A unique feature of plant cell cycle control is that quiescent meristematic and differentiated cells of plants are capable of re-entering the cell cycle. The activation of cell division and transitions between different phases of cell cycle are controlled by a family of cyclin-dependent serine/threonine protein kinases (CDKs). Post-translational regulation of CDKs plays an important role in the maintenance and activation of cell division, and thus controls plastic development of plant tissues. Here I review CDKs and cyclins in plants and describe the current view of CDK regulation by phosphorylation.
An effort was made to generate transgenic plants from embryogenic callus of soybean (Glycine max (L.) Merrill) by using Agrobacterium tumefaciens, strain EHA101 harbouring the binary vector pIG121, which contains neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT) and β-glucuronidase (GUS) genes. Analysis by PCR and Southern blotting analysis of the putative transgenic soybean showed that the plants carried the genes for GUS, NPTII and HPT. The germinating seeds were injected with Agrobacterium tumefaciens, followed by sonication and vacuum infiltration. Analysis of the progeny plants by PCR and Southern hybridization showed that the transformed plants maintained the transferred DNA. Compared with transformation of embryogenic callus, that of germinating seeds was simple and repeatable, since it required no prior tissue culture steps and produced transgenic plants more efficiently. Transgenic plants of kidney bean (Phaseolus vulgaris L.) were also obtained by injection with the Agrobacterium into germinating seeds.
The first intergeneric chimeras between radish and cabbage were successfully synthesized by in vitro grafting method. Regenerated seventy seven chimeras from the original sectorial chimera were investigated on morphological, physiological natures comparing with other interspecific chimeras of Brassica. Chimeral structures were justified and totally classified based on the morphological characteristics, isozymatic band patterns and PCR analysis. In those chimeras, putative four-layered chimeral plants were also found for two vegetatively-propagated generations with general three-layered chimeras. Physiological interactions were typically recognized such as in flowering date, pollen fertility, and pod and seed set. However, genetic changes were not found in the progeny derived from the crosses with both parents.
The genes encoding chalcone synthase (CHS) in the common morning glory (Ipomoea purpurea) comprise a multigene family, and they are divided into two subfamilies. The genes in a subfamily including the CHS-A, CHS-B and CHS-C genes are distantly related to the other known CHS sequences in a phylogenetic tree, whereas the CHS-D and CHS-E genes in another subfamily are more closely related to the well-characterized CHS genes. As an initial step to elucidate biological function of these CHS genes in I. purpurea, the CHS-D and CHS-E cDNAs were expressed in Escherichia coli with different expression systems. The recombinant CHS-D and CHS-E proteins both showed CHS activity to produce naringenin chalcone. These results are discussed with regard to the biological roles of the CHS-D and CHS-E genes in flower pigmentation in I. purpurea. We have also discussed these CHS-D and CHS-E enzyme as members of plant specific polyketide synthases.
A reliable method for the transformation of Pharbitis (Ipomoea) nil is described for the first time. Somatic embryos derived from immature zygotic embryos were used as starting material. These were transformed with Agrobacterium tumefaciens strain GV3101 (pMP90), which harbored the plasmid pIG121Hm that included genes for β-glucuronidase (GUS), kanamycin resistance and hygromycin resistance. Infected embryos were induced to undergo secondary embryogenesis and kanamycin-resistant embryos were selected and allowed to develop into plantlets. All transgenic plants were morphologically normal and fertile. Stable integration and expression of the transgene for GUS were confirmed by histochemical and Southern blotting analysis. Expression of the intron-containing reporter gene for GUS under control of the 35S promoter of cauliflower mosaic virus (CaMV 35S) was detected after staining. Southern hybridization confirmed the stable inheritance of the transgene for GUS.
The basic subunit of glutelin from rice dry seeds were shown to have N-linked sugar chains by lectin staining with PHA-E4, WGA and Con A. NaIO4 and N-glycanase treatments also provided the evidences of N-linked sugar chains. The purified basic subunit was applied to NEPHGE and eight isoforms of it were separated. The fractionated eight isoforms were shown to be nearly homogeneous by C18-silica column chromatography. Each was subject to lectin staining. The eight isoforms were hybridized with PNA, PHA-E4 and Con A. This lectin staining indicated that each of the isoforms of the basic subunit had N-linked sugar chain. This is the first report showing the occurrence of N-linked sugar chain in isoforms of glutelin basic subunit.
