Genetic variation among elite inbred lines suggests potential to breed for BNI-capacity in maize.

Biological nitrification inhibition (BNI) is a plant function where root systems release antibiotic compounds (BNIs) specifically aimed at suppressing nitrifiers to limit soil-nitrate formation in the root zone. Little is known about BNI-activity in maize (Zea mays L.), the most important food, feed, and energy crop. Two categories of BNIs are released from maize roots; hydrophobic and hydrophilic BNIs, that determine BNI-capacity in root systems. Zeanone is a recently discovered hydrophobic compound with BNI-activity, released from maize roots. The objectives of this study were to understand/quantify the relationship between zeanone activity and hydrophobic BNI-capacity. We assessed genetic variability among 250 CIMMYT maize lines (CMLs) characterized for hydrophobic BNI-capacity and zeanone activity, towards developing genetic markers linked to this trait in maize. CMLs with high BNI-capacity and ability to release zeanone from roots were identified. GWAS was performed using 27,085 SNPs (with unique positions on the B73v.4 reference genome, and false discovery rate = 10), and phenotypic information for BNI-capacity and zeanone production from root systems. Eighteen significant markers were identified; three associated with specific BNI-activity (SBNI), four with BNI-activity per plant (BNIPP), another ten were common between SBNI and BNIPP, and one with zeanone release. Further, 30 annotated genes were associated with the significant SNPs; most of these genes are involved in pathways of "biological process", and one (AMT5) in ammonium regulation in maize roots. Although the inbred lines in this study were not developed for BNI-traits, the identification of markers associated with BNI-capacity suggests the possibility of using these genomic tools in marker-assisted selection to improve hydrophobic BNI-capacity in maize.

Enlisting wild grass genes to combat nitrification in wheat farming: A nature-based solution.

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed "biological nitrification inhibition" (BNI). Here, we report the discovery of a chromosome region that controls BNI production in "wheat grass" Leymus racemosus (Lam.) Tzvelev, located on the short arm of the "Lr#3Nsb" (Lr#n), which can be transferred to wheat as T3BL.3NsbS (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3NsbS of L. racemosus We successfully introduced T3BL.3NsbS into the wheat cultivar "Chinese Spring" (CS-Lr#n-SA, referred to as "BNI-CS"), which resulted in the doubling of its BNI capacity. T3BL.3NsbS from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in "BNI-MUNAL" and "BNI-ROELFS." Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input-intensive farming but also can improve adaptation to low N input marginal areas.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Direct production of polyhydroxybutyrate from waste starch by newly-isolated Bacillus aryabhattai T34-N4.

Polyhydroxybutyrate (PHB) is a natural microbial polyester produced by a variety of bacteria and archaea from renewable resources. PHB resembles some petrochemical plastics but is completely biodegradable. It is desirable to identify suitable microbial strains and develop processes that can directly use starch from agricultural wastes without commercial amylase treatment. Here, PHB production using starch from agricultural waste was developed using a newly isolated strain, Bacillus aryabhattai T34-N4. This strain hydrolyzed cassava pulp and oil palm trunk starch and accumulated up to 17 wt% PHB of the cell dry weight. The α-amylase of this strain, AmyA, showed high activity in the presence of cassava pulp starch (69.72 U) and oil palm trunk starch (70.53 U). High expression of amyA was recorded in the presence of cassava pulp starch, whereas low expression was detected in the presence of glucose. These data suggest that starch saccharification by amyA allows strain T34-N4 to grow and directly produce PHB from waste starch materials such as cassava pulp and oil palm trunk starch, which may be used as low-cost substrates.

Suppression of soil nitrification by plants.

Nitrification, the biological oxidation of ammonium to nitrate, weakens the soil's ability to retain N and facilitates N-losses from production agriculture through nitrate-leaching and denitrification. This process has a profound influence on what form of mineral-N is absorbed, used by plants, and retained in the soil, or lost to the environment, which in turn affects N-cycling, N-use efficiency (NUE) and ecosystem health and services. As reactive-N is often the most limiting in natural ecosystems, plants have acquired a range of mechanisms that suppress soil-nitrifier activity to limit N-losses via N-leaching and denitrification. Plants' ability to produce and release nitrification inhibitors from roots and suppress soil-nitrifier activity is termed 'biological nitrification inhibition' (BNI). With recent developments in methodology for in-situ measurement of nitrification inhibition, it is now possible to characterize BNI function in plants. This review assesses the current status of our understanding of the production and release of biological nitrification inhibitors (BNIs) and their potential in improving NUE in agriculture. A suite of genetic, soil and environmental factors regulate BNI activity in plants. BNI-function can be genetically exploited to improve the BNI-capacity of major food- and feed-crops to develop next-generation production systems with reduced nitrification and N2O emission rates to benefit both agriculture and the environment. The feasibility of such an approach is discussed based on the progresses made.

