Hiroko Yamaya

Evidence that a shoot‐derived substance is involved in regulation of the super‐nodulation trait in soybean

Abstract Results of grafting experiments between super-nodulation (or hyper-nodulation) mutants of soybean and their parents reconfirmed that super nodulation is a shoot-controlled phenomenon, suggesting that a systemic regulatory mechanism acts in soybean plants and a specific nodulation-controlling substance (SNS) is synthesized in the shoot and transported to the roots. To search for the SNS involved in the super-nodulation trait of NOD1-3, a mutant of soybean (Glycine max [L.] Merr. cv. Williams), we adopted a bioassay system using plantlets derived from the first trifoliate leaf of the seedlings; this system enabled us to introduce liquid substances continuously into leaves and to assess their effect on root nodulation. Following the application of leaf extract from Williams82 plants lacking visible root nodules, formation of root nodule meristems in NOD1-3 plantlets was repressed on the sixth day after rhizobial inoculation and the number of visible nodules on the eighth day declined to the same level as that in the Williams82 plantlets. Application of NOD1-3 leaf extract resulted in no significant change in the nodulation of both NOD1-3 and Williams82 plantlets. These results suggested that the SNS is a downregulator of nodulation and is responsible for the wild-type (Williams82) phenotype, and that the super-nodulation phenomenon is caused by a paucity of the SNS. The intensity of the repressive effect of the Williams82 leaf extract was not changed by nodulation of the source plants, thus we conclude that visible nodule formation is not required to induce production of the SNS.

Shoot-synthesized nodulation-restricting substances of wild-type soybean present in two different high-performance liquid chromatography peaks of the ethanol-soluble medium-polarity fraction

Abstract The nodule number of leguminous plants is controlled by “autoregulation”, a type of systemic regulation. Although many studies have attempted to identify key substances in this mechanism, none have been identified to date. Previously, we reported that the key substance(s) in this interaction are shoot-synthesized nodulation restricting substance(s) (SNRS), which are contained in the ethanol-soluble medium-polarity fraction of the shoot extract of wild-type soybean cv. Williams82. In the present study, extracts of wild-type soybean were separated by high-performance liquid chromatography (HPLC) and SNRS activity was detected in two different HPLC peaks (P-1, P-2) of the ethanol-soluble fraction. The P-1 and P-2 peaks were also found in the phloem sap of wild-type Williams82, but not in the sap of the hyper-nodulation mutant NOD1-3. A comparison of ultraviolet spectra implied that P-1 and P-2 do not contain salicylic acid, methyl jasmonate, abscisic acid, spermine or spermidine. Our results suggest that P-1 and P-2 contain two or more SNRS(s), although the purity of peaks P-1 and P-2 is still unclear.

Shoot-synthesized nodulation-restricting substances are present in the medium-polarity fraction of shoot extracts from wild-type soybean plants

Abstract Previously, we reported that shoot-synthesized nodulation-restricting substance(s) (SNRS) was present in extracts from shoots of a wild-type soybean, cv. Williams82. In the present paper, we report SNRS activity of fractions prepared from shoot extracts of Williams82. Activity of the ethanol-soluble medium-polarity fraction (MPF), which was prepared by solid-phase extraction of the shoot extract using an octadecyl-bonded silica (ODS) cartridge, was similar to that of the shoot extract. A high-polarity fraction of the extract was separated into subfractions, which were tested for SNRS activity. The anionic plus non-ionic fraction and the cationic plus amphoteric fraction showed no SNRS activity. Our findings indicate that SNRS is present in the MPF of an extract prepared from shoots of Williams82.

Peribacteroid solution of soybean root nodules partly induces genomic loci for differentiation into bacteroids of free-living Bradyrhizobium japonicum cells

Abstract In leguminous root nodules, rhizobia differentiate into morphology specific to symbiosis, called bacteroids. As bacteroids are surrounded with peribacteroid membranes filled with peribacteroid solution (PBS), it is considered that PBS contains substances inducing differentiation of rhizobia into bacteroids. In this study, genome-wide expression profiles of Bradyrhizobium japonicum cells cultured in PBS purified from root nodule of soybean (Glycine max L.) were compared with those of bacteroids using macroarray. PBS treatment preferentially induced regions in a large symbiosis island including various symbiosis relevant genes such as nod, fix, nol and noe, in which 75% of regions were commonly induced in bacteroids, while general repressions outside of the symbiosis island seen in bacteroids were not observed in PBS treated cells. The present results suggest that PBS contained some, but not all, substances inducing expression of the genes which are involved in differentiation into bacteroids.

Biosynthetic origin of the nitrogen atom in cyanamide in Vicia villosa subsp. varia

Abstract Natural cyanamide (NH2CN) has recently been found in three Leguminosae plants: Vicia villosa subsp. varia, Vicia cracca and Robinia pseudo-acacia. As cyanamide has long been thought to be absent in nature, its physiological role and biosynthesis are totally unknown. In the present study, we demonstrated the incorporation of 15N from [15N]nitrate and [15N]ammonium into cyanamide using shoots of V. villosa subsp. varia, which ruled out the possibility that nodules are essential in cyanamide biosynthesis. We also applied [15N2]cyanamide to shoots of V. villosa subsp. varia to monitor its turnover, and detected [15N2]cyanamide in the leaves within 4 h; it was present without detectable degradation for more than 4 days. In contrast, maximum incorporation of 15N into cyanamide molecules was observed after 4 days of feeding the shoots with 15N-labeled inorganic ions and l-[amide-15N]-glutamine, indicating that these nitrogenous compounds are distant precursors of cyanamide. Although the guanidino group of l-arginine (-NH-C(NH2)=NH) and urea (NH2C(=O)NH2) were candidate precursors of cyanamide on the basis of structural similarity, direct incorporation of the guanidino group of l-[13C6,15N4]-arginine and [13C,15N2]urea into cyanamide was not observed. These results eliminated the possibility that cyanamide is biosynthesized by the addition of ammonia to an electrophilic carbon or by the conversion of the tested compounds that were structurally relevant to cyanamide.

Carbon sources of natural cyanamide in Vicia villosa subsp. varia

The 13C labels of [13C]carbon dioxide and D-[13C6]glucose were incorporated into cyanamide (NH2CN) when they were administered to Vicia villosa subsp. varia shoots. In contrast, the administration of sodium [2,3-13C2]pyruvate did not affect the relative area of the [M + 1]+ ion of cyanamide in the gas chromatography–mass spectrometry analysis. [2,3-13C2]Pyruvate was incorporated into organic acids that are part of the citric acid cycle, such as succinate and fumarate, confirming that the shoots absorbed and metabolised it. These observations demonstrated that the carbon atom of cyanamide is derived from any of the carbohydrates that are present upstream of pyruvate in the metabolic pathway.