Using somatic embryogenesis system of Japanese larch that develops embryos composed of embryo-proper and suspensor, the effects of high-cell-density culture and conditioned medium on the somatic embryogenesis were investigated. High-cell-density culture strongly inhibited the somatic embryogenesis. Furthermore, the conditioned medium derived from high-cell-density culture also strongly inhibited the somatic embryogenesis, especially differentiation of the suspensor. The inhibitory effect of the conditioned medium was not attributable to the depletion of nutrients, but to the accumulation of inhibitory factor(s) in the medium. The addition of activated charcoal to high-cell-density culture resulted in the formation of numerous somatic embryos with longer suspensors than in the untreated one. This treatment also resulted in the formation of numerous vacuolated cells-like suspensor on the surface surrounding the embryo-proper. These results indicate that some inhibitory factor(s) that regulates suspensor differentiation are released into the medium of high cell density.
Salicylic acid (SA) is a proposed signal for systemic acquired resistance to pathogen infection, while precise mode of SA translocation is little understood. To study it directly, 14C-SA was fed to the petiole end and the signal was detected autoradiographically. In juvenile plants, the signal moved to the lower stem and root, then the vascular systems of upper, middle and lower leaves within 1 h. In adult plants with 13 developed leaves, it was detected in nodes of 6 upper and 3 lower leaves at 10 min, and all leaves at 1 h along the orthostichy of phyllotaxis after feeding from the 7th leaf. The signal dispersed to the leaf gap, cortex parenchyma, and epidermis. One and 7% of radioactivity recovered was detected as SA β-glucoside at 10 min and 1 h, respectively. Thus, SA can move rapidly at all nodes of plants as an emergency signal within 1 h.
An in vitro culture system of Zinnia mesophyll cells has best used for studies on xylogenesis. Our previous studies with Zinnia cell culture demonstrated the presence of different cell types in the culture and their cell-cell communication. As the first step to investigate such cell-cell communication, we intended to establish a method to separate different cell populations and to culture a specific one. Zinnia mesophyll cells were stained with fluorescein diacetate, and then the fluorescence-positive and negative-cells were separated successfully using flow cytometric cell sorting. The fluorescence-positive cells differentiated into tracheary elements in the presence of an antibiotic, nalidixic acid. These procedures will be useful not only for the analysis of cell-cell interactions in Zinnia culture, but also for separating cells with intact cell walls in other plant species.
Vital protoplasts were isolated from developing dwarf rice endosperms about 10 days post anthesis. A yield of 0.98x105±0.12 protoplasts was obtained from 10 developing grains. These protoplasts were viable for at least three days in the culture medium. Transient expression of the green-fluorescent protein (GFP) was detected after four hours of introduction of the GFP gene by the polyethylene glycol (PEG)-mediated DNA transfer. As expected, the fluorescence of GFP without the targeting sequence was observed diffusely in the cytoplasm. On the other hand, the fluorescence of the fusion protein for rice prolamin followed by the GFP was observed in the small particles and the ER-network. These endosperm protoplasts had no autofluorescence upon excitation with 490 nm (blue) light. The vital protoplasts obtained from this dwarf rice variety are very useful for the physiological, biochemical and molecular biology studies of rice endosperm.
AtRAD51 gene encodes an Arabidopsis homologue of E. coli RecA protein that catalyzes homologous recombination and repair of chromosomal DNA. The AtRAD51 mRNA expression is regulated by DNA damage and cell cycle, but the mechanisms and signal transduction pathway involved in the AtRAD51 transcriptional regulation are largely unknown. In order to investigate regulatory mechanisms of DNA-damage induced gene expression in plants, we carried out functional analysis of the AtRAD51 gene promoter. A 0.7 kb fragment of the 5′-upstream region of AtRAD51 genomic DNA was fused to the firefly luciferase reporter gene and introduced into tobacco cells by microprojectile bombardment and Agrobacterium-mediated transformation. Induction experiment using bleomycin indicated that the AtRAD51 promoter is able to direct gene expression in tobacco cells in response to DNA damage.
Internal ribosome entry sites (IRES) were first identified as sequences in viral genomes that allow cap-independent translation initiation. The IRES sequence from the encephalomyocarditis virus (EMCV) is able to direct internal translation initiation that allows co-expression of multiple genes from one mRNA. Although EMCV-IRES has also been demonstrated to be functional in transgenic plants, the characteristics of the EMCV-IRES in plant cells have not been fully defined. Using a transient expression system by microprojectile bombardment and quantitative reporter gene assay, we have analyzed EMCV-IRES activity in plant cells. We show that the spacing between the IRES sequence and the second cistron is crucial for IRES-dependent translation efficiency. However, the order of cistrons is not an important factor for the EMCV-IRES-dependent translation in plant cells. Studies in different plant species reveal species specificity for EMCV-IRES activity. Analysis of transgenic tobacco plants suggested that the EMCV-IRES-dependent translation is suppressed in roots.
To isolate and analyze salt-stress inducible genes in a halophyte, sea aster (Aster tripolium L.), we screened 5760 Arabidopsis cDNA clones by macroarray procedure using 33P-labeled cDNA targets synthesized from mRNAs isolated from NaCl treated and untreated sea aster seedlings. Seventeen Arabidopsis cDNAs were hetero-hybridized to NaCl inducible sea aster genes. These cDNAs were used as probes to isolate cDNA homologs from a sea aster cDNA library. One of the obtained cDNAs shared 71% amino acids identity with Arabidopsis cysteine protease (AtCysP) and named SaCysP (sea aster CysP). Northern blot analysis revealed that mRNAs corresponding to both SaCysP and AtCysP were induced by salt and osmotic stress in leaves. On the other hand, SASR21 mRNA encoding another CysP in sea aster was irresponsive to these stress in leaves but respond in roots. SaCysP and SASR21 genes may have a tissue-specific function in stress response by modulating their expression levels.
