The production of procyanidin and anthocyanin in grape cells is markedly increased by treatment with salicylic acid. The activity of phenylalanine ammonia-lyase and chalcone-flavanone isomerase increased in close relation to the production of these phenolics. Salicylic acid-induced elevation of these enzyme activities occurred more rapidly than elevation induced by light irradiation. In the cells, the phenolics were degraded in a different manner by salicylic acid treatment than that by light irradiation. Salicylic acid might be used as a simple and effective stimulant for the production of procyanidin as well as anthocyanin in large scale cultures of plant cells.
A gene for a cysteine endopeptidase REP-1, Rep1, is expressed in both the embryo and aleurone layers in germinating rice seeds. The expression of Rep1 was not repressed in the embryo by exogenously applied uniconazole, a gibberellin (GA) biosynthesis inhibitor, even though GA-inducible expression of the gene occurred in the aleurone layers. Serially truncated Rep1 promoters fused to the β-glucuronidase (GUS) reporter gene were employed to identify the cis-regulatory elements involved in the embryo-specific expression. The deletion of the region from -500 to -432 decreased the GUS activity significantly. Linker-scan mutation analysis was carried out to determine the sequences involved in the expression of Rep1 in embryos, which showed that the region from -500 to -432 contained at least two positive regulatory elements.
We constructed a binary vector to improve the transformation efficiency and transgene expression in chrysanthemum plants in an Agrobacterium-mediated system. Most of the sequences unnecessary for maintaining plasmids in bacteria were removed from the pBI121 vector, and an expression cassette of the neomycin phosphotransferase II gene, in which a point mutation was repaired, and an expression cassette of β-glucuronidase (GUS) with a short 5’ un-translated sequence of β-glucanase from soybean, were set within a transferred DNA. This vector was used to transform leaf discs of chrysanthemum plants, resulting in the transformation efficiency increasing several times compared with pBI121 and the activity of GUS increasing prominently. We also introduced a double-stranded RNA-specific ribonuclease (pac1) from Schizosaccharomyces pombe by using this novel vector to confer simultaneous tolerance against virus and viroid diseases. Using this technique we obtained more than 80 transgenic chrysanthemum plants, and found that half of them expressed a moderate amount of pac1 protein as detected by Western blot analysis.
In present-day biology, a facile gene delivery system is required to study gene function in a high throughput way. The use of plant virus vectors that can introduce foreign or extra endogenous genes into plants attracts much attention. It is advantageous in that researchers need only minimal experience and results can be obtained in a short period of time. Here we describe infectious cDNA clones of the tomato mosaic tobamoviruses (ToMV) that were modified for facile insertion of foreign genes and which retained the ability to multiply in plants. This vector, named TocJ, was able to harbor the GFP gene from Aequorea victoria and systemically spread in some Solanaceous plants. TocJ would be suitable for the overexpression of foreign genes and the analysis of gene functions in various Solanaceous plants.
Microsporogenesis is a highly organized indispensable event for the sexual reproduction of higher plants. The process includes a series of organ development, cell differentiation and meiosis. We examined the functional genes involved in microsporogenesis by random sequencing using a cDNA library from lily zygotene stage microsporocytes and isolated two cDNAs encoding novel gene products. Deduced amino acid sequences of both gene products, designated M355 and M404 contained a cluster of basic amino acid residues that may constitute a nuclear loclization signal. RT-PCR analysis indicated that the temporal and spatial expression of M355 and M404 is associated with early stages of microsporogenesis or meiosis. Transient expression of GFP fusion proteins in onion epidermal cells revealed nuclear localization activity of both proteins. These results suggest that M355 and M404 are involved in a nuclear event during the progression of microsporogenesis in lily.
When hypocotyl sections of Raphiolepis umbellata L. were cultured in a woody plant medium containing 8.9μM benzylaminopurine and 1.1μM α-naphthaleneacetic, adventitous shoots were formed. Fully developed plants were obtained in 3 months of the culture. A chimeric plasmid pENiR harboring cDNA of Arabidopsis thaliana nitrite reductase gene (nii) under the control of an enhanced promoter consisting of sequences from both cauliflower mosaic virus 35S promoter and tobacco mosaic virus omega regions and a nopaline synthase terminator was constructed and introduced to the hypocotyl sections by particle bombardment. A plasmid pCH bearing hygromycin phosphotransferase gene (hph) was cointroduced. Transformed shoots were selected for hygromycin resistance. Polymerase chain reaction analysis showed the presence of hph gene in 16 out of 2572 bombarded hypocotyl sections, and two of which were found to bear Arabidopsis nii gene. Translation of this gene in transgenic plants was confirmed by Western blot analysis.
