Differential expression of β‐conglycinin genes in nodulated and non‐nodulated isolines of soybean

Abstract

Nodulated (T202) and non‐nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7‐day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS‐PAGE. The β‐subunit of β‐conglycinin could scarcely be detected in the non‐nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′‐ and α‐subunits of β‐conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β‐subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β‐subunit in the non‐nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)‐subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β‐subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations. Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse‐grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO3–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO3–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N2 fixation by the non nodulated T201 plants. However, when adequate NO3 was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO3 was supplied, the subunit proportion of the seed storage protein was similar in non‐nodulating and nodulating isolines. On the other hand, when plants received a low level of NO3 (2 mM), the β‐conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO3–the proportion of β‐conglycinin increased markedly. The results indicate that non‐nodulated T201 has a normal, non‐defective, β‐subunit gene and that limited N availability decreases accumulation of β‐conglycinin, whereas high N availability increases the proportion of β‐conglycinin in soybean seeds, irrespective of whether N was derived from N2 fixation or from NO3 absorption.

Distribution and Mobilization of Sulfur during Soybean Reproduction

“…In fact, the increased seed protein levels of plants fed ammonium was due almost entirely to the P subunit, as glycinin's contribution to total seed dry weight remained unchanged. Ohtake et aL (1996) found a similar repression of P-subunit mRNA accumulation in a field-grown, non-nodulating, soybean when compared with its nodulating sister isoline.…”

Effect of short-term application of nitrogen on the accumulation of β-subunit of β-conglycinin in nitrogen-starved soybean (Glycine maxL.) developing seeds

“…1994). By supplying a sufficient level of nitrogen to non-nodulating plants, the seeds were able to accumulate the f3-subunit (Ohtake et al 1997; Krishnan et al 2000). Moreover, the accumulation of f3-conglycinin, especially the f3-subunit was positively correlated with the seed total nitrogen concentration (Ohtake at al.…”

“…non-nodulating isoline, T20 1) plants were cultivated hydroponically in a programmed incubator (TAITEC LX-3000, Tokyo, Japan) under the following conditions: 12 h of illumination (336 /-Lmol m-2 S-I) at a temperature of 28 a C, and 12 h of darkness at 18T. The chemical composition of the culture medium was the same as that reported in the previous paper (Ohtake et al 1997). The plants were grown in a culture solution containing 0.5 mM sodium nitrate, which was replaced twice per week.…”

“…Seed proteins were extracted from about 20 mg of seed powder after homogenization with 200 fLL of 0.1 M phosphatebuffered saline (pH 7.0) (Ohtake et al 1997). The homogenates were clarified by centrifugation at 15,000 x 9 for 15 min.…”

“…Fifty mg of seed powder was extracted with 80% (v / v) ethanol for sugar analysis. The concentration of trimethylsilyl-derivates of glucose, fructose, and sucrose in seeds were analyzed by gas chromatography (Hitachi Gas Chromatograph 163; OV-17 column) (Ohtake et al 1997).…”