Kunihiko Ojima

Cellular localization of NADH-dependent glutamate-synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants

Tissue and cellular localization of NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) in the unexpanced leaf blades and young grains of rice (Oryza sativa L.) was investigated using tissue-print immunoblot and immunocytological methods with an affinity-purified anti-NADH-GOGAT immunoglobulin G. Tissue-print immunoblots showed that the NADH-GOGAT protein was mostly located in large and small vascular bundles of the unexpanded blades. When the cross-sections (10μ in thickness) prepared from the paraffin-embedded blades were stained with the antibody, the NADH-GOGAT protein was detected in vascular-parenchyma cells and mestome-sheath cells. In developing grains, the NADH-GOGAT protein was detected in both phloem- and xylem-parenchyma cells of dorsal and lateral vascular bundles, and in the nucellar projection, nucellar epidermis, and aleurone cells. On the other hand, ferredoxin (Fd)-dependent GOGAT (EC 1.4.7.1) was located mainly in mesophyll cells of the leaf blade and in chloroplast-containing cross-cells of the pericarp of the grains. The spatial expression of these GOGAT proteins indicates distinct and non-overlapping roles in rice plants. In the leaf blades and young grains, NADH-GOGAT could be involved in the synthesis of glutamate from the glutamine that is transported through the vascular system from roots and senescing tissues.

Changes in the Content of Two Glutamate Synthase Proteins in Spikelets of Rice (Oryza sativa) Plants during Ripening

Nitrogen accumulation in the apical spikelets on the primary branches of the main stem of rice plants have been studied during the ripening process (0–35 d after flowering). The level of NADH-dependent glutamate synthase (GOGAT) protein and activity increased 4- and 6-fold, respectively, in the first 15 d after flowering. Maximum levels of NADH-GOGAT were found at that time when the spikelets had just begun to increase in dry weight and to accumulate storage proteins. Subsequently, both the level of NADH-GOGAT protein and its activity in spikelets declined rapidly. Although changes in ferredoxin (Fd)-dependent GOGAT paralleled changes in NADH-GOGAT, the relative abundance of NADH-GOGAT protein in the spikelets was about 3 times higher than that of Fd-GOGAT from 5 to 15 d after flowering. When the chaff (lemma and palea) was separated from the spikelets 10 d after the flowering, 16% of the NADH-GOGAT protein was found in the chaff and 84% in the young grain tissues (endosperm, testae, aleurone tissues, and embryo). On the other hand, Fd-GOGAT protein was distributed 52% in the chaff and 48% in the young grain tissues in spikelets of the same age. Activity of NADP-isocitrate dehydrogenase, which may generate the 2-oxoglutarate required for the GOGAT reactions, was much higher than that of total GOGAT activities on a spikelet basis during the ripening process. These results suggest that in rice plants NADH-GOGAT is responsible for the synthesis of glutamate from the glutamine that is transported from senescing tissues to the spikelets.

Nitrogen Accumulation in the Inferior Spikelet of Rice Ear during Ripening

Abstract Nitrogen accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet), of the ear on the main stem of rice plant (Oryza sativa L. var. Sasanishiki) was characterized during grain filling. In the superior spikelet, the accumulation of dry matter and nitrogen which started immediately after lowering, proceeded rapidly, and reached the maturation level at 20 d after heading. In the inferior spikelet, however, the amount of dry matter and nitrogen accumulation was minimal immediately after flowering. It increased when grain filling of the superior spikelet was almost completed. 15N-labeled ammonia was administered to the plants at different stages of ripening and the amount of incorporation in the spikelets was analyzed at harvest. The labeled nitrogen administered at the early stages of ripening was the main source of the labeled nitrogen incorporated in the superior spikelet. However, the labeled nitroge...

Physiological response of root tip of alfalfa to low pH and aluminium stress in water culture

In acid volcanic soils, plant roots are thought to be injured by acidity (low pH) and/or solubilized aluminium (Al) ions. An attempt was made to separate the effects of low pH from those of Al on the elongation and viability of alfalfa (Medicago sativa L.) radicles in water culture. Root elongation was irreversively curtailed by 20 hours treatment at pH 4.0 without Al or 20 mmol m-3 Al at pH 5.0. Viability of surface cells of root tips was detected as a degrading activity of fluorescein diacetate (FDA) by cellular esterases and subsequent accumulation of derived fluorescein within cells. Large numbers of the surface cells lost their viability after four hours exposure at the low pH. In contrast, surface cells maintained both FDA degrading activity and ability to accumulate fluorescein 20 h after initial exposure to the Al solution (20 mmol Al m-3, pH 5.0). These results suggest that there are some significant differences in the mechanisms of phytotoxicity to alfalfa root between the two stress factors.

