Barley Cultivar Sarab 1 Has a Characteristic Region on the Thylakoid Membrane That Protects Photosystem I under Iron-Deficient Conditions.

The barley cultivar Sarab 1 (SRB1) can continue photosynthesis despite its low Fe acquisition potential via roots and dramatically reduced amounts of photosystem I (PSI) reaction-center proteins under Fe-deficient conditions. We compared the characteristics of photosynthetic electron transfer (ET), thylakoid ultrastructure, and Fe and protein distribution on thylakoid membranes among barley cultivars. The Fe-deficient SRB1 had a large proportion of functional PSI proteins by avoiding P700 over-reduction. An analysis of the thylakoid ultrastructure clarified that SRB1 had a larger proportion of non-appressed thylakoid membranes than those in another Fe-tolerant cultivar, Ehimehadaka-1 (EHM1). Separating thylakoids by differential centrifugation further revealed that the Fe-deficient SRB1 had increased amounts of low/light-density thylakoids with increased Fe and light-harvesting complex II (LHCII) than did EHM1. LHCII with uncommon localization probably prevents excessive ET from PSII leading to elevated NPQ and lower PSI photodamage in SRB1 than in EHM1, as supported by increased Y(NPQ) and Y(ND) in the Fe-deficient SRB1. Unlike this strategy, EHM1 may preferentially supply Fe cofactors to PSI, thereby exploiting more surplus reaction center proteins than SRB1 under Fe-deficient conditions. In summary, SRB1 and EHM1 support PSI through different mechanisms during Fe deficiency, suggesting that barley species have multiple strategies for acclimating photosynthetic apparatus to Fe deficiency.

Effects of Nodulation on Metabolite Concentrations in Xylem Sap and in the Organs of Soybean Plants Supplied with Different N Forms.

The effects of nodulation on N metabolism in soybean plants supplied with various forms of N are not fully understood. Ureides are the principal forms of N transported from nodules, but nitrate and asparagine are the primary N compounds transported from roots supplied with NO3-. In this research, the effects of 1-day treatments of NO3-, NH4+, urea, or NO3- + NH4+ on N metabolite concentrations in xylem sap and each organ were compared between nodulated and non-nodulated soybeans. Capillary electrophoresis and colorimetry were used for the analysis. In the xylem sap of the nodulated plants with an N-free solution, ureides were the major N metabolites, followed by asparagine and glutamine. Ureides concentrations were much lower in the xylem sap of the non-nodulated soybeans. In the NO3- treatment, the concentrations of ureides in the xylem sap of the nodulated plants decreased compared to the control plants. In the NH4+, urea, and NO3- + NH4+ treatments, the concentrations of asparagine and glutamine increased significantly compared with the control and NO3- treatments. Similar changes with the N treatments were observed between the nodulated and non-nodulated soybeans, suggesting that nodulation does not have significant effects on the metabolism of absorbed N in roots.

"Live-Autoradiography" Technique Reveals Genetic Variation in the Rate of Fe Uptake by Barley Cultivars.

Iron (Fe) is an essential trace element in plants; however, the available Fe in soil solution does not always satisfy the demand of plants. Genetic diversity in the rate of Fe uptake by plants has not been broadly surveyed among plant species or genotypes, although plants have developed various Fe acquisition mechanisms. The "live-autoradiography" technique with radioactive 59Fe was adopted to directly evaluate the uptake rate of Fe by barley cultivars from a nutrient solution containing a very low concentration of Fe. The uptake rate of Fe measured by live autoradiography was consistent with the accumulation of Fe-containing proteins on the thylakoid membrane. The results revealed that the ability to acquire Fe from the low-Fe solution was not always the sole determinant of tolerance to Fe deficiency among barley genotypes. The live-autoradiography system visualizes the distribution of ß-ray-emitting nuclides and has flexibility in the shape of the field of view. This technique will strongly support phenotyping with regard to the long-distance transport of nutrient elements in the plant body.

Suppressive effect of the deep placement of lime nitrogen on N2O emissions in a soybean field.

