Keiki Okazaki

Rapid characterization of plant mutants with an altered ion‐profile: a case study using Lotus japonicus

Legumes are second only to cereals in their importance to humans, and study of their functional genomics of nutrition and other trace elements is crucial for agricultural production and food fortification. We describe here an ionomic screening experiment carried out to investigate the accumulation of 15 elements in shoots of mutants of Lotus japonicus, a good genetic tool for legume study.Approximately 2000 mutagenized M2 plants were cultivated in a novel low-cost high-throughput system and their elemental profiles were determined by inductively coupled plasma mass spectroscopy (ICP-MS).After triple-checking the element concentrations in M2 or M3 plant shoots, 31 mutants with altered elemental profiles were identified. Surprisingly, the number of genes regulating essential elements was similar to the number regulating nonessential elements. Magnesium (Mg) and nickel (Ni) were correlated in a number of mutants.Further investigation suggested that phosphorus (P) and cobalt (Co) might be involved in the ion homeostasis network of Mg and Ni.The results suggested that the pathways for element uptake or translocation were highly linked through the ion transport-related genes. Ionomics proved to be a powerful functional genomics tool for determining genes related to ion homeostasisin this study.

Gas chromatography–mass spectrometry associated global analysis of rice root exudates under aseptical conditions

Abstract Components of rice root exudates were surveyed by gas chromatography–mass spectrometry (GC-MS) and the effect of phosphate (P) starvation was investigated. Rice seedlings were aseptically grown in controlled environments under P-supplied (+P) or P-deficient (–P) conditions. Root exudates were collected from the culture solution 4 and 8 days after treatment (D4 and D8, respectively), which contain the first and second 4-day intervals. The collected solution was lyophilized and then eluted with methanol. In the present study, primary metabolites, such as sugars, amino acids, organic acids and fatty acids, were mainly targeted. After derivatization with methoxyamine hydrochloride and N-methyl-N-trifluoroacetamide, the samples were analyzed by GC-MS. Sugars and various amino acids (such as aspartic acid and l-isoleucine) were mainly detected and the components changed with the growth stage rather than the P content in the culture solution. The relative amount of sugars (such as glucose and fructose) and amino acids (such as aspartic acid and l-isoleucine) decreased in the D8 samples. Of the 78 detected compounds, orthogonal partial least-squares discrimination analysis (O-PLS-DA) showed that four compounds were related to the P content of the nutrient solution and 58 were related to the sampling date (days after treatment). The results suggest that the physiological change during seedling development was unexpectedly large and that the response to the environment was rather small.

Maintenance of soybean yield with reduced phosphorus application by previous cropping with mycorrhizal plants

Abstract Previous cultivation of arbuscular mycorrhizal (AM)-host plants promotes AM colonization, phosphorus (P) uptake and the growth of succeeding AM-host plants. Three field experiments were conducted to investigate whether reduced application rates of P fertilizer could maintain soybean (Glycine max (L.) Merr. cv. Tsurumusume) yields when fields were previously cropped with AM-host plants. In the experiments, soybean was cropped after growing both AM-host (wheat, sunflower, vetch, maize and azuki bean) and non-AM-host plants (radish, white mustard, sugar beet and buckwheat) or fallow with different rates of P fertilizer (0–200 kg P2O5 ha−1). The results showed that previous cropping with AM-host plants increased AM colonization of soybean roots, soybean growth, shoot P content and yield compared with either previous cropping with a non-AM host or fallow treatment. Soybean yields following AM-host plants did not decrease with reductions in the P application rate from 150 to 50 kg P2O5 ha−1, although soybean yields decreased in some cases after non-AM-host plants or fallow treatment. A general linear model analysis revealed that soybean yield following AM-host plants was less affected by a reduction in P application rate than plants following non-AM-host plants. As a result, P application rates can be reduced from 150 kg P2O5 ha−1 (the rate recommended by the Hokkaido Government) to 50 kg P2O5 ha−1 for soybean cultivation in soils after AM-host plants.

