In order to estimate whether mastoparan 7 specifically activates plant heterotrimeric G proteins, comparisons were made between the responses of a rice normal cultivar and a rice dwarf mutant, Daikoku d1, which has a mutation in the gene of the α subunit of a heterotrimeric G protein, to mastoparan 7. First, we found that mastoparan 7 induced the activity of the α-amylase via the heterotrimeric G protein in rice embryo-less half seeds. This observation indicated that mastoparan 7 activated the G proteins in plant cells, as does it in mammalian cells. Second, mastoparan 7 seemed to cause damages in rice cells in a G protein-independent manner under some experimental conditions. We propose that mastoparan 7 should carefully be used for study on the plant G protein-mediated signaling pathways.
For chromosome duplication of Potato virus Y (PVY)-resistant haploid mutants obtained by the combination treatment of tobacco anther culture and ion beam exposure, pith tissue culture was performed. Thirty-five plants were obtained by pith tissue culture and subsequent acclimatization. Their chromosome number ranged from 22 to 96, and 16 of them had 48 chromosomes. These plants produced fertile seeds by self-pollination. When plants derived from self-pollinated seeds were inoculated with PVY, resistant and susceptible plants were observed. Of the plants derived from the most resistant mutant in the haploid generation, about 50% was resistant to PVY.
Inosine diphosphatase (IDPase) isoforms associated with Golgi membranes were studied in sycamore cell culture. These enzyme isoforms were solubilized with Triton X-100 and purified by chromatography using DEAE-Toyopearl and SOURCE-S columns. The isoforms were separated into two distinguishable fractions (peak 1 and 2) by SOURCE-S column chromatography. Furthermore the peak 1 contained at least two isoform bands detected by native-PAGE analysis. The apparent molecular sizes of these three isoforms were estimated by both gel filtration and SDS-PAGE to be 50 kDa, indicating that the Golgi membrane-bound IDPase has a monomeric structure. These IDPase isoforms required divalent cations (Ca2+, Mg2+, Co2+, Mn2+) for their hydrolyzing activity, and were inhibited by ATP. IDP, UDP, and GDP were effective substrates for these enzymes. It is clearly indicated that the sycamore Golgi membrane-bound IDPase is a nucleoside diphosphatase.
The calluses were indeced from turnips, Brassicae campestris L. which were susceptible and resistant to Plasmodiophora brassicae in Murashige-Skoog’s agar medium supplemented with 1.0 ppm 6-benzylaminopurine and 0.5 ppm α-naphthalene acetic acid. When a 10 μl water suspension containing 104 resting spores of P. brassicae was placed on the surface of the calluses, about 3 × 105 zoosporangium-like spheroids (SLS) were recovered from the susceptible calluses after 24 h of the treatment but no SLS was found in the resistant callus. On 6th day after the treatment, the SLS increased to about 4 × 106 in the susceptible callus. Upon inoculation of resistant callus with 104 resting spores, the phenylalanine ammonia lyase (PAL) activity increased about 4-fold after 20 h, however, such increase was not observed in the susceptible callus during the same period. The consitutive PAL activity of susceptible callus was roughly one 6th of that of resistant callus.
A simple regeneration protocol has been developed for two millets, Eleusine coracana and Echinochloa crusgalli. The plantlet regeneration in both the millets is via somatic embryogenesis as evidenced by histological studies. In the case of E. coracana, up to 340 plants could be regenerated per 100 seed calli while up to 2266 plants could be regenerated per 100 seed calli of E. crusgalli. Subsequently, the regenerating seed callus as well as leaf segments from these two millets have been used as explants for assessing the suitability of five gene promoter constructs for transformation via biolistic means. Transient GUS histochemical as well as spectrofluorometric assays reveal the high efficiency of Ubiquitin I gene promoter from maize in terms of bringing about maximum GUS activity in both the millets. The activity of Ubiquitin I promoter from maize was highest in leaf lamina followed by leaf sheath and seed callus. Other four promoters were found to be much less efficient for both millets.
A sequence homologous to a gene encoding succinate dehydrogenase subunit 4 (sdh4) was found in pea mitochondrial genome but non-functional. A functional sdh4 gene was isolated from pea nuclear genome, suggesting gene transfer from the mitochondrion to the nucleus during evolution. As compared with mitochondrial sdh4 genes, the pea nuclear sdh4 gene has an extension in its N-terminal region, suggesting encoding a protein targeting signal to mitochondria. There are five introns and six exons in the pea nuclear sdh4 gene. Sequence homology to mitochondrial sdh4 is observed in the exon 6 but not in the other five exons, suggesting mitochondrial sequence was integrated downstream of the five exons. Comparison of nuclear sdh4 genomic sequences from pea, Arabidopsis and rice suggests that the number and location of introns are different among the three. Therefore, sdh4 genes may have evolved in the nuclei independently among plant species.
Poly[(R)-(−)-3-hydroxyalkanoate] (PHA) synthase from Aeromonas caviae FA440 and β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha H16 were targeted into tobacco plastid by utilizing a plastid-targeting signal peptide derived from the tomato ribulose-bisphosphate carboxylase (Rubisco) small subunit. The resulting tansgenic tobacco plant expressed all of the introduced genes, and GC-MS analysis of chloroform extract of tobacco leaves demonstrated that the transgenic plant produced poly[(R)-(−)-3-hydroxybutyrate] (PHB). The productivity of PHB in plastids was about 100-fold greater than that in cytoplasm without any apparent deleterious effects on growth and seed production. Intracellular localization of PHB in the leaf of the transgenic plant was examined under epifluorescence microscopy after Nile blue A staining, and it was proven that PHB is formed in chloroplasts around the inside of cell walls.
Clear 180-bp nucleosomal DNA laddering in cultured rice cells was induced only by an incompatible strain of Pseudomonas avenae. This supports the current view that the hypersensitive cell death of a plant is programmed cell death. The flagellin-deficient mutant of the incompatible strain did not induce DNA laddering, suggesting that DNA laddering was mediated by incompatible flagellin perception.
A genomic clone encoding a karasurin precursor was isolated from root tubers of Trichosanthes kirilowii var. japonica. The sequence analysis indicates that karasurin-A and karasurin-C exhibiting abortifacient and ribosome-inhibiting activities are produced from a common karasurin precursor by proteolitically cleaving at a different site. Northern hybridization analysis shows that the karasurin gene was expressed only in root tubers. An efficient system to overproduce the recombinant karasurin-A as fused protein with the maltose-binding protein was established.
Transgenic rice plants of Taiwanese japonica rice cultivar, Taichung 65, were obtained by co-cultivating scutellum calli with an Agrobacterium tumefaciens strain, EHA101, that carried a binary vector harboring the luciferase (Luc) gene driven by the CaMV35S promoter. The transformation efficiency of Taichung 65 was similar to that obtained by the methods routinely used for a transformable cultivar, Notohikari. There was no correlation between the length of the culture period (21 to 30 days) and the transient transformation efficiency in Taichung 65 and in Notohikari. In the T0- and T1-generations, the transgenes were integrated and stably expressed, indicating that the transgene was inherited to the next generation. The copy number of integrated transgene varied from one to three in the T1-transformants, which was confirmed by Southern blot analysis. Moreover, approximately 60% of the T1-transformants of Taichung 65 showed the LUC-positive phenotype. These results suggest that, in addition to Notohikari, Taichung 65 is a practical, transformable cultivar.