Genetic polymorphisms in Japanese fragrant landraces and novel fragrant allele domesticated in northern Japan.

Rice fragrance is an important characteristic for Southeast Asian consumers, and fragrant landraces from Japan were first recorded in the 17th century. Principal component analysis clearly showed that Japanese fragrant landraces were genetically different from non-Japanese fragrant landraces. Japanese fragrant landraces were composed of six clades, none of which carried the most common fragrance mutation, an 8-bp deletion in exon 7 of Badh2. Fragrant landraces comprised two major groups carrying different Badh2 mutations. One group carried a known SNP at exon13 and the other a SNP at the exon1-intron1 junction as splicing donor site. The latter was considered to be a potential splicing mutant group as a novel allele at Badh2. Heterozygosity (He) scores in the two fragrant groups were not significantly different from non-fragrant landraces and modern cultivars. However, lower He scores were found around the Badh2 locus in the two groups. The potential splicing mutant group showed a more extended haplotype than the E13 SNP group. A likely causal factor responsible for loss of function is a novel splicing mutation allele that may have been generated quite recently. The fragrance allele has dispersed as a result of out-crossing under local environmental conditions.

A PCR-based marker for a locus conferring the aroma in Myanmar rice (Oryza sativa L.).

Aromatic rice is an important commodity for international trade, which has encouraged the interest of rice breeders to identify the genetic control of rice aroma. The recessive Os2AP gene, which is located on chromosome 8, has been reported to be associated with rice aroma. The 8-bp deletion in exon 7 is an aromatic allele that is present in most aromatic accessions, including the most popular aromatic rice varieties, Jasmine and Basmati. However, other mutations associated with aroma have been detected, but the other mutations are less frequent. In this study, we report an aromatic allele, a 3-bp insertion in exon 13 of Os2AP, as a major allele found in aromatic rice varieties from Myanmar. The insertion is in frame and causes an additional tyrosine (Y) in the amino acid sequence. However, the mutation does not affect the expression of the Os2AP gene. A functional marker for detecting this allele was developed and tested in an aroma-segregating F(2) population. The aroma phenotypes and genotypes showed perfect co-segregation of this population. The marker was also used for screening a collection of aromatic rice varieties collected from different geographical sites of Myanmar. Twice as many aromatic Myanmar rice varieties containing the 3-bp insertion allele were found as the varieties containing the 8-bp deletion allele, which suggested that the 3-bp insertion allele originated in regions of Myanmar.

Root metabolic response of rice (Oryza sativa L.) genotypes with contrasting tolerance to zinc deficiency and bicarbonate excess.

Plants are routinely subjected to multiple environmental stresses that constrain growth. Zinc (Zn) deficiency and high bicarbonate are two examples that co-occur in many soils used for rice production. Here, the utility of metabolomics in diagnosing the effect of each stress alone and in combination on rice root function is demonstrated, with potential stress tolerance indicators identified through the use of contrasting genotypes. Responses to the dual stress of combined Zn deficiency and bicarbonate excess included greater root solute leakage, reduced dry matter production, lower monosaccharide accumulation and increased concentrations of hydrogen peroxide, phenolics, peroxidase and N-rich metabolites in roots. Both hydrogen peroxide concentration and root solute leakage were correlated with higher levels of citrate, allantoin and stigmasterol. Zn stress resulted in lower levels of the tricarboxylic acid (TCA) cycle intermediate succinate and the aromatic amino acid tyrosine. Bicarbonate stress reduced shoot iron (Fe) concentrations, which was reflected by lower Fe-dependent ascorbate peroxidase activity. Bicarbonate stress also favoured the accumulation of the TCA cycle intermediates malate, fumarate and succinate, along with the non-polar amino acid tyrosine. Genotypic differentiation revealed constitutively higher levels of D-gluconate, 2-oxoglutarate and two unidentified compounds in the Zn-efficient line RIL46 than the Zn-inefficient cultivar IR74, suggesting a possible role for these metabolites in overcoming oxidative stress or improving metal re-distribution.