Deep placement of slow-release nitrogen (N) fertilizers improves the growth and yield of soybean with a high N use efficiency. This study examined the effectiveness of deep placement of lime nitrogen (LN) in reducing N2O emissions in a soybean field and compared it with conventional fertilization. Before sowing soybeans, the starter N fertilizer (16 kg-N ha-1 ammonium sulfate) was mixed in the surface soil and the following four treatments were installed: the control with only the starter N (CT), conventional top-dressing of 60 kg-N ha-1 coated urea (CV), deep placement (20 cm depth) of 100 kg-N ha-1 urea (DU), and deep placement (20 cm depth) of 100 kg-N ha-1 LN (DL). The seasonal patterns of N2O emission rates measured using the closed chamber method differed among the treatments: in CT, N2O emissions were relatively low; in CV, N2O emissions derived from the top-dressed coated urea were observed from 91 days after sowing; in DU and DL, deeply-placed N was converted to N2O in the early growth stages. The cumulative N2O emissions in DL (1.8 kg-N ha-1) during the soybean cultivation period were significantly lower than those in DU (3.1 kg-N ha-1) and CV (2.8 kg-N ha-1), and slightly higher than CT (1.2 kg-N ha-1). The magnitude of N2O emissions was significantly lower in DL than DU, indicating that the choice of N fertilizer is important to reduce N2O emissions. Focusing on N2O emissions per unit coarse grain yield of soybeans, the value in DL was 0.45 g-N kg-1, which was significantly lower than 0.74 g-N kg-1 in CV. In conclusion, the deep placement of LN has the potential to be a sustainable farming method that can promote yields and reduce N2O emissions in soybean cultivation for high yield with N fertilization.

Application of Nitrate, Ammonium, or Urea Changes the Concentrations of Ureides, Urea, Amino Acids and Other Metabolites in Xylem Sap and in the Organs of Soybean Plants (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

Enhancement of Photosynthetic Iron-Use Efficiency Is an Important Trait of Hordeum vulgare for Adaptation of Photosystems to Iron Deficiency.

Leaf iron (Fe) contents in Fe-deficiency-tolerant plants are not necessarily higher than that in Fe-deficiency-susceptible ones, suggesting an unknown mechanism involved in saving and allowing the efficient use of minimal Fe. To quantitatively evaluate the difference in Fe economy for photosynthesis, we compared the ratio of CO2 assimilation rate to Fe content in newly developed leaves as a novel index of photosynthetic iron-use efficiency (PIUE) among 23 different barley (Hordeum vulgare L.) varieties. Notably, varieties originating from areas with alkaline soil increased PIUE in response to Fe-deficiency, suggesting that PIUE enhancement is a crucial and genetically inherent trait for acclimation to Fe-deficient environments. Multivariate analyses revealed that the ability to increase PIUE was correlated with photochemical quenching (qP), which is a coefficient of light energy used in photosynthesis. Nevertheless, the maximal quantum yield of photosystem II (PSII) photochemistry, non-photochemical quenching, and quantum yield of carbon assimilation showed a relatively low correlation with PIUE. This result suggests that the ability of Fe-deficiency-tolerant varieties of barley to increase PIUE is related to optimizing the electron flow downstream of PSII, including cytochrome b6f and photosystem I.

Characterization of Rhizobia for the Improvement of Soybean Cultivation at Cold Conditions in Central Europe.

In central Europe, soybean cultivation is gaining increasing importance to reduce protein imports from overseas and make cropping systems more sustainable. In the field, despite the inoculation of soybean with commercial rhizobia, its nodulation is low. In many parts of Europe, limited information is currently available on the genetic diversity of rhizobia and, thus, biological resources for selecting high nitrogen-fixing rhizobia are inadequate. These resources are urgently needed to improve soybean production in central Europe. The objective of the present study was to identify strains that have the potential to increase nitrogen fixation by and the yield of soybean in German soils. We isolated and characterized 77 soybean rhizobia from 18 different sampling sites. Based on a multilocus sequence analysis (MLSA), 71% of isolates were identified as Bradyrhizobium and 29% as Rhizobium. A comparative analysis of the nodD and nifH genes showed no significant differences, which indicated that the soybean rhizobia symbiotic genes in the present study belong to only one type. One isolate, GMF14 which was tolerant of a low temperature (4°C), exhibited higher nitrogen fixation in root nodules and a greater plant biomass than USDA 110 under cold conditions. These results strongly suggest that some indigenous rhizobia enhance biological nitrogen fixation and soybean yield due to their adaption to local conditions.

Recent Advances in Carbon and Nitrogen Metabolism in C3 Plants.

