Susumu Arima

Hydrogen peroxide spraying alleviates drought stress in soybean plants

To ascertain the effect of exogenously applied hydrogen peroxide (H2O2) on drought stress, we examined whether the spraying of soybean leaves with H2O2 would alleviate the symptoms of drought stress. Pre-treatment by spraying leaves with H2O2 delayed foliar wilting caused by drought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content of drought-stressed leaves pre-treated with H2O2 was higher than that of leaves pre-treated with DW. Therefore, we analyzed the effect of H2O2 spraying on photosynthetic parameters and on the biosynthesis of oligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress, the net photosynthetic rate and stomatal conductance of leaves pre-treated with H2O2 were higher than those of leaves pre-treated with DW. In contrast to DW spraying, H2O2 spraying immediately caused an increase in the mRNA levels of d-myo-inositol 3-phosphate synthase 2 (GmMIPS2) and galactinol synthase (GolS), which encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In addition, the levels of myo-inositol and galactinol were higher in H2O2-treated leaves than in DW-treated leaves. These results indicated that H2O2 spraying enabled the soybean plant to avoid drought stress through the maintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide biosynthesis rather than by rapid stomatal closure.

Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling.

Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plants endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.

Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plants

We have introduced an intact maize gene for phosphoenolpyruvate carboxylase (PEPC) into C3 plants, rice (Oryza sativa cv. Kitaake). Most transgenic rice plants showed high-level expression of the maize gene. PEPC was two to three times more active in the leaves of some transgenic plants than in maize leaves. In transformants, the sensitivity of photosynthesis to O2 inhibition was reduced with the increased activity of the maize C4 PEPC. However, the alleviation of O2 inhibition was not due to an increase in the partial direct fixation of atmospheric CO2 via the enhanced maize PEPC, but rather due to the reduced stimulation of photosynthesis with a subatmospheric O2 level. Pi feeding to the leaves restored CO2 assimilation rate under the subatmospheric O2 condition, and consequently, the O2 inhibition in the transformants increased to a level comparable to that of the non-transformants. These results suggested that the O2-insensitive photosynthesis in the PEPC transformants was caused by a Pi limitation of photosynthesis. The activities of two key enzymes for sucrose synthesis, SPS and FBPase, and the sucrose and starch content were reduced in the leaves of the transformants. On the other hand, the dark respiration rate and the malate content in the leaves increased in the transformants. These results indicated that enhanced PEPC activity led to a decrease in the availability of Pi in chloroplast via a reduction of the activities of the key enzymes responsible for Pi recycling and it also caused an increased consumption of the substrate (triose-phosphate) in respiration, but not in sucrose biosynthesis.

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.

Red/far red light controls arbuscular mycorrhizal colonization via jasmonic acid and strigolactone signaling

Establishment of a nitrogen-fixing symbiosis between legumes and rhizobia not only requires sufficient photosynthate, but also the sensing of the ratio of red to far red (R/FR) light. Here, we show that R/FR light sensing also positively influences the arbuscular mycorrhizal (AM) symbiosis of a legume and a non-legume through jasmonic acid (JA) and strigolactone (SL) signaling. The level of AM colonization in high R/FR light-grown tomato and Lotus japonicus significantly increased compared with that determined for low R/FR light-grown plants. Transcripts for JA-related genes were also elevated under high R/FR conditions. The root exudates derived from high R/FR light-grown plants contained more (+)-5-deoxystrigol, an AM-fungal hyphal branching inducer, than those from low R/FR light-grown plants. In summary, high R/FR light changes not only the levels of JA and SL synthesis, but also the composition of plant root exudates released into the rhizosphere, in this way augmenting the AM symbiosis.

Effect of abscisic acid on symbiotic nitrogen fixation activity in the root nodules of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μM ABA, we obtained mutants that not only showed increased root nodule number, but also enhanced nitrogen fixation. The mutant was designated enf1 (enhanced nitrogen fixation 1) and was confirmed to be monogenic and incompletely dominant. In long-term growth experiments with M. loti, although some yield parameters were the same for both enf1 and wild-type plants, both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared than those traits in wild-type seeds and plants. The augmentation of the weight and N content of the enf1 plants most likely reflects the increased N supplied by the additional enf1 nodules and the concomitant increase in N fixation activity. We determined that the endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than that of wild-type seedlings. When wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), which results in reduced ABA content, the N fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide (NO) in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation, but also nitrogen fixation activity by decreasing NO production in nodules.

