Hideo Sasakawa

Citrate Secretion Coupled with the Modulation of Soybean Root Tip under Aluminum Stress. Up-Regulation of Transcription, Translation, and Threonine-Oriented Phosphorylation of Plasma Membrane H+-ATPase

The aluminum (Al)-induced secretion of citrate has been regarded as an important mechanism for Al resistance in soybean (Glycine max). However, the mechanism of how Al induces citrate secretion remains unclear. In this study, we investigated the regulatory role of plasma membrane H+-ATPase on the Al-induced secretion of citrate from soybean roots. Experiments performed with plants grown in full nutrient solution showed that Al-induced activity of plasma membrane H+-ATPase paralleled secretion of citrate. Vanadate and fusicoccin, an inhibitor and an activator, respectively, of plasma membrane H+-ATPase, exerted inhibitory and stimulatory effects on the Al-induced secretion of citrate. Higher activity of plasma membrane H+-ATPase coincided with more citrate secretion in Al-resistant than Al-sensitive soybean cultivars. These results suggested that the effects of Al stress on citrate secretion were mediated via modulation of the activity of plasma membrane H+-ATPase. The relationship between the Al-induced secretion of citrate and the activity of plasma membrane H+-ATPase was further demonstrated by analysis of plasma membrane H+-ATPase transgenic Arabidopsis (Arabidopsis thaliana). When plants were grown on Murashige and Skoog medium containing 30 μm Al (9.1 μm Al3+ activity), transgenic plants exuded more citrate compared with wild-type Arabidopsis. Results from real-time reverse transcription-PCR and immunodetection analysis indicated that the increase of plasma membrane H+-ATPase activity by Al is caused by transcriptional and translational regulation. Furthermore, plasma membrane H+-ATPase activity and expression were higher in an Al-resistant cultivar than in an Al-sensitive cultivar. Al activated the threonine-oriented phosphorylation of plasma membrane H+-ATPase in a dose- and time-dependent manner. Taken together, our results demonstrated that up-regulation of plasma membrane H+-ATPase activity was associated with the secretion of citrate from soybean roots.

Salt tolerance of Casuarina equisetifolia and Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia

Abstract Effects of NaCl on the seed germination and growth of Casuarina equisetifolia seedlings and multiplication of the Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia were examined. The germination rate of the seeds markedly decreased as the NaCl concentration increased and germination did not occur at 300 mM NaCl. The fresh weight of both shoots and roots of the seedlings treated with NaCl for 6 weeks apparently decreased as the NaCl concentration increased. However, root nodules were formed by inoculation with the Frankia Ceq1 strain in some seedlings treated with 300 mM NaCl and the viability of the seedlings at 500 mM NaCl was almost the same as that of the seedlings not subjected to the NaCl treatment. The Na+ concentration in the shoots sharply increased with the elevation of the NaCl concentration in the ambient solution, but the level was approximately 300 mM even in the seedlings treated with 500 mM NaCl for 6 weeks. On the other hand, the increase of the Na+ concentration in the roots by the NaCl treatment was much smaller than that in the shoots and the level was less than 150 mM. The growth of the free-living Frankia Ceq1 strain was approximately linearly suppressed as the NaCl concentration in the medium increased and the hyphae became somewhat thicker and shorter or disintegrated in the medium containing NaCl at a concentration above 150 mM. The Na+ concentration in the cells increased as the NaCl concentration in the medium increased, but the level was maintained at less than 30 mM even in the medium containing 500 mM NaCl. The cells whose growth was suppressed by the NaCI treatment grew actively again at almost the same rate as the control cells (not subjected to the NaCl treatment) when they were transferred to NaCl-free medium. These results strongly suggested that both C. equisetifolia seedlings and Frankia Ceq1 strain are highly tolerant to salt and this symbiotic system is useful for the recovery of the vegetation in areas with severe salt accumulation.

Salt tolerance of Elaeagnus macrophylla and Frankia Ema1 strain isolated from the root nodules of E. macrophylla.