Aroma Characteristic and Volatile Profiling of Carrot Varieties and Quantitative Role of Terpenoid Compounds for Carrot Sensory Attributes

The aroma characteristics and volatile profiles of 14 carrot varieties were investigated by sensory evaluations and gas chromatography-mass spectrometry volatile analyses. The sensory map obtained by principal components analysis showed that the sensory attributes comprised 3 categories: sour/green, overall carrot/harsh/ink-like, and fruity/fresh/sweet. The Kuroda type is characterized by lower intensities of overall carrot/harsh/ink-like and fruity/fresh/sweet notes. Furthermore, volatile profiling indicated that this type did not have significantly higher amounts of volatiles. Partial least squares regression analysis determined the quantitative contributions to ink-like, harsh, and fruity carrot aromas; monoterpenes had significant positive correlations with these attributes, while bisabolene isomers had negative correlations. The aroma attribute intensity and contents of volatiles and nutritional compounds are relatively low in the Kuroda type than in other carrot types. This type may be useful for reducing carrot harshness during the development of new carrots with good eating qualities.

Varietal difference in radiocesium uptake and transfer from radiocesium deposited soils in the genusAmaranthus

Abstract Within Amaranthaceae, 33 different varieties, including local varieties from Japan, were grown in 2012 in a field in the town of Iino in the Fukushima prefecture, which is located approximately 51 km north of Tokyo Electric Power Company, Fukushima Daiichi Nuclear Power Plant (FDNPP). The contamination level of the soil was 2770 ± 140 Bq kg−1 dry weight (134Cesium (Cs) + 137Cs, average ± SE), and the field was also cultivated in 2011. There was a significant varietal difference in the dry weight production, radiocesium accumulation and transfer factor (TF) of radiocesium from the soil to the plant. The ratio of the lowest TF to the highest TF was approximately 3. Because the ratio of 137Cs to 133Cs was significantly positive, radiocesium seems to be absorbed in a manner similar to that of 133Cs. It is suggested that the varietal difference in the behavior of radiocesium uptake mainly depends on its genetic background rather than on environmental factors.

Metabolite profiling of spinach (Spinacia oleracea L.) leaves by altering the ratio of NH+ 4/NO− 3 in the culture solution

Abstract To observe the effect of different forms of N, we have applied metabolic profiling using gas chromatography mass spectrometry to evaluate the metabolite composition of spinach. The aim of the present study was to find an appropriate indicator of overall metabolic response to N source. The effect of the NH+ 4/NO− 3 ratio on spinach tissue was investigated, comparing two cultivars that differed in their ability to use N. There was wide variation in NH+ 4/NO− 3 absorption without any distinct growth inhibition in either cultivar. Statistical analysis revealed that the metabolites could be broadly divided into two types, correlating either positively or negatively with NH+ 4 uptake in the culture solution. Principal component analysis (PCA) was an effective tool that extracted the indexes of the effect of the ratio of N forms as PCA scores. Close relationships between metabolite profiles and NH+ 4 uptake were observed, indicating that metabolic profiling is able to distinguish the invisible metabolic change in mature leaves of spinach and may provide a new tool for the investigation of quality features or rhizosphere nutritional condition.

Metabolite profiling of Komatsuna (Brassica rapa L.) field-grown under different soil organic amendment and fertilization regimes

Among agricultural soil fertility management options, the environmental benefits of organic amendments have recently drawn particular attention. However, little information exists about their effects on crop metabolites or quality. Field plots of Komatsuna (Brassica rapa L. var. perviridis) were planted in a fractional factorial design with the following treatments: soil amendments of cattle manure (0, 2 or 4 kg m−2), wheat straw (0, 0.05 or 0.1 kg m−2), fast release nitrogen (N) (0, 6 or 12 g N m−2 of ammonium sulfate), slow release N (0, 3 or 6 g N m−2 of coated ammonium nitrate), phosphorus (P) [0, 5 or 10 g phosphorus pentoxide (P2O5) m−2 of lime superphosphate] and potassium (K) [0, 6 and 12 g potassium oxide (K2O) m−2 as potassium sulfate]. Metabolite profiling was carried out using a gas chromatograph/mass spectrometer (GC/MS), which yielded 62 and 67 metabolites in the leaves and the petioles, respectively. Metabolite peak areas were subjected to principal component analysis (PCA). The first component accounted for 44.1% of the total variance and bore a close relationship to N. The third component accounted for 8.8% of the total variance and was used to distinguish between different levels of manure application. An analysis of variance (ANOVA) of treatment factor effects on individual metabolites showed that the three most significant factors, from highest to lowest, were N absorption, manure amendments and slow release N. The effects of the manure amendments were not fully explained by its attendant N, P or K inputs. This result raises the question as to what mechanisms may bring about the metabolic changes caused by the manure amendment. The current findings will serve to direct further studies on the relationship between crop quality and cultivation procedures and will lead to more efficient quality control methods.