A PCR-based marker for a locus conferring aroma in vegetable soybean (Glycine max L.).

Vegetable soybean (Glycine max L.) is an important economic and nutritious crop in South and Southeast Asian countries and is increasingly grown in the Western Hemisphere. Aromatic vegetable soybean is a special group of soybean varieties that produce young pods containing a sweet aroma, which is produced mainly by the volatile compound 2-acetyl-1-pyrroline (2AP). Due to the aroma, the aromatic vegetable soybean commands higher market prices and gains wider acceptance from unfamiliar consumers. We have previously reported that the GmAMADH2 gene encodes an AMADH that regulates aroma (2AP) biosynthesis in soybeans (Arikit et al. 2010). A sequence variation involving a 2-bp deletion in exon 10 was found in this gene in all investigated aromatic varieties. In this study, a codominant PCR-based marker for the aroma trait in soybeans was designed based on the 2-bp deletion in GmAMADH2. The marker was verified in five aromatic and five non-aromatic varieties as well as in F(2) soybean population segregating for aroma. The aromatic genotype with the 2-bp deletion was completely associated with the five aromatic soybean varieties as well as the aromatic progeny of the F(2) population with seeds containing 2AP. Similarly, the non-aromatic genotype was associated with the five non-aromatic varieties and non-aromatic progeny. The perfect co-segregation of the marker genotypes and aroma phenotypes confirmed that the marker could be efficiently used for molecular breeding of soybeans for aroma.

Deficiency in the amino aldehyde dehydrogenase encoded by GmAMADH2, the homologue of rice Os2AP, enhances 2-acetyl-1-pyrroline biosynthesis in soybeans (Glycine max L.).

2-Acetyl-1-pyrroline (2AP), the volatile compound that provides the 'popcorn-like' aroma in a large variety of cereal and food products, is widely found in nature. Deficiency in amino aldehyde dehydrogenase (AMADH) was previously shown to be the likely cause of 2AP biosynthesis in rice (Oryza sativa L.). In this study, the validity of this mechanism was investigated in soybeans (Glycine max L.). An assay of AMADH activity in soybeans revealed that the aromatic soybean, which contains 2AP, also lacked AMADH enzyme activity. Two genes, GmAMADH1 and GmAMADH2, which are homologous to the rice Os2AP gene that encodes AMADH, were characterized. The transcription level of GmAMADH2 was lower in aromatic varieties than in nonaromatic varieties, whereas the expression of GmAMADH1 did not differ. A double nucleotide (TT) deletion was found in exon 10 of GmAMADH2 in all aromatic varieties. This variation caused a frame-shift mutation and a premature stop codon. Suppression of GmAMADH2 by introduction of a GmAMADH2-RNAi construct into the calli of the two nonaromatic wild-type varieties inhibited the synthesis of AMADH and induced the biosynthesis of 2AP. These results suggest that deficiency in the GmAMADH2 product, AMADH, plays a similar role in soybean as in rice, which is to promote 2AP biosynthesis. This phenomenon might be a conserved mechanism among plant species.

Simple, selective, and rapid quantification of 1-deoxynojirimycin in mulberry leaf products by high-performance anion-exchange chromatography with pulsed amperometric detection.

1-Deoxynojirimycin (DNJ) occurs in mulberry and other plants and is a highly potent glycosidase inhibitor reported to suppress blood glucose levels, thus preventing diabetes. Derivatization is required for quantification of DNJ upon use of spectral detection methods. Because of this difficulty, the DNJ contents of mulberry-based food products are rarely stated, even if DNJ is their active component. A simple, selective, and rapid method of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to quantify DNJ in mulberry-based food products was developed. Stability testing of DNJ under heat treatment was also performed. A water extract of mulberry tea sample was subjected to HPAEC-PAD in a CarboPac MA1 column with a sodium hydroxide gradient. DNJ was clearly separated at a retention time of 7.26 min without interference and was selectively detected in the water extract. The detection limit was 5 ng. Heat stability studies suggested that DNJ was heat stable. HPAEC-PAD was not subject to interference, was highly selective for DNJ, and was superior to other high-performance liquid chromatography (HPLC) techniques in terms of sample preparation, resolution, and sensitivity. The method allowed simple, selective, and rapid analysis of DNJ in food matrices and might be useful for development of mulberry-based food products. Heat treatment could be an option for sterilizing mulberry-based products.