C and N are the most important essential elements constituting organic compounds in plants. The shoots and roots depend on each other by exchanging C and N through the xylem and phloem transport systems. Complex mechanisms regulate C and N metabolism to optimize plant growth, agricultural crop production, and maintenance of the agroecosystem. In this paper, we cover the recent advances in understanding C and N metabolism, regulation, and transport in plants, as well as their underlying molecular mechanisms. Special emphasis is given to the mechanisms of starch metabolism in plastids and the changes in responses to environmental stress that were previously overlooked, since these changes provide an essential store of C that fuels plant metabolism and growth. We present general insights into the system biology approaches that have expanded our understanding of core biological questions related to C and N metabolism. Finally, this review synthesizes recent advances in our understanding of the trade-off concept that links C and N status to the plant's response to microorganisms.

Effects of Different Chemical Forms of Nitrogen on the Quick and Reversible Inhibition of Soybean Nodule Growth and Nitrogen Fixation Activity.

It has been reported that supply of nitrate to culture solution rapidly and reversibly inhibits nodule growth and nitrogen fixation activity of soybean. In this study, the effects of ammonium, urea, or glutamine on nodule growth and nitrogen fixation activity are compared with that for nitrate. Soybean plants were cultivated with a nitrogen-free nutrient solution, then 1 mM-N of nitrate, ammonium, glutamine, or urea were supplied from 12 DAP until 17 DAP. Repression of nodule growth and nitrogen fixation activity at 17 DAP were observed by ammonium, urea, and glutamine like nitrate, although the inhibitory effects were milder than nitrate. The removal of nitrogen from the culture solutions after nitrogen treatments resulted in a recovery of the nodule growth. It was found that the glutamine treatment followed by N-free cultivation gave highest nitrogen fixation activity about two times of the control. Tracer experiments with 15N and 13C were performed to evaluate the translocation of N and C to the different tissues. Culture solutions containing a 15N-labeled nitrogen source were supplied from 21 DAP, and the whole shoots were exposed to 13CO2 for 60 min on 23 DAP, and plants were harvested on 24 DAP. The percentage distribution of 15N in nodules was highest for ammonium (1.4%) followed by glutamine (0.78%), urea (0.32%) and nitrate (0.25%). The percentage distribution of 13C in the nodules was highest for the control (11.5%) followed by urea (5.8%), glutamine (2.6%), ammonium (2.3%), and nitrate (2.3%). The inhibitory effects of nitrogen compounds appeared to be related to a decrease in photoassimilate partitioning in the nodules, rather than 15N transport into the nodules. The free amino acid concentrations after nitrogen treatments were increased in the nodules and leaves by nitrate, in the roots by ammonium, in the stems by urea, and the roots, stems, and leaves by glutamine treatment. The concentrations of asparagine, aspartate, and glutamine were increased after nitrogen treatments. By the long-term supply of nitrogen for 2-weeks, nitrate significantly increased the lateral roots and leaf growth. The long-term supply of urea and glutamine also promoted the lateral roots and leaf growth, but ammonium suppressed them.

Transcriptome and Metabolome Analyses Reveal That Nitrate Strongly Promotes Nitrogen and Carbon Metabolism in Soybean Roots, but Tends to Repress It in Nodules.

Leguminous plants form root nodules with rhizobia that fix atmospheric dinitrogen (N2) for the nitrogen (N) nutrient. Combined nitrogen sources, particular nitrate, severely repress nodule growth and nitrogen fixation activity in soybeans (Glycine max [L.] Merr.). A microarray-based transcriptome analysis and the metabolome analysis were carried out for the roots and nodules of hydroponically grown soybean plants treated with 5 mM of nitrate for 24 h and compared with control without nitrate. Gene expression ratios of nitrate vs. the control were highly enhanced for those probesets related to nitrate transport and assimilation and carbon metabolism in the roots, but much less so in the nodules, except for the nitrate transport and asparagine synthetase. From the metabolome analysis, the concentration ratios of metabolites for the nitrate treatment vs. the control indicated that most of the amino acids, phosphorous-compounds and organic acids in roots were increased about twofold in the roots, whereas in the nodules most of the concentrations of the amino acids, P-compounds and organic acids were decreased while asparagine increased exceptionally. These results may support the hypothesis that nitrate primarily promotes nitrogen and carbon metabolism in the roots, but mainly represses this metabolism in the nodules.