Abstract Effects of NaCl on the seed germination and growth of Elaeagnus macrophylla seedlings and multiplication of Frankia Ema1 strain isolated from the root nodules of E. macrophylla were examined. The germination rate of seeds was not reduced by N aCI at 50 mM, but was reduced with further increase of the concentration, and germination did not occur at 200 mM NaCl. Root nodules were formed in the seedlings treated with 100 mM NaCl. The fresh and dry weight of the seedlings slightly decreased by the increase of the concentration of NaCl and the rate of decrease was 10–20% at NaCl concentrations higher than 100 mM. However, Na+ concentrations in the shoot increased up to 300 mM by treatment with N aCI at a concentration above 50 mM for 30 d and in some plants the lower leaves fell. The growth of free-living Frankia was markedly suppressed and the hyphae became thick and short in the solution at 100 mM NaCl. This trend was more obvious at 200 mM NaCl. Na+ concentration in the cells in the medium with 200 mM NaCl was lower than 20 mM and the cells underwent multiplication again when they were transferred to the medium which did not contain N aCl. These results indicate that the salt tolerance of E. macrophylla seedlings was not very high and the threshold for NaCl ranged between 50 and 100 mM, whereas Frankia Ema1 strain was able to survive in a much higher salt environment.

Nitrogen accumulation and changes in natural 15N abundance in the tissues of legumes with emphasis on N2 fixation by stem-nodulating plants in upland and paddy fields

Abstract Several legumes were grown during the summer season, with particular interest in the growth and N2 fixation by two stem-nodulating legumes (Sesbania rostrata and Aeschynomene indica) under upland and paddy conditions. The variations of natural 15N abundances in plant parts were investigated, and from the differences in the values in N2-fixing legumes and non-fixing plants (non-nodulating soybeans and paddy weeds), the % of fixed N in N2 -fixing plants was calculated. In upland fields, soybean, mungbean, crotalaria, and two stem-nodulating plants gained by N2 fixation as much as 46-100% N in total N or 8-43 g N.m-2 after 116-137 days of growth. In paddy fields, A. indica and S. rostrata gained by N2 fixation as much as 93 and 78% N, or 12.7 and 28.1 g N-m-2, respectively. In IRRI paddy fields (Philippines), a low natural abundance of 15N was also detected in S. rostrata as compared to rice plants, suggesting the large contribution of N2 fixation. In both stem-nodulating legumes, the stem and root ...

Isolation of endophytic Frankia from root nodules of Casuarina equisetifolia and infectivity of the isolate to host plants

Abstract Attempts were made to isolate endophytic Frankia from the root nodules formed on Casuarina equisetifolia grown in a mixture of vermiculite and field soil obtained from the Okayama University Campus. The isolation of Frankia was performed by macerating a nodule lobe directly in a test tube containing modified Qmod medium with casamino acids and sodium pyruvate as nitrogen and carbon sources. One colony, which showed hyphal and sporangial characteristics similar to those of Frankia, appeared in a test tube after 1-month incubation. The root nodules were successfully formed on the roots of the host plants by inoculation of the isolate which was multiplied using the original Qmod medium containing yeast extract, peptone and propionate. The acetylene reduction activity (ARA) obtained was 6.29 ± 1.57 µmol C2H4 h−1 (g F.W. nod.)−1. Accordingly, we identified the isolate as an effective Frankia strain for C. equisetifolia and designated it as Ceq1. Since this strain also formed root nodules on Alnus sieboldiana and Myrica rubra, it was considered to have a wider host range than that of Frankia strains which had been previously isolated from Casuarina nodules. However, the values of the nodule fresh weight and ARA in A. sieboldiana and M. rubra were much lower than those in C. equisetifolia and only half of the plants tested were nodulated in M. rubra. This strain did not form root nodules on Elaeagnus macrophylla. Frankia vesicles, which are the specialized structures where nitrogenase is located, were not observed in the free-living culture grown in Qmod medium containing abundant nitrogen and in the nodules newly formed by inoculation of the Ceq1 strain.

