The scutellar epithelium of rice seeds shows a variety of activities under different physiological conditions. The epithelial cells cease activity soon after the start of germination, but in the seminal root culture system, they retain their haustorial property for a long period. Furthermore, on solid medium with added 2, 4-D, they divide to form callus masses that lack a cuticular layer. The two-step method of callus culture results in a higher frequency of shoot-bud formation that is related to topographical variation on the callus surface. The development of a compact callus with white and green patches is a morphological prerequisite for shoot-bud formation. The cells constituting a callus mass behave differently according to the positional information determined by the internal and external environment. The external appearance of the callus largely depends on the extracellular matrix. The callus surface consists of a fragile slime-like membrane, firm fibrils, and a thin epicuticular layer. Finally, it is suggested that the newly formed cuticular layer on the callus surface is essential for shoot-bud formation. Consequently, we propose that the topographical characteristics of the callus mass play an important role in shoot-bud formation.
Rice is one of the most important crops in the world, and thus as a model plant it has been the subject of much research. But many kinds of rice species are not easy to handle in a laboratory space. To investigate the dwarf rice’s suitability for experimental work, we analyzed the characteristics of Hosetsu-dwarf. Hosetsu-dwarf has small body size, a short life cycle, and normal seed formation. We established a standard method for its hydroponic cultivation. We found as well that its endosperm polypeptide compositions and the morphological observation were not unusual. Thus we propose that Hosetsu-dwarf is an excellent tool for basic studies of seed development.
A successful transformation procedure using Agrobacterium has been established for the most important ornamental statice, Limonium sinuatum Mill. Cell suspension cultures derived from friable calli, which were originated from leaf segments, were infected with Agrobacterium tumefaciens strain EHA105 (pIG121Hm) and co-cultivated for 2 days. The concentration of acetosyringone in co-cultivation medium and the growth stage of host plant cell used for infection were important factors for obtaining high transient GUS expression. After selection on medium with 75mgl-1 kanamycin, approximately 20 putative transgenic callus lines with kanamycin-resistance and GUS expression were obtained from 1g F. W. of cell cultures infected. Adventitious shoots were induced from 5 different putative transgenic callus lines on the regeneration medium containing 75mgl-1 kanamycin, and finally 16 transgenic plants which showed GUS expression in leaves were obtained. Presence of NPT II and GUS genes was confirmed by PCR analysis in the genomic DNAs of transgenic callus. Integration of GUS gene into the genome of transgenic plantlets derived from different transgenic callus lines was detected by Southern blot analysis.
Three isoforms of polyphenol oxidase (I-a, I-b and II) were purified from betacyanin-producing callus cultures of Portulaca grandiflora, by ammonium sulfate precipitation followed by successive column chromatographies of DEAE cellulose, Sephacryl S-200, hydroxyapatite and Mono-Q. The molecular masses of the monomeric polyphenol oxidase I-a and I-b were calculated to be 66 and 65 kDa, respectively, when analyzed by SDS-PAGE. Polyphenol oxidase II appeared to be a heterodimer of two subunits with molecular masses of 25 and 27 kDa. The optimal pH for polyphenol oxidase activity of I-a and I-b was 5.0, and for II was 6.0. These enzymes were markedly inhibited by sulfhydryl binding reagents, ascorbate, kojic acid, copper-chelating reagents and Fe2+. L-DOPA (L-3, 4-dihydroxyphenylalanin) was a good substrate for these enzymes, although no activity with tyrosine and hydroquinone was observed. The apparent Km values of polyphenol oxidase I-a, I-b and II for DOPA were 2.0, 2.2 and 3.5mM, respectively. No monophenol oxidase (tyrosine hydroxylation) activity by these enzymes was observed.
We isolated a cDNA encoding a novel GT-box binding protein from Arabidopsis, designated GT-4. The predicted open reading frame encodes a protein of 372 amino acids and the predicted protein sequence revealed the presence of a putative DNA-binding domain with 80% homology to the trihelix region of previously described light-responsive element binding protein, GT-1. Reverse transcription-PCR analysis showed that GT-4 transcripts are present in all tissues tested in light-grown plants, but light-regulated expression of GT-4 mRNA was observed only in etiolated seedlings. Electro-mobility shift assay using recombinant protein revealed that the GT-box-binding specificity of GT-4 is almost identical to that of GT-1. Transient expression of GT-4::GFP fusion protein in onion epidermal cells revealed the presence of a nuclear localization signal within the GT-4 protein. These results suggest the possibility that GT-4 is involved in GT-box-mediated gene expression by recognizing target sequences closely related to GT-1 binding sites.
We isolated a novel receptor-like kinase with leucine-rich repeat by a differential screening for genes that were specifically expressed in inflorescence shoot apices of Arabidopsis, and named it IRK (inflorescence and root apices receptor-like kinase) after its expression pattern and structural features. IRK contains sequentially from the amino- to carboxy-terminus: a potential signal peptide for membrane targeting, a leucine-rich repeat between two conserved cystein motifs, a transmembrane domain, and a serine/threonine kinase domain. The kinase domain expressed in E. coli showed an autophosphorylation activity in vitro, indicating that IRK encodes an active protein kinase. The IRK protein fused to green fluorescence protein was targeted to plasma membrane, indicating that IRK is a membrane-localized receptor kinase. IRK was expressed not only at shoot apices but also in root tips and developing flowers. Thus, IRK seems to be a receptor-like kinase with a relatively general function in inflorescence and root apices for an unknown signaling mechanism.
