Ethylene is a gaseous plant hormone which gives cues to various developmental processes and stress responses in plants. Plants regulate their ethylene sensitivity for programmed development and responses to environmental stresses. After the successful isolation of the Arabidopsis ethylene receptor gene ETR1, ethylene receptor genes have been found in various plant species. Characterization of ethylene receptor genes provides clues to understanding how plants regulate their ethylene sensitivity. This knowledge also gives us potential methods for engineering the ethylene sensitivity of plants. Genetically-engineered plants with reduced ethylene sensitivity will be potential resources for improving crop performance. Moreover, those transgenic plants will provide further information to elucidate the mechanism of how plants regulate their ethylene sensitivity. The recent progress in genetic and protein analyses of ethylene receptors is summarized in this review. The possible strategies for altering the ethylene sensitivity of plants using the ethylene receptor genes are also discussed.
Cell suspension cultures of Lithospermum erythrorhizon, a medicinal plant, produce red naphthoquinone pigments, which are derivatives of shikonin, when cultured in the production medium M9 in darkness, whereas light as well as ammonium ion which is a major component of Linsmaier and Skoog's medium, completely inhibit shikonin production. In the non-pigmented cells, a large amount of shikonin precursor p-hydroxybenzoic acid is accumulated as its O-glucoside instead of shikonin. An overview of the regulation of shikonin biosynthesis and the localization of important intermediates is presented in this review. We also discuss the effect of light in more detail, and characterize the dark-inducible genes (LEDI) which have been isolated from shikonin-producing cells, as well as the establishment of stable transformation of this plant. Furthermore, the latest results of functional analyses of LEDI genes in hairy root cultures are also reported.
Plants have an excellent mechanism for responding to pathogen invasion by specific and inducible defense reactions, which is often apparent as the so-called hypersensitive reaction (HR). The molecular dissection of the mechanism of HR is difficult since the biological system involves intricate interactions between plant and pathogen. A simplified system, in which suspension-cultured plant cells are synchronously challenged with a single molecular inducer of HR, elicitor, would greatly facilitate studies of the molecular mechanism of HR. Several components involved in elicitor signaling have recently been identified and characterized using the experimental system.
Genetic challenge to express higher carbonic anhydrase (CA) activity in the cytoplasm of tobacco mesophyll cells was carried out by an improved promoter (El2 Ω) with a cDNA of partially modified mouse CA. In leaves of T1 progenies of transgenic tobacco (ffCA plant), the expressed mouse CA peptide was confirmed with 1, 5-3.2 times higher than the transgenic tobacco having foreign mouse CA controlled by simple CaMV 35S promoter (fCA plant). The ffCA plants manifested higher photosynthetic carbon assimilation (PCA) rate under ambient CO2 condition than wild type plants. The chlorophyll fluorescence measurement analysis revealed that the ffCA plant's improved PCA rate is related to the reduction of mesophyll CO2 transfer resistance (Rr) with little change in the stomatal resistance (Rs) or mesophyll CO2 fixation resistance (Rx). Moreover, ffCA plants showed a higher growth rate under high light condition.
Resistance gene-like fragment ADG2 has been shown to co-segregate with Ryadg, which controls extreme resistance to potato Y potyvirus (PVY) in Solanum tuberosum subsp. andigena (Hämäläinen et al. 1997, 1998). Applicability tests of ADG2 as a selection tool for resistance to PVY were carried out by genomic Southern analysis on a broad range of cultivars and breeding lines of potato and some other solanaceous species. A new marker band (10.5kb) associating with Ry was found in addition to previously reported 3.5kb marker band. While the 3.5kb marker was specifically linked to Ryadg with extremely high association (96%), the newly identified 10.5kb marker band was detected in genotypes containing Ryadg and other Ry genes. Some signals with ADG2 were also observed in other species within family Solanaceae inferring with the diversity of the corresponding chromosome region (s) among solanaceous species.
The effect of different concentrations of sucrose, glucose and fructose on in vitro rooting of shoots of Japanese persimmon that had been induced from selected rootstocks was studied. When the shoots of Japanese persimmon strain “No.3” were cultured on autoclaved half-strength Murashige and Skoog medium (MS medium) containing 0.2M fructose, roots were induced in 94% of the shoots after 45 days of culture whereas only 30% of rooting was obtained on media containing the same concentration of sucrose or glucose. However, filter-sterilized fructose had no stimulative effect on rooting. The autoclaved medium containing 0.2M fructose showed consistently high percentages of rooting (80%<) of the shoots for 9 rootstock strains tested, whereas only 3 strains responded to the culture on medium containing 0.1M sucrose following 250mg/l IBA pretreatment.
This work was undertaken to investigate the relationship between water status and sucrose metabolism of suspension culture of carrot (Daucus carota L.) cells. Embryogenic callus was induced on carrot hypocotyl in vitro. The cell suspension culture was then produced from this callus. In suspension culture, water potential and pH of the culture solution were decreased immediately after culture initiation to -0.62MPa and 4.5 respectively, but gradually increased from 2 days after culture initiation. Sucrose in suspension culture solution was found to be immediately hydrolyzed into glucose and fructose after the initiation of culture and disappeared within 2 days from culture initiation. The disappearance of sucrose from the culture solution within 2 days of culture initiation corresponds with increases in the activity of cell-wall-bound acid invertase. These results indicate that the decline of water potential of the culture solution at the early stages of carrot suspension culture was associated with sucrose metabolism as a result of an increase in acid invertase activity.