Characterization of two tobacco cell lines selected to grow in the presence of either ionic Al or insoluble Al-phosphate

A tobacco cell line tolerant to ionic A1 (AIT) was selected at a frequency of 10-6 to 10-7 by plating the wild-type cells on agar medium containing 2.5 mm AICI3 and 0.1 mM phosphate at pH 4.0. Another cell line which is able to grow with insoluble Al-phosphate as a sole source of phosphate (IPU) was also selected by subculturing the wild-type cells for more than 10 passages in suspension culture in the presence of 2 mM phosphate and 4 mM A1C13 at pH 5.0. The IPU cell line also tolerated ionic A1 but the AIT line showed poor growth with insoluble Al-phosphate as a sole source of phosphate. Both cell lines excreted citrate into the media. The rate of citrate excretion by the IPU cell line, on a fresh weight basis, was higher than that by the AIT and wild-type cell lines.

Aluminum-tolerance of carrot (daucus carota L.) plants regenerated from selected cell cultures

Abstract Aluminum-tolerant variants from cell cultures of carrot (Daucus carota L. cv. MS Yonsun) were selected by exposing them to excess ionic aluminum. The medium contained A1CI3 and 0.1 mM phosphate at pH 4.0. Under ionic aluminum stress (0.5-1.0 mM), 282 calli were selected. Twenty-eight fertile plants were regenerated from 7 selected calli. Based on the root elongation tests of the seedlings, the seeds displayed aluminum-tolerance. In the case of the cell line H7-08, the selection occurred at a frequency of ca. 6 × 10-7 when the materials were plated on agar medium containing 0.5 mM aluminum.

Enhancement of cell separation by colchicine in cell suspension cultures of soybean

SummaryWhen cell suspension of soybean was cultured in the presence of colchicine (1.0x10-4 to 1.0x10-3 M), the degree of cell separation was greatly increased. This promotion of cell separation was accompanied with swelling of cells and modification of cell form. The treated suspension consisted predominantly of single cells and a few small aggregates, and could be repeatedly subcultured in the presence of 1.0x10-4 M colchicine, but could not at 1.0x10-3 M.

Characteristics of ammonium uptake by rice cells in suspension culture

Abstract As a model system that does not involve long distance transport, cell cultures of rice (Oryza sativa L. cv Sasanishiki) were used to investigate the characteristics of NH4 + uptake. The cells, which had been subcultured for 8 d in normal R2 medium containing 5 mm NH4 + and 40 mm NO3 -, were pre-cultured for another 2.5 d without nitrogen and then used as the materials. In some experiments, the cells were further treated with 10.μm of methionine sulfoximine (MSX) for 3 h in the medium lacking nitrogen to inhibit the endogenous glutamine synthetase activity. Uptake of NH4 + was determined by measuring the consumption of NH4 + from 25 mL of an uptake medium containing 25 mm 2-morpholinoethanesulfonic acid-NaOH (pH 5.6), 100 μm CaCl2, 100 μm NH4Cl, and 0.5 g fresh weight cells. When the uptake was followed as a function of time, the cells treated with MSX rapidly absorbed NH4 + during the first 20 min and then the absorption leveled off. The cells without MSX treatment absorbed the ion at the same ra...

Studies on the greening of cultured soybean and Ruta cells: III. Effects of minorelement deficiency on the growth and photosynthetic activities of Ruta cells

Abstract (1) The green cells of Ruta graveolens took up sucrose from the medium more promptly in the light than in darkness, and the final dry weight of the cultures in the light was nearly two times of that in darkness. (2) The chlorophyll production of Ruta cells in the light increased remarkably at the later stage of the culture period when the concentration of sucrose in the medium decreased and the growth rate of the cells declined. (3) From an estimation concerned with the dry matter production and contents of minorelements in their deficiency cultures, the order in the comparative requirements of five elements for growth was infered as follows: Zinc, Iron > Manganese > Copper > Molybdenum (4) The effects of iron deficiency in the successive subculture appeared on chlorophyll production at the early stage and on the growth in terms of dry weight and photosynthetic CO2 fixation. (5) The effects of manganese deficiency were observed more easily on photosynthetic CO2 fixation and the Hill activity than...

STUDIES ON THE GREENING OF CULTURED SOYBEAN AND RUTA CELLS : II. Photosynthetic Activities of the Cultured Green Cells

Investigations were performed to determine some properties related to photosynthesis in the cultured green cells of soybean (Glycine max L. Merr) and Ruta (Ruta graveolens L.). The results obtained are summarized as follows. 1) The fixation activity of CO2 was determined by the incorpora tion of 14C from Na2 14CO3. There was no great difference in the amount of photosynthetic CO2 fixation, per chlorophyll unit, between the cultured green cells of Ruta and soybean leaves. In the cultured green cells of soybean also, the CO2 fixation activity was found to be significant, although the activity was relatively lower than that in soybean leaves. 2) The porphyrin synthesis ability of the cultured green cells was examined in a cell free system, and two porphyrins, uroporphyrin and coproporphyrin, which might have been converted from the added δ-aminolevurinic acid, were detected by thin-layer chromatography. 3) The existence of Fraction I protein and its function were confirmed with analytical ultracentr...