STEM NODULATION ON Aeschynomene indica PLANTS BY ISOLATED RHIZOBIA

Stem nodulation on Aeschynomene indica was first reported 30 years ago (l). The nodule have been observed only on the submerged lower parts of stems. Recently, this species has been found to bear nodules on the aerial parts of stems under natural conditions, which fix dinitrogen as indicated by their acetylene reducing activities (7). EAGLESHAM and SZALAY (3) reported more recently that a single inoculation of the stems of Aeschynomene plants with a rhizobial strain isolated from a submerged stem nodule on an A. indica induced the formation of green N2-fixing aerial stem nodules. However, the mechanism of this stem nodulation had remained unclear. The objectives of this study were: (a) to compare the ability of stem nodulation between rhizobial strains isolated from root and stem nodules; and (b) to examine the effects of sucrose and yeast extract on stem nodulation.

Host range of Frankia strains isolated from actinorhizal plants growing in Japan and their relatedness based on 16S rDNA

Abstract The host range of seven Frankia strains isolated from the root nodules of five actinorhizal plants in four genera growing in Japan and their relatedness based on 16S rDNA were examined. The Ceq1 strain isolated from Casuarina equisetifolia formed many nodules with high nitrogen fixation activity on the source host, C. equisetifolia. The Ceq1 strain formed nodules on Alnus sieboldiana, Alnus hirsta and Myrica rubra, but their nodulation ratio and nitrogen fixation activity were very low. The Asi1 strain isolated from A. sieboldiana, the Ahi1 strain isolated from A. hirsta, and the Mru1, Mru2 and Mru8 strains isolated from M. rubra formed many nodules on both Alnus species and M. rubra. The Ema2 strain isolated from Elaeagnus macrophylla formed nodules on both Elaeagnus species and M. rubra. Based on the aspect of nodulation and nitrogen fixation activity, Ceq1 strain is an effective strain only for Casuarina, five strains of Asi1, Ahi1, Mru1, Mru2 and Mru8 are effective strains for both Alnus and Myrica, and Ema2 strain is an effective strain for both Elaeagnus and Myrica. The results of phylogenetic analyses based on 16S rDNA sequences showed that seven strains were separated into two major clusters. Cluster 1 was further grouped into two subclusters, 1a and 1b. The Asi1, Ahi1, Mru1, Mru2 and Mru8 strains belonged to 1a and the Ceq1 strain belonged to cluster 1b. The Ema2 strain was classified into cluster 2. This phylogenetic classification of Frankia based on 16S rDNA sequences appears to be consistent with the host range determined using an inoculation test.

Different dynamics of genome content shuffling among host-specificity groups of the symbiotic actinobacterium Frankia

BackgroundFrankia is a genus of soil actinobacteria forming nitrogen-fixing root-nodule symbiotic relationships with non-leguminous woody plant species, collectively called actinorhizals, from eight dicotyledonous families. Frankia strains are classified into four host-specificity groups (HSGs), each of which exhibits a distinct host range. Genome sizes of representative strains of Alnus, Casuarina, and Elaeagnus HSGs are highly diverged and are positively correlated with the size of their host ranges.ResultsThe content and size of 12 Frankia genomes were investigated by in silico comparative genome hybridization and pulsed-field gel electrophoresis, respectively. Data were collected from four query strains of each HSG and compared with those of reference strains possessing completely sequenced genomes. The degree of difference in genome content between query and reference strains varied depending on HSG. Elaeagnus query strains were missing the greatest number (22–32%) of genes compared with the corresponding reference genome; Casuarina query strains lacked the fewest (0–4%), with Alnus query strains intermediate (14–18%). In spite of the remarkable gene loss, genome sizes of Alnus and Elaeagnus query strains were larger than would be expected based on total length of the absent genes. In contrast, Casuarina query strains had smaller genomes than expected.ConclusionsThe positive correlation between genome size and host range held true across all investigated strains, supporting the hypothesis that size and genome content differences are responsible for observed diversity in host plants and host plant biogeography among Frankia strains. In addition, our results suggest that different dynamics of shuffling of genome content have contributed to these symbiotic and biogeographic adaptations. Elaeagnus strains, and to a lesser extent Alnus strains, have gained and lost many genes to adapt to a wide range of environments and host plants. Conversely, rather than acquiring new genes, Casuarina strains have discarded genes to reduce genome size, suggesting an evolutionary orientation towards existence as specialist symbionts.

