Effects of phosphate on the Ca2+ uptake and the sucrose-controlled secretion of α-amylase molecules in cultured rice cells were investigated. Phosphate markedly stimulated Ca2+ uptake into rice cells, particularly at the outer cell layer of the cell cluster. Phosphate also increased the synthesis and extracellular liberation of α-amylase II-4 in sucrose-supplemented cells. The distribution pattern of enzyme in rice cell clusters induced by phosphate was similar to that of Ca2+ uptake. Phosphate did not increase the level of mRNA of α-amylase II-4, indicating that phosphate stimulates the translation and posttranslational secretory processes of α-amylase II-4 in the presence of sucrose. Furthermore, phosphate enhanced both the Ca2+ uptake and α-amylase II-4 synthesis in the microsomes. These results strongly suggested that the ratio of phosphate to sugar is important for regulating the Ca2+ uptake, and that phosphate and sugar precisely coordinate the Ca2+-mediated synthesis and extracellular liberation of α-amylase II-4.
Peptidyl plant growth factor phytosulfokine-α (PSK-α) is known to stimulate both cell proliferation and differentiation, the mechanism for which, however, has yet to be elucidated. We here investigated the effects of PSK-α on cell cycle regulation using non-embryogenic cell cultures of carrot. Flow cytometric analysis of cell nuclear DNA content showed that the addition of PSK-α (1x10-7 M) to a medium containing a low concentration (4.5x10-7 M) of 2, 4-dichlorophenoxyacetic acid (2, 4-D) promoted progression of the G1 cell cycle. Similar results were obtained with a high 2, 4-D (4.5x10-6 M) medium lacking PSK-α. PSK-α appeared to be involved in cell cycle progression outside the G1 phase. RT-PCR using cell cycle-related genes from carrot showed that the amounts of the transcripts of these genes were increased by the addition of PSK-α, and the levels of which were very similar to those with high 2, 4-D medium. The timing of the transcript increase roughly corresponded to that of the cell cycle progression. PSK-α seemed to stimulate re-entry of quiescent cells into the cell cycle. We propose a mechanism that PSK-α stimulates cell proliferation through the cell cycle regulation.
A cDNA clone, named as SbCHS cDNA, encoding chalcone synthase (CHS, EC 188.8.131.52) was isolated from a cDNA library derived from hairy root cultures of Scutellaria baicalensis Georgi by screening with a 1.4 kbp full length CHS cDNA of Phaseolus vulgaris as the probe. Complete nucleotide sequence of the SbCHS cDNA contained 1170-base pair open reading frame encoding 390 amino acid residues. The deduced amino acid sequence of SbCHS cDNA exhibited 82.1% identity with CHS of P. vulgaris. SbCHS mRNA expression in S. baicalensis hairy roots was unusually reduced by UV light irradiation, wounding, and yeast extract, as shown by Northern blot analysis. Greater formation of naringenin chalcone (100%) than of pinocembrin chalcone (67.4%) was observed by using thin-layer chromatography (TLC) to assess the enzymatic activity of recombinant SbCHS expressed in Escherichia coli.
Indole acetic acid (IAA) level and nitrilase activity were measured in infected and healthy turnips, Brassica campestris L., subsp. Rapa, over a time course to confirm increases in IAA levels upon infection by Plasmodiophora brassicae and to investigate underlying mechanisms. Healthy and infected seedlings were assayed from 20 to 90 days after sowing. IAA levels in both roots fluctuated similarly over days 20-35. By day 45, IAA in infected roots increased to five-fold over healthy roots then decreased to the level of healthy roots by day 90. Nitrilase activity was negligible on days 20 and 25 but increased thereafter in infected and healthy roots. However, activity in healthy roots decreased substantially by day 40 while infected roots showed a continued increase to day 45 then decreased to a low level. These findings suggest that IAA concentration increases after P. brassicae infection, possibly due to IAA synthesis via pathways involving nitrilase.
The expression of the thiamin-binding protein gene in sesame was found only in seeds during maturation, not in seeds during germination, or in roots, stems and leaves. The amount of mRNA of the thiamin-binding protein increased with the development of sesame seeds. The level of mRNA was maximum in the mature seeds. Also, the thiamin-binding activity from seed proteins increased along with seed development. On the other hand, immunohistochemical analysis using an antibody against the protein and an immunogold-silver enhancement kit detected the thiamin-binding protein only in the albumen cells of seeds. These results suggest that in sesame, the thiamin-binding protein is synthesized in developing seeds and accumulated in the albumen of the seeds.
The wide use of transgenic crops has raised some concern about their escape into the field and movement of transgene to unintended hosts. Thus, it is essential to keep track of the transgenic plants in the field. Here, we show that alteration of leaf morphology, which is caused by knotted1-type homeobox genes can be a useful marker. We examined transgenic tobacco expressing the homeobox gene that exhibited ‘curved’, ‘wrinkled’, or ‘dwarf’ phenotype whose foliar malformations were easily distinguished from wild-type plants with the naked eye. Expression of the homeobox genes could have various effects on some growth parameters of the host plants. However, the growth of ‘curved’-type transgenic plants was not much different from that of wild-type plants, suggesting that the ‘curved’ phenotype is a useful marker for assessing the whereabouts of transgenes/transformants in the field.