The differential display technique was used to isolate genes expressed during yeast extract-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures, and a cDNA clone whose deduced amino acid sequence is similar to plant cytosolic NADP-malic enzymes was identified and designated LeME. The heterologously-expressed LeME oxidized L-malate in presence of NADP, but it did not catalyze formation of 4-hydroxyphenyllactic acid from 4-hydroxyphenylpyruvate in presence of NADPH, one of the crucial biosynthetic steps leading to rosmarinic acid. The potential role of LeME in elicitor-inducible rosmarinic acid biosynthesis is discussed.
Cloning of the expressing gene in the target cell is important for understanding the molecular mechanism of specific gene expression. Laser micro-dissection techniques allow the precise separation of a single target cell from morphologically distinct cells. We describe here an excellent method for separating a single cell or a cell cluster consisting of a few cells from a small target area of plant tissues. In rice endosperm tissues, the aleurone layer and starchy endosperm tissue are differentiated from a single endosperm mother cell. We succeeded in dissecting a specific cell area from endosperm and aleurone tissue (7 days after flowering). This information will be useful for studying cell differentiation and developing mechanisms of endosperm tissue. RT-PCR analysis for mRNAs for actin and glutelin confirmed the purity of the dissected cells.
The regeneration of multiple shoots from hypocotyl sections of Pittosporum tobira was studied on woody plant medium supplemented with differing concentrations of thidiazuron and naphthaleneacetic acid. Maximally 75% of hypocotyl sections formed multiple shoots when using the medium containing 10μM thidiazuron and 3.2μM naphthaleneacetic acid. Multiple shoots were transferred to a shoot-elongation medium followed by transfer to rooting medium. Plantlets formed were acclimatized, and complete plants were regenerated about 10 months after the initiation of the culture. A plasmid vector pANiR, which bears the cDNA of the nitrite reductase (NiR) gene from Arabidopsis thaliana under the control of cauliflower mosaic virus 35S promoter and nopaline synthase terminator, was introduced - together with pCH-bearing hygromycin resistance gene - into hypocotyl sections of Pittosporum tobira using a particle gun. A number of hygromycin-resistant calli were obtained, from which shoots were regenerated. The integration of Arabidopsis NiR cDNA into the genome of Pittosporum tobira was detected by the polymerase chain reaction method, confirming that these shoots were transgenic.
Protoplasts of Undaria pinnatifida and Petalonia binghamiae were cultured for two and one months, respectively, in the presence of various levels of Mg. Under Mg-free conditions in ASP12NTA medium all protoplasts died. The percentage of filaments per surviving protoplast of U. pinnatifida increased from 0.47% in 50mM Mg to 5.33% in 200mM Mg. Under these conditions the cell number per filament also increased from 154 to 513. The percentage of filaments per surviving protoplast of P. binghamiae increased from 77% in 50mM Mg to 95% in 150mM Mg, while the number of cells per filament increased from 5.8 to 60.8. At concentrations higher than 100mM Mg, the Mg and chlorophyll a concentrations were almost equal to those in the thalli.
Identifying genes involved in salt-stress responses in mangrove plants is one of the most critical steps in elucidating the salt-resistance mechanisms in these plants. In this study, we screened cDNAs whose mRNA amounts were regulated by salt-stress using cDNA Representational Difference Analysis (cDNA RDA) from suspension-cultured cells of a mangrove, Bruguiera sexangula. Four cDNA fragments whose mRNA amounts were up regulated by salt-stress were isolated. Database searches revealed that these cDNAs encode proteins similar to endo-polygalacturonase, alcohol dehydrogenase, and two putative Arabidopsis proteins. In addition, eight cDNA fragments whose mRNA amounts were down regulated by salt-stress were also isolated. Further characterization of these cDNAs will likely be important in elucidating the salt-resistance mechanisms in mangrove plants.
We studied the effects of various treatments given before or at inoculation with Agrobacterium tumefaciens strain EHA105 (pIG121Hm) on the transient GUS gene expression in leaf segments of Saintpaulia ionantha Wendl. Sonication and vacuum infiltration treatments in the presence of Agrobacterium had no positive effect on GUS expression. In contrast, the explants cultured for 3 to 5 weeks on shoot induction medium containing 0.5mgl-1 NAA and 0.5mgl-1 BA, prior to Agrobacterium inoculation markedly increased transient GUS expression. The addition of acetosyringone to the co-cultivation medium enhanced the GUS expression. A similar enhancing effect was observed in all of the 4 cultivars examined, suggesting the wide applicability of the pre-culture treatment.
To isolate genes responsive to N-acetylchitooligosaccharide elicitor in suspension-cultured rice cells, we screened rice ESTs by DNA microarray analysis, and were able to identify novel elicitor-responsive genes. Sequencing analysis of three ESTs revealed that the up-regulated genes include a transcription factor, Myb, small G-protein, Rac, and calmodulin. The results indicated the genome-wide change of gene expression in response to N-acetylchitooligosaccharide elicitor.