Promotion of root elongation and ion uptake in rice seedlings by 4,4,4-trifluoro-3-(indole-3-)butyric acid

Abstract We examined the mode of action of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA) on rice seminal root elongation, the most effective application method of TFIBA to plants and the effect of TFIBA on ion uptake by rice seedlings. Elongation of the seminal root of rice was promoted approximately 80% by a continuous supply of 10−5 mol L−1 TFIBA, whereas elongation was inhibited approximately 40% by 10−4 mol L−1 TFIBA. The epidermal and cortical cells at 5 mm and 30 mm from the root apex were vertically elongated by 60% and 30%, respectively, by a continuous supply of 10−5 mol L−1 TFIBA. The large vertical elongation of root cells apparently resulted in 15% thinning of the root diameter and inhibition of root hair formation. When rice seeds were soaked in 10−5 mol L−1 or 10−4 mol L−1 TFIBA for 48 h during germination and then cultured in distilled water without TFIBA, the roots were elongated significantly by 40% only in the seedlings treated with 10−4 mol L−1 TFIBA. No inhibition of root hair formation was observed in these seedlings. Uptake of K+ and NO− 3 decreased drastically in the roots whose elongation was increased by 80% by a continuous supply of 10−5 mol L−1 TFIBA. In contrast, uptake of both ions increased significantly in the roots whose elongation was increased by 40% with a supply of TFIBA only during seed germination. These results strongly suggest that treatment with TFIBA during germination promotes root elongation without inhibiting root hair formation and increases ion uptake by seedlings.

ORIGINAL ARTICLE: Host range of Frankia strains isolated from actinorhizal plants growing in Japan and their relatedness based on 16S rDNA

Abstract Corrigendum The publisher would like to draw the readers attention to an error in the following article: Nagashima Y, Tani C, Yamamoto M and Sasakawa H: Host range of Frankia strains isolated from actinorhizal plants growing in Japan and their relatedness based on 16S rDNA. Soil Sci. and Plant. Nutr., 54, 379–386. On page 383, table 3 was published incorrectly. The article is reprinted on the following pages in its entirety with the corrected table. The publisher apologizes for this error and any confusion it may have caused. Readers are advised to cite the original article (citation details above) and not this Corrigendum. The host range of seven Frankia strains isolated from the root nodules of five actinorhizal plants in four genera growing in Japan and their relatedness based on 16S rDNA were examined. The Ceq1 strain isolated from Casuarina equisetifolia formed many nodules with high nitrogen fixation activity on the source host, C. equisetifolia. The Ceq1 strain formed nodules on Alnus sieboldiana, Alnus hirsta and Myrica rubra, but their nodulation ratio and nitrogen fixation activity were very low. The Asi1 strain isolated from A. sieboldiana, the Ahi1 strain isolated from A. hirsta, and the Mru1, Mru2 and Mru8 strains isolated from M. rubra formed many nodules on both Alnus species and M. rubra. The Ema2 strain isolated from Elaeagnus macrophylla formed nodules on both Elaeagnus species and M. rubra. Based on the aspect of nodulation and nitrogen fixation activity, Ceq1 strain is an effective strain only for Casuarina, five strains of Asi1, Ahi1, Mru1, Mru2 and Mru8 are effective strains for both Alnus and Myrica, and Ema2 strain is an effective strain for both Elaeagnus and Myrica. The results of phylogenetic analyses based on 16S rDNA sequences showed that seven strains were separated into two major clusters. Cluster 1 was further grouped into two subclusters, 1a and 1b. The Asi1, Ahi1, Mru1, Mru2 and Mru8 strains belonged to 1a and the Ceq1 strain belonged to cluster 1b. The Ema2 strain was classified into cluster 2. This phylogenetic classification of Frankia based on 16S rDNA sequences appears to be consistent with the host range determined using an inoculation test.