We isolated a full-length cDNA clone, TaGB1, encoding the β subunit of heterotrimeric G-protein from common wheat, Triticum aestivum cv. S615. The predicted amino acid sequence deduced from the cDNA was 70-89% and >50% homologous to those of the β subunits of plants and animals, respectively. The TaGB1 exhibited the higest homology of 89% with the rice G-protein β subunit gene and preserved the most essential seven repeats of the “WD-40” motif, which are commonly found in all plant and animal G-protein β subunits. Southern hybridization revealed that the wheat genome contains a single-copy gene for the β subunit of heterotrimeric G-protein. The analysis of the expression for Gβ in wheat showed that TaGB1 mRNA was expressed in all of the organs tested and exhibited high degrees of mRNA accumulation in spikes and internodes as well as dark-grown seedlings. It was also observed by RT-PCR that the transcript of TaGB1 was expressed throughout the ancestral genomes of wheat.
A single nucleotide polymorphic sequence (SNP) in the trnL-F intergenic spacer region of chloroplast DNA that discriminate Musa acuminata (AA) cytoplasm from Musa balbisiana (BB) cytoplasm has been found. This mutation was initially amplified using a pair of universal primers and converted into dCAPS (derived cleaved amplified polymorphic sequence) markers. Using these markers in combination with flow cytometric analysis, ‘Pisang Klutuk’ (syn. ‘Pisang Awak’) was found to be a triploid ABB cultivar (not a wild BB accession as previously classified). A 15-bp long deletion was also found within the trnL-F spacer sequence of M. acuminata subspecies banksii. This mutation will be useful as a specific marker for the cytoplasm derived from this subspecies. These maternally inheritable chloroplast DNA mutations discovered in this study are vital for better understanding the origins of banana cytoplasm to clarify the lineage of banana cultivars and the contribution of wild progenitors to cultivated types.
Green fluorescent protein (GFP) has been used extensively as a novel non-invasive reporter in the investigation of issues such as promoter function, transformation marker recognition, and sub-cellular localization analysis. A plant-optimized synthetic GFP modified via the replacement of the serine to threonine at position 65 [sGFP(S65T)] has achieved high levels of expression with no toxic side-effects in many plants. It would be useful to develop a non-invasive selection system based only upon GFP expression levels. A trapping vector was constructed using an sGFP(S65T) and a quantitative fluorescence imaging system was used for non-invasive screening of trapped tobacco cultured cells. Putative trapped lines could be identified using this system. Moreover, the GFP products from the candidate trapped lines could be quantitatively visualized directly via SDS-PAGE using the fluorescence imaging system. We conclude that the non-invasive selection system described here could be a powerful new tool for plant biotechnology.
Microsegments from peppermint, Mentha piperita, were placed on supplemented Murashige & Skoog medium and infected with Agrobacterium rhizogenes. The hairy roots resulted from peppermint were cultured on various media in order to regenerate plants. The hairy roots formed a callus on Gamborg B5 medium containing 1μM 1-naphthaleneacetic acid (NAA), 10μM N-(2-chloro-4-pyridyl)-N’-phenylurea (4-CPPU), and 10% coconut powder in the dark. Shoots were regenerated from the calli cultured on 1/2 Murashige and Skoog medium containing 1μM NAA and 10μM 4-CPPU during a 16-h photoperiod. All plants were recovered following rooting of the shoots in B5 medium without hormones. The polymerase chain reaction (PCR) analysis of the genomic DNA showed that all regenerated plants had the region from the rolA to rolB gene.
A three-dimensional (3-D) analysis technique was developed to visualize the whole nodule and Nitrogen fixation (N2-fix) zone in a stem nodule from Sesbania rostrata infected with symbiotic rhizobium, Azorhizobium caulinodans ORS571. The reconstructed N2-fix zone of the stem nodule was analyzed by arranging the ratio of opacity, by cutting at vertical and level direction, and by extraction in a personal computer. The N2-fix zone was a ring shape, surrounding the vascular bundle. This is the first report that shows whole distribution of N2-fix zone in S. rostrata stem nodule.
The expression of the Oryza sativa selenium-binding protein homolog (OsSBP), which was previously shown to be transcriptionally activated in fungus-infected or elicitor-treated rice leaves, was studied in the response of endogenous signaling molecules and generators of reactive oxygen species. OsSBP expression increased rapidly in response to jasmonic acid (JA) and salicylic acid (SA), both of which play important roles as endogenous signaling molecules that induce resistance to pathogens. OsSBP also responded to abscisic acid (ABA) and paraquat, both of which cause increased generation of reactive oxygen species. The deduced OsSBP protein has a bis (cystenyl) sequence motif CxxC, which acts as an active redox center controlling oxidation / reduction of protein in vivo. This is the first report about the molecular characterization of OsSBP in rice.