The utilization of nitrate in microspore culture of Brassica napus was examined. The microspores developed into embryos in a medium containing glutamine as a sole source of nitrogen (medium A) as well as in a medium containing nitrate and glutamine (medium C). On the other hand, no embryo could develop from the microspores in a medium with nitrate as a sole source of nitrogen (medium B). Transfer experiments from one medium to another during various periods of culture indicated that the microspores cultured in medium A stopped growing as soon as they were transferred to medium B. On the other hand, the microspores cultured in medium B did not show any change even after transfer to medium A on the 4th day of culture. Although the amount of nitrate in medium B increased within the first 14 days, the increase was less pronounced in medium C. The activity of nitrate reductase in vivo was not detected in the developing androgenic embryos before the 8th day of culture, whereas 15 days-old embryos showed a higher activity of nitrate reductase than leaf. From these results it was concluded that nitrate was not used for early androgenic embryogenesis in B. napus before 15 days of culture because of the lack of nitrate reductase activity. Negative effect of the presence of nitrate on androgenic embryogenesis was not observed, either. On the contrary, the presence of nitrate promoted the growth of heart-shaped or more developed embryos after rapid and complete consumption of glutamine in the culture medium C. Nitrate in the medium for microspore culture of B. napus was considered to be an ideal source of nitrogen at the later stages of androgenic embryogenesis.
Exogenous DNA uptake and transient expression of the GUS reporter gene was studied in wheat (Triticum aestivum L.) zygotic embryos employing a simple procedure of cellular permeabilization by membrane interactive agents like saponin and toluene. Uptake of expression vectors with different promoters was detected in mature and immature embryos. The frequency of GUS expression was detected to be higher in mature embryos than immature embryos probably due to a certain degree of disorganization of the plasmalemma during the dehydrated state of the embryos. Of the various permeabilizing agents employed, saponin and toluene were detected as potential membrane permeabilizers without adversely affecting embryo viability. Variations in GUS gene expression were not significant among different genotypes of T. aestivum CPAN1676, PBW343 and HD2329, thus indicative of the relative genotype independence of this process. The results support the proposed idea of direct DNA uptake as an alternate and simple method for inserting foreign DNA into plant cells and its use for transformation of higher plants.
To compare transformation frequency and to investigate the developmental alterations in transgene expression, two cut-flower chrysanthemums ‘Yamabiko’ (spray-type) and ‘New Summer Yellow’ (standard-type) were transformed with three disarmed Agrobacterium tumefaciens strains C58C1, MP90 and LBA4404, all strains having the pBI121 plasmid. No marked difference was observed in transformation efficiency among cultivar/bacterial strain combinations. β-glucuronidase (GUS) activity levels in transformants were fairly low and varied among the different transgenic lines, ranging from 30 to 250pmol min-1 (mg protein)-1. GUS expression was not observed in the transgenic lines transformed with strain LBA4404. The alterations of GUS activity levels were examined for a long term, and it was observed that GUS activities reduced to zero level in most of transgenic lines 12 months after the inoculation of bacteria.
The cDNA and genomic clones from sweet potato encoding granule-bound starch synthase I (GBSSI) were isolated and characterized. The sequence analysis of the cDNA shows that the sweet potato GBSSI mature protein is comprised of 531 amino acids and that the precursor has a transit peptide of 77 amino acids. A comparison of the cDNA and genomic sequences suggested that the sweet potato GBSSI gene has 14 exons and 13 introns, the first of which is located in the untranslated region. This gene is considered to be a low-copy gene based on genomic Southern analysis.
A cDNA encoding for a thiol protease was isolated from Matricaria chamomilla. The cDNA contained an open reading frame consisting of 501 amino acids, which had three active site motifs of thiol protease.
Stable fasciation appeared in in vitro rhizome cultures of Cymbidium kanran Makino. Fasciated rhizomes had ridge-like flat rhizomes with elliptical epidermal cells, stomates and rhizoids. Most axillary buds of fasciated rhizomes were inactive resulting in rare branching. Fasciated rhizomes did not respond to cytokinins which were effective in the shoot conversion of non-fasciated normal rhizomes.
A gene for Brazil nut 2S albumin, a methionine-rich protein, was fused to a promoter region of the canavalin gene. This chimeric gene was introduced into shoot apices of embryonic axes of French bean by particle bombardment and seeds were obtained from plants regenerated from the bombarded axes. Protein in the transgenic seeds was analyzed by immunoblotting with an antiserum against Brazil nut 2S albumin. A 12-kDa immunoreactive polypeptide was detected and its amount in the seed was estimated to comprise less than 1% of the soluble protein.