Effect of nitrate on nodule and root growth of soybean (Glycine max (L.) Merr.).

The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

Enhancement of the nitrogen fixation efficiency of genetically-engineered Rhizobium with high catalase activity.

The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H2O2). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H2O2 in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H2O2 content concomitantly with the enhancement of leghemoglobins contents.

Rapid quantification of cyanamide by ultra-high-pressure liquid chromatography in fertilizer, soil or plant samples.

A rapid and simple method for determination of cyanamide in fertilizer, soil and plants has been developed. In this method, cyanamide is extracted with 2% acetic acid and the extract separated by centrifugation. It is then purified by passing through a membrane filter. The extract was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and the derivatized compound separated by ultra-high-pressure liquid chromatography. It is then detected with a UV detector at 260 nm by the same method as is used for amino acid analysis. The proposed method is fast, simple and cheap and also has good selectivity and sensitivity for the determination of cyanamide in a wide range of biotic and abiotic materials.

Properties and substrate specificities of proteolytic enzymes from the edible basidiomycete Grifola frondosa.

Highly active proteolytic enzymes are found in the fruiting bodies of Grifola frondosa. The general properties and substrate specificities of these proteases from G. frondosa (ProGF) were studied. The optimal pH for ProGF activity was pH 3 or 7 using hemoglobin or Hammersten casein as a substrate, respectively. The ProGF exhibited over 70% of maximal activity within the pH range of 4.5-8.5. In terms of temperature, the ProGF were maximally active at 55 degrees C, while over 80% of maximal activity was observed within the range of 50-75 degrees C. These proteases were substrate-specific, mainly cleaving at Ala(14)-Leu(15), Tyr(16)-Leu(17), and Pro(28)-Lys(29) bonds, with occasional cleavage of Phe(24)-Phe(25) bonds in the oxidized insulin B-chain. The ProGF also liberated hydrophobic amino acids, such as valine, leucine, and phenylalanine, using the oxidized insulin B-chain as a substrate. When soy protein was used as a substrate, valine, leucine, phenylalanine, and tyrosine were selectively released from the hydrolysate. Thus, over the time course of incubation, the peptide concentration increased as the average peptide chain length decreased. These results indicate that the ProGF include both endopeptidases recognizing leucine, phenylalanine, and lysine at the P1' position, and aminopeptidases preferentially releasing hydrophobic and aromatic amino acids such as valine, leucine, phenylalanine, and tyrosine.

Nitrogen Fixation and Translocation in Young Sugarcane (Saccharum officinarum L.) Plants Associated with Endophytic Nitrogen-Fixing Bacteria.

The tracer (15)N(2) was used to investigate sites of N(2) fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to (15)N(2)-labeled air in a 500 mL plastic cylinder. Plants fed (15)N(2) for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from (15)N(2) was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanol-insoluble fractions in each plant part, and the (15)N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.

The autoregulation of nodulation mechanism is related to leaf development.

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.

Large-scale analysis of gene expression profiles during early stages of root nodule formation in a model legume, Lotus japonicus.

Gene expression profiles during early stages of formation of symbiotic nitrogen-fixing nodules in a model legume Lotus japonicus were analyzed by means of a cDNA array of 18,144 non-redundant expressed sequence tags (ESTs) isolated from L. japonicus. Expression of a total of 1,076 genes was significantly accelerated during the successive stages that represent infection of Mesorhizobium loti, nodule primordium initiation, nodule organogenesis, and the onset of nitrogen fixation. These include 32 nodulin and nodulinhomolog genes as well as a number of genes involved in the catabolism of photosynthates and assimilation of fixed nitrogen that were previously known to be abundantly expressed in root nodules of many legumes. We also identified a large number of novel nodule-specific or enhanced genes, which include genes involved in many cellular processes such as membrane transport, defense responses, phytohormone synthesis and responses, signal transduction, cell wall synthesis, and transcriptional regulation. Notably, our data indicate that the gene expression profile in early steps of Rhizobium-legume interactions is considerably different from that in subsequent stages of nodule development. A number of genes involved in the defense responses to pathogens and other stresses were induced abundantly in the infection process, but their expression was suppressed during subsequent nodule formation. The results provide a comprehensive data source for investigation of molecular mechanisms underlying nodulation and symbiotic nitrogen fixation.

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules.

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.