Genotypic adaptation of soybean to late sowing in southwestern Japan.

Abstract In southwestern Japan, late sowing of soybean is an option that may avoid the damage caused by excessive soil moisture during the rainy season. We investigated the adaptability to late sowing using 17 genotypes from 5 countries. The seeds were sown on 15 – 20 July, and 2 – 5 August in the normal and late sowing seasons, respectively, in an upland field in Karatsu, southwestern Japan in 2009, 2010 and 2011. Late sowing reduced the seed yield by 28.1% and 21.8% on average in the 2009 and 2011 experiments, respectively, whereas it had almost no effect in 2010, in which the temperature was high from sowing to flowering. Seed yield was not improved by increasing the growth period with a longer juvenile growth stage in the genotypes that originated in tropical areas. In the late sowing, seed yield was significantly correlated with the reproductive period from flowering to maturity and pod number, but not with the vegetative period from sowing to the end of leaf expansion or seed size. Soybean cultivars Caviness (USA), Parana and IAS-5 (Brazil), and Akisengoku and Akiyoshi (Japan) showed higher productivity in both types of sowing season, and their seed yields were less reduced by late sowing. These genotypes generally have larger pod number and seed number or longer seed filling periods, but they have medium-size seeds. Our results indicate that the seed yield in late sowing could be improved by the selection of adaptive genotypes that have larger seed number and/or longer seed filling periods.

Efficient in vitro germination and shoot proliferation of chilling-treated water chestnut (Trapa japonica Flerov) embryonal explants

SummaryEmbryonal explants from water chestnut (Trapa japonica Flerov) seeds germinated with high efficiency following a 40-d cold treatment at 5°C on half-strength MS (Murashige and Skoog) medium supplemented with 2.7 μM N6-benzyladenine (BA), 0.5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM gibberellic acid (GA3). Control and chill-treated (different durations) embryonal explants were cultured onto media which contained half-strength MS medium supplemented with different concentrations and combinations of cytokinins [BA, thidiazuron (TDZ), kinetin, zeatin], auxin (NAA) and GA3. A liquid half-strength MS medium with 1.1 μM BA and 0.5 μM NAA resulted in the best shoot proliferation of control or chill-treated explants, and the addition of 0.5 μM GA3 stimulated axillary shoot elongation. Germination and shoot proliferation were always greater for chill-treated explants compared with control explants under the same culture conditions. Shoots produced in vitro rooted 100% of the time in a liquid half-strength MS medium with 1.1 μM BA, 0.5 μM NAA and 1.1 μM indole-3-butyric acid, and the regenerated plantlets were established successfully in a water chestnut paddy field.

Varietal Difference in Nitrogen Redistribution from Leaves and Its Contribution to Seed Yield in Soybean

A large amount of nitrogen is redistributed from vegetative organs to the seeds during seed filling in soybean (Glycine max [L.] Merrill). However, the effect of nitrogen redistributed from leaves on the seed yield production is not clear. We evaluated the varietal difference in nitrogen redistribution and its contribution to the seed yield. Ten soybean cultivars were cultivated under conventional conditions in the field in Saga, Japan. The plant samples were collected at various reproductive stages, and then the nitrogen contents in each part were determined. The redistributed nitrogen was estimated by the difference in the nitrogen contents of leaves between the plants at the R5 and R7 stages. The nitrogen content of leaves began decreasing after R5 stage in all cultivars, indicating the start of nitrogen redistribution. About 13.8% to 37.9% of the total nitrogen in the seeds was estimated to have been redistributed from the leaf tissues in the ten cultivars. The seed yield was correlated positively with the amount of redistributed nitrogen from leaves but neither with the nitrogen concentration in the leaves at R5 nor with the proportion of redistributed nitrogen in the seeds. However, in high seed yielding years, 2008 and 2009, the seed yield was not associated with nitrogen redistribution; and the lowest nitrogen redistribution was associated with a relatively high seed yield in Tamahomare. Our results indicated that redistribution of a large amount of nitrogen does not always contribute to high seed yielding, implying the direct nitrogen uptake during seed filling could be more important factor for high seed yielding depending on the cultivars.