A cationic peroxidase gene (Shpx6a) of a forage legume species, Stylosanthes humilis, was transferred to poplar (Populus tremula) in antisense orientation under the control of 35S RNA promoter of cauliflower mosaic virus. Transformed plants were regenerated on selective media after co-cultivation of poplar stem explants with Agrobacterium tumefaciens and integration of transgenes was confirmed by PCR and Southern hybridization analyses. Analyses of selected transgenic plants showed reductions in total leaf peroxidase activity which was 50% to 70% of that measured in untransformed control plants. Transgenic poplar plants with reduced leaf peroxidase activity had 10-20% lower lignin content than control plants. Although which isoform of poplar peroxidase (s) has been inhibited by 35S-Shpx6a antisense construct is not clearly known, our results suggested the possibility of manipulation of lignin content through inhibition of lignin-specific peroxidases.
This report demonstrates the presence of S-RNases in style of self-incompatible Japanese plum (Prunus salicina Lindl.) and presents information about cDNA sequences encoding the S-RNases. Two stylar glycoprotein spots that were found in highly basic zone of 2D-PAGE gel with Mr of about 30kDa shared similarity in N-terminal amino acid sequence and immunological characteristics with other rosaceous S-RNases and appeared to be S-RNases of Japanese plum. Deduced amino acid sequences from the cDNA clones encoding these glycoproteins contained two active sites of T2/S type RNases and five regions conserved among other rosaceous S-RNases. Genomic DNA blot and RNA blot analyses using the cDNA clones as probes and phylogenetic analysis based on the deduced amino acid sequences from the cDNAs all indicated that the cDNA clones encoded S-RNase corresponding to the stylar glycoproteins detected on 2D-PAGE gel. Phylogenetic analysis based on the deduced amino acid sequences from the cDNAs supported the hypothesis that the S-RNases of Rosaceae have diverged after the divergence of subfamilies but before the divergence of species.
We have established an efficient Agrobacterium-mediated transformation method in some leading varieties of Japonica rice (Oryza sativa, L.), including Koshihikari. Scutellum calli were induced from mature seeds on our revised medium, KA1, with high frequencies (50 to 70%) and were used for co-culture with Agrobacterium tumefaciens EHA101 which carries binary vector harboring either β-glucuronidase (GUS) gene or synthetic green fluorescent protein (sGFP (S65T)) gene driven by CaMV 35S promoter. Scutellum calli at 3 weeks old were highly efficient for the regeneration of transformants. The transformation efficiencies ranged from 15 to 34 % in seven leading varieties of nonglutinous rice. The presence of the foreign genes in the genome was confirmed by southern blot analysis, and the expression of sGFP (S65T) gene was detected in several tissues of transformants with bright fluorescent signals under a fluorescent microscopy. The present study demonstrates the usefullness of sGFP (S65T) gene as a reporter in transformed rice plants.
The luc gene, from firefly, was ligated into the multiple cloning site of the pBI121 plasmid and was then incorporated into Agrobacterium tumefaciens strain LBA4404. This vector containing a CaMV 35S promoter, a NOS terminator, a NPTII gene and a NOS promoter was used to transform ‘Hibush’ eggplant (Solanum melongena). Genomic DNA from the transformants was analyzed by assay of both PCR and southern blotting, which confirmed the presence of an expected luc fragment. Four transformants were chosen for an extended LUC assay. During the course of one year, LUC activity fluctuated, but was maintained at the initial activity even after 50 weeks. Expression of the luc gene in transgenic calli revealed that fluctuation of the LUC activity was due to changes in the environmental temperature. Luminescence from transgenic eggplant was detected using image analysis and light emission was found to be intense throughout the whole plant.
cDNA encoding serine Carboxypeptidase gene of Matricaria chamomilla was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA end techniques. The cloned cDNA contained an open reading frame consisting of 501 amino acids, which had two active site motifs of serine Carboxypeptidase and high homology with other plant carboxypeptidases. The M. chamomilla Carboxypeptidase was found to have leucine zipper structure at the N terminal region, suggesting that this enzyme functions at dimer form. Nine N-myristoylation sites in the enzyme let us anticipate that this enzyme localizes at a membrane or lipid layer.
Towards developing a rapid and reliable in vitro regeneration system for the propagation of mulberry, culture response of Morus multicaulis cv. Goshoerami and M. indica cvs. K2, RFS175 and S1, was examined. The presence of a cytokinin was essential for bud break. Thidiazuron (TDZ) at a concentration of 0.1mgl-1 was found to be more effective than BAP for bud break and shoot proliferation in M. indica cvs. RFS175 and k2, whereas 0.5mgl-1 TDZ was better than BAP in M. indica cv. S1. Although TDZ enhanced the efficiency of shoot multiplication in M. indica, 2.5mgl-1 BAP was found to be superior in the case of M. multicaulis. TDZ not only significantly reduced the days required for bud break but also increased the percentage of bud breaks and the number of shoots per explant in M. indica. In vitro rooting of Morus shoots improved significantly by adding activated charcoal to culture media. A significant increase in the percentage of rooting and a decrease in the days required for rooting were observed by using 0.05% activated charcoal in M. multicaulis cv. Goshoerami and M indica cv. S1, and 0.1% activated charcoal in M. indica cvs. K2 and RFS175. In vitro-raised plantlets were successfully acclimatized and transferred to the field.
Cotyledons isolated from immature embryos of 11 varieties of soybean were cultured on embryogenesis media containing 2, 4-D at 40mgl-1 and sucrose at 10, 20, 40, or 60gl-1. The number of somatic embryos per cotyledon was counted after 5 weeks of culture. Analysis of variance showed that the effects of genotype, sucrose concentration, and genotype×sucrose interaction were all highly significant. Further analysis of the genotype×sucrose interaction showed that the heterogeneity of regression among the varieties was significant. The variety mean and regression were significantly correlated, indicating that the 2 factors are not under separate genetic control. Nevertheless, these results suggest that embryogenic response to 2, 4-D and NAA seems to be under separate genetic control in soybean.