl ITC and methyl ITC. These results suggest that the administration of ITCs to Arabidopsis at high doses has an herbicidal effect by inducing oxidative burst-like responses, but that administration at lower doses enhances the expression of specific GST genes in Arabidopsis.

Stimulatory effects of aluminum on the growth of cultured roots of tea

Abstract The effect of Al on the growth of excised tea (Camellia sinensis L.) roots differentiated from stem segments in vitro was investigated in shaking liquid culture. 1) The optimum initial pH for tea root growth in modified MS medium was 5.5. Growth of main and lateral roots was affected by the difference in NAA concentrations. Main root growth was stimulated by 5×10-7 and 1×10-6 M NAA, and lateral root growth was stimulated by 5 × 10-6 and 1 × 10-5 M NAA. 2) Main root growth showed a progressive increase for 3 weeks, then became stationary. Lateral roots began to grow at 2 weeks, with a progressive increase over the following 6 weeks. 3) The growth of the tea roots in the main or lateral root growth medium was stimulated by the supply of Al and P together, but was not affected when Al and P were supplied separately. 4) The pH of the culture medium decreased gradually to about 4.0 during the shaking culture, and remained stable. Growth of roots supplied with Al was stimulated after the decrease of me...

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase

Abstract; Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 μg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 μl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 107 cells per gram of fresh weight alga.

Variation in amylase activities in radish (Raphanus sativus) cultivars.

The radish (Raphanus sativus) is a root vegetable of the Brassicaceae family which shows amylolytic activity in the taproot. However, there is little information about differences in these amylolytic activities among radish cultivars. We analyzed the amylase activities and starch contents of 7 kinds of radish cultivars. The Koshin cultivar showed the highest amylase activity, with a level approximately 6 times higher than that of the Sobutori cultivar, which had the lowest. Cultivars with higher amylase activities showed higher starch contents. These results suggest that there are intraspecies variations in amylolytic activities in radishes, and positive correlations between amylase activity and starch content.

Drought-regulated expression of prolyl-tRNA synthetase genes in radish (Raphanus sativus) seedlings

Abstract We cloned a cDNA that encodes a radish (Raphanus sativus) prolyl-tRNA synthetase (RsProRS1) by screening drought-suppressed genes from an EST library of radish seedlings. The predicted amino acid sequences suggested that RsProRS1 was very similar to one of the two Arabidopsis prolyl-tRNA synthetases. RsProRS1 belongs to the eukaryote/archaeon-like prolyl-tRNA synthetase (ProRS), because it lacked an insertion domain between motifs 2 and 3, which is characteristic of prokaryotic-like ProRSs. RsProRS1 seemed to be organellar targeted since it had a putative signal sequence at N-terminus. We also isolated a cDNA fragment of another type of prolyl-tRNA synthetase (RsProRS2) by using sequence information of the Arabidopsis genome. In radish seedlings, the proline content increased by dehydration but decreased by rehydration. The gene expression of both RsProRSs was suppressed by dehydration but enhanced by rehydration, like the proline dehydrogenase gene expression. Opposite regulations were found for the Δ1-pyrroline-5-carboxylate synthetase gene expression. These results suggest that proline accumulation in radish during drought stress can be regulated not only by proline biosynthetic and metabolic genes, but also by ProRS genes which supply proline for protein synthesis.

Toxicity of cationic polymer flocculants to higher plants. I: Seedling assay

Effects of Cu and cationic polymer flocculants on hydroponically cultured plants were studied. In the presence of 1 μM Cu, more than 25 mg/liter of a cationic flocculant with an anion exchange capacity (AEO) of 3.99 me/liter caused growth reduction and chlorosis in turnip (Brassica rapa L.). Root browning also occurred at the flocculant concentration of more than 12.5 mg/liter. The plants treated with the flocculant contained higher levels of Cu in the fibrous roots and lower levels of Fe in the leaves as compared to the contents of control plants. When the medium Cu concentration was increased to 10 μM, 40 mg/liter of the flocculant caused severe toxic symptoms in turnip. Copper and the flocculant additively affected Cu content of fibrous roots. Even at a low concentration of Cu (0.1 μM), the growth of turnip, sesame (Sesamum indicum L.), cucumber (Cucumis sativus L.), and wheat (Triticum aestivum L.) was inhibited by the flocculant with this order of sensitivity. Results of dialysis of CuCl2, solution i...

Characterization of cadmium-tolerant carrot cells in response to cadmium stress

Abstract Non-tolerant (CW) and Cd-tolerant (CCdl) carrot (Daucus carota L.) cells were compared for their response to cadmium (Cd). The CCdl cells were induced by selection from the CW cells on a B5 medium containing 100 µM Cd. The CCdl cells were more tolerant to Cd than the CW cells. In an 80 µM Cd treatment, the growth rate (8 d/O d) based on the fresh weight of the CCdl cells and CW cells was 69 and 24%, respectively, compared to the controls. Cd stress led to the decrease of the total glutathione content and to the increase of the contents of non-protein thiols (t-SH, phytochelatins) and Cd in both types of cells. Under Cd stress, the contents of t-SH and Cd of the CW cells were higher than those of the CCdl cells. The CW cells in the 80 and 160 µM Cd treatments accumulated Cd with the increase in time. In contrast, the Cd content in the CCdl cells in all the treatments and in the CW cells in the 40 µM Cd treatment gradually decreased after 1 d. In an additional experiment, both types of cells pretreated with 20–80 µM Cd for 1 d were transferred to a Cd-free medium. The CCdl cells in all the pretreatments continuously excluded Cd over a 4-d period, unlike the CW cells. Therefore, a larger depletion of Cd was found in the CCdl cells, which may be partially attributed to Cd tolerance.