Virus-induced gene silencing (VIGS) can be used to study gene function by mediating sequence-specific mRNA degradation and suppressing the expression of endogenous target genes. We previously demonstrated that the TocJ vector based on the tomato mosaic tobamoviruses (ToMV) was able to multiply, spread systemically and express green fluorescence protein in Solanaceous plants. TocJ harbouring fragments of endogenous genes could induce VIGS of the parental gene expression, but also induced viral infection symptoms. In this study, an attenuated strain of ToMV, L11A, was used to construct a ToMV vector in order to reduce the virus-induced symptoms. This new vector, named LcJ, was able to spread systemically and mediated VIGS of endogenous genes without visible symptoms. We propose that the use of this attenuated strain for the construction of virus vectors is beneficial for the induction of VIGS.
We have developed a method for comprehensive analysis of sugar phosphates by high performance anion exchange chromatography with pulsed amperometric detection coupled with a titanium dioxide column as a trap-column to remove sample matrices. Levels of sugar phosphates and a nucleotide phosphate from Arabidopsis thaliana grown at three different inorganic phosphate (Pi) concentrations in nutrient media or from Pi-related Arabidopsis mutants were investigated. Fructose-6-P, Galactose-1-P, Glucose-1-P, Glucose-6-P and Mannose-6-P apparently increased in proportion to increases in the in vivo level of Pi in wild type plants. In contrast, levels of Sucrose-6-P and UDP-Glucose decreased as the in vivo Pi levels increased. Responses of the former sugar phosphates except Mannose-6-P to the in vivo Pi levels in shoots of the mutants were similar in the wild type plants. However, Sucrose-6-P and UDP-Glucose responded differently between the wild type and mutant plants.
Interspecific hybrids of Nicotiana langsdorffii × N. tabacum showed hybrid lethality when they were transferred to 24°C after cultured at 34°C for 50 days. Characteristic features of programmed cell death (PCD) were detected in these plants, but no such features were observed when seedlings were transferred to higher than 26°C. Chromatin condensation and nuclear fragmentation were observed in protoplasts isolated from yellow and brown leaves of hybrid seedlings expressing lethality. Electrophoresis of total DNA isolated from the leaves of hybrid seedlings showed a DNA ladder patterns, suggesting nucleosomal fragmentation of DNA. These results suggest that PCD is accompanied by temperature-dependent lethality of hybrids between N. langsdorffii and N. tabacum.
Three isozymes of chitinase were induced by a fungal elicitor in cultured tobacco BY-2 cells. The most acidic one, designated TBC-3, was described here. The molecular mass of TBC-3 was estimated to be 28.5 kDa. The N-terminal amino acid sequence of TBC-3 was analyzed and a homology search was performed. Fifteen amino acids at the N-terminus showed 60% homology to class II chitinases from other plants but not higher than 27% homology to already known chitinases from tobacco. Therefore, TBC-3 is thought to be a novel tobacco chitinase that may be classified into class II.
Calli derived from mature zygotic embryos and seedling explants of Larix gmelinii were used to investigate for organogenesis. After 4 weeks of culture on half-strength DCR medium containing BA, numerous adventitious buds formed on the callus tissues which were derived from zygotic embryos, but none formed on calli generated from seedling explants. Rates of callus proliferation and adventitious bud formation were higher when the callus was transferred to medium supplemented with activated charcoal. To induce shoot elongation, multiple shoots were separated and transferred to medium with 0.05% activated charcoal. To induce adventitious root formation, elongated shoots of 1.5-2.0 cm in length were cultured in half-strength MS medium containing 0.3 mg l-1 NAA and 0.3 mg l-1 IBA. To assess genetic homogeneity and somatic variation, chromosomes of regenerated plantlets were observed. No numerical or structural changes to the chromosomes were found in regenerated plantlets using this system.
Transformed plants of Egyptian clover (berseem clover, Torifolium alexandrinum L.) were obtained from hairy roots induced by infection with Agrobacterium rhizogenes strain DC-AR2 (harboring mikimopine-type pRi1724). Among established 16 hairy root lines showing growth variations on MS medium containing 0.5 mg l-1 NAA, two lines with moderate or slow proliferation regenerating shoots spontaneously. The presence of T-DNA of pRi1724 in the genome of the regenerated shoots was confirmed by the DNA gel blot analysis. The regenerated shoots repeatedly formed shoots but scarcely formed roots on hormone-free MS. Whole plants were obtained from the shoots when they were cultured on Florialite for two months. The regenerated plants displayed the hairy root syndrome such as dwarfing and alterations of leaf shape. We propose here that hairy root-mediated regeneration is one of the effective tools for obtaining transgenic plants of Egyptian clover.
It has been proposed that the suspensor has important roles in early embryogenesis in seed plants. However, the roles of the suspensor are not well understood, because the development of zygotic embryos normally occurs deep within both the endosperm and the maternal cells. In this paper, we report the development of an in vitro culture system to investigate the roles of the suspensor in the development of the embryo proper, using a somatic embryogenesis system with Japanese larch (Larix leptolepis GORDON). Our results indicate that the suspensor is essential for the normal development of somatic embryos of this species. This method provides a useful experimental system to investigate the interactions between the embryo proper and the suspensor.