Metabolite profiling of Raphanus sativus L. to evaluate the effects of manure amendment

Abstract Organic soil amendments attract considerable attention, given their potential to promote nutrient recycling and contribute to reducing CO2 emissions. To evaluate their effects on quality characteristics of agricultural products, we comprehensively analyzed the metabolite compositions of radish (Raphanus sativus L.) leaves and roots using gas chromatography mass spectrometry (GC/MS). A field experiment arranged in a split-plot design investigated the effect of three levels of composted dairy manure amendments (0, 2 and 4 kg m−2; main plot) factorially combined with three levels of inorganic nitrogen (N) fertilization (0, 6 and 12 g N m−2 as ammonium sulfate; subplots) on radish metabolites. For both leaf and root metabolites, a principal component analysis was applied to all 124 metabolite peaks revealed by the GC/MS analysis. The first principal component accounted for 46.3% of the total variance and indicated a close relationship between metabolite profiles and inorganic-N (Ninorg) application rates, whereas the second principal component, accounting for 14.6% of the total variance, pointed to a close relationship between the metabolite profiles and the manure application rates. Leaf metabolites were more sensitive to inorganic-N and manure rates than root metabolites. Amino acids in roots and leaves showed a close relationship with Ninorg application rates, whereas the response to the level of manure was not clear. Organic acids in leaves were closely related to both Ninorg and manure levels. With increased amounts of applied manure, the concentrations of malic acid, myo-inositol-phosphate and sucrose decreased, whereas the concentrations of shikimic acid, arabinose and L-methionine increased. The observation that radish metabolites clearly differed under different application rates of both Ninorg and manure will prove useful in improving and distinguishing the quality of agricultural products grown using organic fertilizers.

Sensory and metabolic profiles of “Fuji” apples (Malus domestica Borkh.) grown without synthetic agrochemicals: the role of ethylene production

Flavors of “Fuji” apple cultivated with or without synthetic agrochemicals were compared using quantitative descriptive analyses (QDA) and metabolite profiling for 3 seasons. Experimental plots included conventional crops (with agrochemicals) and organic crops (without agrochemicals) at our institute and organic and conventional farms. Additionally, mass market samples were analyzed. Organic apples were weak in sweetness and floral characteristics and had enhanced green and sour flavors. Most esters and sugars were present in lower concentrations in organic than in conventional apples. Close relation of principal component 1 of QDA and metabolite profiles, to ethylene production suggested that ethylene is considerably involved in flavor synthesis. Reduced ethylene associated with immaturity accounted for insufficient flavor synthesis and weak aroma and flavor attributes of organic apples. Furthermore, organic apples from the farm were more flavorsome than those from the institute in 2012, suggesting possible recovery of ethylene production after a long organic cultivation period. Organic “Fuji” apples, which were weak in sweet and floral characteristics and had lower levels of esters, were accounted for by smaller ethylene.

Metabolite Profiling of Mizuna (Brassica rapa L. var. Nipponsinica) To Evaluate the Effects of Organic Matter Amendments

Organic matter amendment is an essential agricultural protocol to improve soil function and carbon sequestration. However, the effect of organic matter amendments on crop quality has not been well-defined. This study applied gas chromatography-mass spectrometry to investigate the metabolite profiling of mizuna ( Brassica rapa L. var. Nipponsinica) with different organic matter amendments with respect to quality and quantity. Principal component analysis showed that 33.4, 15.6, and 6.6% of the total variance was attributable to the plant N concentration, fast-release organic fertilizer (fish cake), chicken droppings), and rapeseed cake), and manure application (fresh and dried), respectively. The peak areas of 18 and 15 compounds were significantly altered under organic fertilizer and manure amendment, respectively, compared with pure chemical fertilizer amendment. The compounds altered with manure amendment were similar to those reported in previous studies using other species. This study is the first to show clear metabolic alterations in plants through the amendment of fast-release organic fertilizer. Mizuna is a unique plant species that responds to both organic fertilizer and manure. These observations are useful to clarify the effect of organic matter amendment and quality control in farming systems using organic matter.