“Cinerarin” is a polyacylated anthocyanin that is responsible for the blue coloration of cineraria (Senecio cruentus) flowers. We isolated a full-length cDNA (Sc3MaT) encoding a putative anthocyanin acyltransferase from S. cruentus. The Sc3MaT cDNA was expressed in Escherichia coli cells and the expression product was purified to homogeneity and functionally characterized. The Sc3MaT could catalyze the regiospecific malonyl transfer from malonyl-CoA (Km, 61μM) to pelargonidin 3-O-glucoside (Km, 11μM) to produce pelargonidin 3-O-6”-O-malonylglucoside with a kcat value of 8.8 s-1. The specificities for acyl donors and acceptors were highly restricted to malonyl-CoA and anthocyanidin 3-O-glucoside, respectively. Therefore, it may be concluded that Sc3MaT is a malonyl-CoA:anthocyanidin 3-O-glucoside-6”-O-malonyltransferase. The other enzymatic properties of Sc3MaT were comparable with those of known anthocyanin acyltransferases. Because a reaction product of Sc3MaT, delphinidin 3-O-6”-O-malonyglucoside, constitutes a part of cinerarin, Sc3MaT is probably involved in the cinerarin biosynthesis in this plant.
An improved and efficient system for the expression of GUS in the shoots of 3-day-old rice seedlings was successfully developed using vacuum Agroinfiltration. Over 70% of inoculated shoots in the two japonica cultivars and one indica cultivar tested showed transient expression of GUS under the control of a CaMV 35S promoter interrupted by an intron. The GUS directed by a rice light-harvesting Chlorophyll-a/b-binding protein Cab promoter was also expressed in rice shoots via vacuum Agroinfiltration. Compared to particle bombardment method to test the efficiency of various promoters, transient GUS expression in rice via Agroinfiltration is more precise for assaying the inducible Cab promoter as well as the CaMV 35S constitutive promoter.
We isolated promoter sequences of the Cry j 1 gene-which encodes an allergenic protein in the pollen of Japanese cedar-by an inverse-PCR technique using genomic DNA as the template. One of the isolated promoter sequences conferred pollen-associated expression on a fusion construct that included a gene for β-glucuronidase (GUS) in transgenic tobacco plants. Histochemical analysis of GUS expression using four deletion mutants of this promoter sequence revealed that expression differed among pollen grains at different stages of maturation and that a 223-bp 5’-untranslated region of the gene was sufficient for spatial and temporal expression of the gene in pollen and pollen tubes. Our results indicate that the promoter sequence might be adequate for active pollen-associated expression in tobacco, as it is in Cryptomeria japonica.
The Dof protein family is a group of plant transcription factors carrying highly conserved 52 residues referred to as the Dof domain, and belongs to the C4 zinc-finger transcription factors. We isolated various PsDof genes by PCR using the Dof-domain nucleotide sequence of the PsDof1 gene from a cDNA library of elicitor-treated pea epicotyls, and these isolated PsDof genes were then classified phylogenetically. Since the obtained genes (PsDof1 to PsDof7) were scattered over various positions of the phylogenetic tree, they were expected to perform various functions as Dof-type transcription factors. From their positions in the tree, it is expected that the PsDof2 and PsDof5 genes are defense related, as is the PsDof1 gene.
The synthesis of capsaisinoid monoglucoside, a key material for the preparation of the capsaicinoid oligosaccharide, using plant cultured cells has been investigated. Among the cultured cells tested, only the cells of Phytolacca americana glucosylated capsaicin and 8-nordihydrocapsaicin into the corresponding monoglucoside in good yields. Thus, the one-step glucosylation of capsaicinoids has been achieved using the cells of P. americana.
When leaf segments of a tomato cultivar ‘Ponderosa’ were inoculated with Agrobacterium rhizogenes MAFF07-20001 carrying the binary vectors pRi and pBI121/sGFP, adventitious roots were developed from calli formed at the edges of the segments. Primordial roots were obtained with green fluorescence under blue light and elongated vigorously on hormone-free medium without loss of the green fluorescence. They were easily distinguishable from the non-fluorescing roots on the same segments. Successful integration of the sGFP and rol C genes into the chromosome of tomato roots was confirmed by polymerase chain reaction and Southern hybridization. The present method enables us to evaluate the hairy root formation without subculture, isolation and DNA analysis. All commercial cultivars available in Japan (24 cultivars) and 14 breeding lines of tomato were tested by this method. All but two breeding lines produced the hairy roots. Thus, the present method is useful for hairy root production in tomato.