Fukumi Sakai

Protein synthesis in tobacco mesophyll protoplasts induced by tobacco mosaic virus infection

Abstract Tobacco mesophyll protoplasts inoculated in vitro with tobacco mosaic virus (TMV) were used to study protein synthesis induced or stimulated by virus infection. Ultraviolet irradiation of inoculated protoplasts sufficiently reduced host protein synthesis without markedly affecting TMV replication. In addition to viral coat protein, incorporation of amino acids into at least two high-molecular-weight proteins were demonstrated in infected protoplasts. One of these was unrelated to coat protein, consisted of a single polypeptide of about MW 140,000 and was present in association with some cellular structure less dense than nuclei or chloroplasts. The other protein with molecular weight of about 180,000 also appeared to be unrelated to coat protein, since it was labeled with amino acids not contained in the coat protein. Synthesis of the 140,000-MW protein followed a time course very similar to that of viral RNA replication and attained a maximum incorporation rate 4 hr earlier than coat protein synthesis. Production of progeny virus particles closely followed the course of coat protein synthesis.

A stilbene synthase from Japanese red pine (Pinus densiflora): implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis.

Stilbene synthase (STS) and chalcone synthase (CHS) are plant-specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently isolated from Pinus densiflora three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in Escherichia coli and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX (Ki = 6 μM) as well as PDSTS2 (Ki = 13 μM), which presumably maintains the balance between the stilbenoid and flavonoid biosyntheses. PDSTS3 was insensitive to product inhibition. We identified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees.

RNA and protein synthesis in protoplasts isolated from tobacco leaves

Abstract RNA and protein synthesis in the protoplasts isolated from tobacco mesophyll were studied by measuring the incorporation of labeled precursors. 1. 1. Incorporation of [ 14 C]uracil into protoplast RNA was accounted for almost entirely by RNA synthesis in the nucleus. Little radioactivity was incorporated into rRNAs of the chloroplasts. 2. 2. Incorporation of [ 14 C]uracil was inhibited by actinomycin D, chromomycin A 3 and 2-thiouracil, but not by rifampicin and daunomycin. 3. 3. Rate of protein synthesis was found to be 2 · 10 −6 nmole leucine per protoplast per h, corresponding to a turnover rate of 0.5 % per h. 4. 4. Incorporatio of [ 14 C]leucine was inhibited by cycloheximide, blasticidin S and enomycin. Only partial inhibition was obtained with chloramphenicol and puromycin. 5. 5. [ 14 C]Leucine was incorporated into both cytoplasmic and chloroplast protein. Incorporation into cytoplasmic protein was inhibited by cycloheximide, while that into chloroplast protein was sensitive to chloramphenicol. 6. 6. Average half-life of mRNA was estimated to be 4 h.

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

SummaryA high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.

Induction of a novel cytochrome P450 (CYP93 family) by methyl jasmonate in soybean suspension-cultured cells

We isolated a cDNA encoding a novel cytochrome P450 (CYP93A1) from soybean suspension-cultured cells that had been treated with methyl jasmonate (MeJA). The amino acid sequence of the gene product had 30-40% identity with those of other plant P450s. The protein contained the heme-binding domain which is highly conserved among plant P450s. Transcription of the cytochrome P450 gene in soybean cells was induced by 30 microM MeJA even in the presence of cycloheximide, and reached maximum level 6 h after MeJA treatment. This is the first report of a plant cytochrome P450 gene whose transcription is induced by MeJA even without protein synthesis.We isolated a cDNA encoding a novel cytochrome P450 (CYP93A1) from soybean suspension‐cultured cells that had been treated with methyl jasmonate (MeJA). The amino acid sequence of the gene product had 30–40% identity with those of other plant P450s. The protein contained the heme‐binding domain which is highly conserved among plant P450s. Transcription of the cytochrome P450 gene in soybean cells was induced by 30 μM MeJA even in the presence of cycloheximide, and reached maximum level 6 h after MeJA treatment. This is the first report of a plant cytochrome P450 gene whose transcription is induced by MeJA even without protein synthesis.

Formation of nuclear polyhedral bodies and nuclear polyhedrosis virus of silkworm in mammalian cells infected with viral DNA

Abstract The infectious DNA of nuclear polyhedrosis virus of silkworm ( Bombyx mori Linne) caused production of characteristic nuclear polyhedral bodies in cultured FL cells of human amniotic origin, which did not support virus growth. The activity of the viral DNA in producing polyhedral bodies in FL cells was lost by pretreatment of the inoculum with DNase and by heating, but RNase was inactive. The polyhedral bodies formed in the cells were identical with those formed in cells of the silkworm with respect to their morphological and serological character. The polyhedral bodies isolated from FL cells and dissolved in alkali caused nuclear polyhedrosis when injected into silkworm pupae.

The nucleic acid of nuclear-polyhedrosis virus of the silkworm

Viral DNA was isolated from virus particles of nuclear-polyhedrosis of the silkworm, Bornbyx mori L., with 0·1 M -Na 2 CO 3 -NaHCO 3 buffer solution (pH 10·0), and then fractionated and purified on methylated albumin columns. Viral DNA showed a single peak on methylated albumin column fractionation as well as in analytical centrifugation, and the S 20,w of viral DNA was 13·1 s. The purified viral DNA was infectious. While DNase-treated DNA lost its infectivity, RNase-treated DNA was unaffected. The evidence that the ratio in viral DNA of adenine to thymine as well as of guanine to cytosine was equal to unity, that the DNA gave a sharp melting curve and that heat-denatured DNA was observed to react with formaldehyde showed that the DNA isolated was double-stranded DNA.

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

Abstract We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 106 cells/g fresh weight cells and then cultured at a concentration of 2.5×105 cells/ml in NH4NO3-free Murashige and Skoog (MS) medium supplemented with 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 µM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 µM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 µM kinetin or 1 µM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants.

Clonal structure of natural populations of Cryptomeria japonica growing at different positions on slopes, detected using RAPD markers

Abstract Cryptomeria japonica D. Don (Japanese cedar), an economically important timber species endemic to Japan, is dominant on ridges and upper slopes in cool-temperate natural forests of Kyoto Prefecture. Recruitment of sexual progeny in the area near the Japan Sea is extremely rare, and propagation occurs predominantly through clonal growth by layering. The development pattern that occurs with layering and the resulting complexity of the population structure make it difficult to identify distinct clones, even by excavation. Therefore, we used the randomly amplified polymorphic DNA (RAPD) technique to examine the clonal structure of upper- and lower-slope plots established in two C . japonica populations in Kyoto Prefecture. A total of 263 plants sampled from four plots were analyzed using 10 arbitrarily chosen decamer primers, which produced 50 highly reproducible RAPD bands. There was a different clonal structure in upper- and lower-slope plots. Lower-slope plots were made up of a small number of genets with many ramets, while upper-slope plots were made up of a large number of genets with a few ramets. Clonal diversity measured using PD , Simpson’s D , and Fager’s E was higher in the upper-slope plots. These results show that natural C . japonica populations maintain relatively high clonal variation, compared with other clonal plant species, and that repeated seedling recruitment occurred more frequently in upper-slope plots than in lower-slope plots.