Purification and identification of 1-deoxynojirimycin (DNJ) in okara fermented by Bacillus subtilis B2 from Chinese traditional food (Meitaoza).

This study was to purify an alpha-glucosidase inhibitor from okara (soy pulp) fermented by Bacillus subtilis B2 and to identify its chemical structure. Membrane dialysis, active charcoal, CM-Sepharose chromatography, and preparative thin-layer chromatography (TLC) were used in the purification, while positive mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectrometry were used in the identification. The MS and NMR data showed that the purified alpha-glucosidase inhibitor was 1-deoxynojirimycin (DNJ) with a molecular weight of 163 Da. This is the first time that DNJ was isolated from foods fermented with Bacillus species. Okara fermentation with B. subtilis B2 might be used to produce a food-derived DNJ product as a functional food for diabetic patients.

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity.

*Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. *A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low [Zn](ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. *There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low [Zn](ext), correlating with altered expression of root-specific auxin-responsive genes. *Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low [Zn](ext); these traits are potential breeding targets.

Detection, isolation and characterization of a root-exuded compound, methyl 3-(4-hydroxyphenyl) propionate, responsible for biological nitrification inhibition by sorghum (Sorghum bicolor).

Nitrification results in poor nitrogen (N) recovery and negative environmental impacts in most agricultural systems. Some plant species release secondary metabolites from their roots that inhibit nitrification, a phenomenon known as biological nitrification inhibition (BNI). Here, we attempt to characterize BNI in sorghum (Sorghum bicolor). In solution culture, the effect of N nutrition and plant age was studied on BNI activity from roots. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the inhibitory effect of root exudates. One major active constituent was isolated by activity-guided HPLC fractionations. The structure was analysed using NMR and mass spectrometry. Properties and the 70% inhibitory concentration (IC(70)) of this compound were determined by in vitro assay. Sorghum had significant BNI capacity, releasing 20 allylthiourea units (ATU) g(-1) root DW d(-1). Release of BNI compounds increased with growth stage and concentration of supply. NH4+ -grown plants released several-fold higher BNI compounds than NO3- -grown plants. The active constituent was identified as methyl 3-(4-hydroxyphenyl) propionate. BNI compound release from roots is a physiologically active process, stimulated by the presence of NH4+. Methyl 3-(4-hydroxyphenyl) propionate is the first compound purified from the root exudates of any species; this is an important step towards better understanding BNI in sorghum.

Nitrification Inhibitors from the root tissues of Brachiaria humidicola, a tropical grass.

Nitrification inhibitory activity was found in root tissue extracts of Brachiaria humidicola, a tropical pasture grass. Two active inhibitory compounds were isolated by activity-guided fractionation, using recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi. The compounds were identified as methyl-p-coumarate and methyl ferulate, respectively. Their nitrification inhibitory properties were confirmed in chemically synthesized preparations of each. The IC50 values of chemically synthesized preparations were 19.5 and 4.4 microM, respectively. The ethyl, propyl, and butyl esters of p-coumaric and ferulic acids inhibited nitrification, whereas the free acid forms did not show inhibitory activity.

Precursors of 2-acetyl-1-pyrroline, a potent flavor compound of an aromatic rice variety.

The biological formation of a potent flavor compound, 2-acetyl-1-pyrroline, in the aromatic rice variety (Khao Dawk Mali 105) was studied in seedlings and callus of the rice. Concentrations of 2-acetyl-1-pyrroline were determined by GC-MS-SIM using an isotope dilution method. Increases in concentration occurred when proline, ornithine, and glutamate were present in solution, with proline increasing the concentration by more than 3-fold compared to that of the control. Results of tracer experiments using (15)N-proline, (15)N-glycine, and proline-1-(13)C indicated that the nitrogen source of 2-acetyl-1-pyrroline was proline, whereas the carbon source of the acetyl group was not the carboxyl group of proline. 2-acetyl-1-pyrroline was formed in the aromatic rice at temperatures below that of thermal generation in bread baking, and formed in the aerial part of aromatic rice from proline as the nitrogen precursor.