Metabolomics Reveals the Effects of Nitrogen/Phosphorus/Potassium (NPK) Fertilizer Levels on Cucumber Fruit Raised in Different Nutrient Soils.

Fertilizers are widely used to improve the quality of fruits and vegetables. However, the overuse of fertilizers has become an issue because it causes environmental problems and negatively affects productivity and fruit quality. In this study, we examined the effects of nitrogen, phosphorus, and potassium (NPK) fertilizer levels on the metabolism of cucumber fruit in low- and high-nutrient soils using mass-spectrometry-based metabolomics approaches. Cucumber metabolite content was notably different depending on the initial soil nutrient status. Most amino acids and phenylpropanoids were abundant in the cucumbers raised in low-nutrient soil, whereas organic acids, some amino acids (aspartate, glutamate, and ornithine), and carbohydrates were comparatively higher in fruits from high-nutrient soil. The fertilizer supply resulted in an alteration in the metabolite profile, while no change in fruit yield was observed in either low- or high-nutrient soils. Fertilizer treatment perturbed the metabolite contents in cucumbers from low-nutrient soil. In contrast, treatment with higher concentrations of fertilizer in high-nutrient soil increased phenylpropanoid content in the cucumbers, while most metabolites decreased. In conclusion, fertilization levels should be carefully determined, considering culture conditions such as the original soil status, to increase product yield and fruit quality and avoid environmental problems.

Effects of limited water supply on metabolite composition in tomato fruits (Solanum lycopersicum L.) in two soils with different nutrient conditions.

Effect of water supply to metabolites in tomato fruit was compared in two soils with different nutrient conditions, i.e., either limited or excess. Two types of soil nutrient condition, type A: nutrient-limited and type B: nutrient-excess, were prepared as follows; type A is a low nutrient-containing soil without a replenishment of starved nitrogen and phosphorous, type B is a high nutrient-containing soil exceeding the recommended fertilization. Soil water was adjusted either at -30 kPa (sufficient) or -80 kPa (limited). For harvested tomato fruits, we examined primary and secondary metabolites using non-targeted mass spectrometry based metabolomics. The fruit production and leaf SPAD were greatly dependent on soil nutrient levels, by contrast, the level of lycopene remained unchanged by different levels of water and nutrient supply. The perturbation of metabolites by water supply was clear in the nutrient-excess soil. In particular, limited water supply strongly decreased primary metabolites including sugars and amino acids. We demonstrated that water stress differently shifted primary metabolites of tomato fruits in two soils with different nutrient conditions via non-targeted mass spectrometry-based metabolomics. In conclusion, we suggest that the limited water supply in soils with surplus nutrient is not a recommendable way for tomato 'cv. Super Dotaerang' production if fruit nutritional quality such as sugars and amino acids is in the consideration, although there was no disadvantage in fruit yield.

Combined Effects of Nutrients × Water × Light on Metabolite Composition in Tomato Fruits (Solanum Lycopersicum L.).

Tomato cultivation in the greenhouse can be facilitated by supplemental light. We compared the combined effects of nutrients, water, and supplemental light (red) on tomato fruit quality. To do this, three different nutrient conditions were tested, i.e., (1) low N, (2) standard N, and (3) high N. Water was supplied either at -30 kPa (sufficient) or -80 kPa (limited) of soil water potential. Supplemental red LED light was turned either on or off. The metabolites from tomato fruits were profiled using non-targeted mass spectrometry (MS)-based metabolomic approaches. The lycopene content was highest in the condition of high N and limited water in the absence of supplemental light. In the absence of red lighting, the lycopene contents were greatly affected by nutrient and water conditions. Under the red lighting, the nutrient and water conditions did not play an important role in enhancing lycopene content. Lower N resulted in low amino acids. Low N was also likely to enhance some soluble carbohydrates. Interestingly, the combination of low N and red light led to a significant increase in sucrose, maltose, and flavonoids. In high N soil, red light increased a majority of amino acids, including aspartic acid and GABA, and sugars. However, it decreased most of the secondary metabolites such as phenylpropanoids, polyamines, and alkaloids. The water supply effect was minor. We demonstrated that different nutrient conditions of soil resulted in a difference in metabolic composition in tomato fruits and the effect of red light was variable depending on nutrient conditions.

Characterization of Metabolic Changes under Low Mineral Supply (N, K, or Mg) and Supplemental LED Lighting (Red, Blue, or Red-Blue Combination) in Perilla frutescens Using a Metabolomics Approach.

In order to achieve premium quality with crop production, techniques involving the adjustment of nutrient supply and/or supplemental lighting with specific light quality have been applied. To examine the effects of low mineral supply and supplemental lighting, we performed non-targeted metabolite profiling of leaves and stems of the medicinal herb Perilla frutescens, grown under a lower (0.75×) and lowest (0.1×) supply of different minerals (N, K, or Mg) and under supplemental light-emitting diode (LED) lighting (red, blue, or red-blue combination). The lowest N supply increased flavonoids, and the lowest K or Mg slightly increased rosmarinic acid and some flavonoids in the leaves and stems. Supplemental LED lighting conditions (red, blue, or red-blue combination) significantly increased the contents of chlorophyll, most cinnamic acid derivatives, and rosmarinic acid in the leaves. LED lighting with either blue or the red-blue combination increased antioxidant activity compared with the control group without LED supplementation. The present study demonstrates that the cultivation of P. frutescens under low mineral supply and supplemental LED lighting conditions affected metabolic compositions, and we carefully suggest that an adjustment of minerals and light sources could be applied to enhance the levels of targeted metabolites in perilla.

Effects of Nutrient and Water Supply During Fruit Development on Metabolite Composition in Tomato Fruits (Solanum lycopersicum L.) Grown in Magnesium Excess Soils.

Tomato cultivation in the greenhouse or field may experience high surplus salts, including magnesium (Mg2+), which may result in differences in the growth and metabolite composition of fruits. This study hypothesized that decreasing the supply of nutrients and/or water would enhance tomato fruit quality in soils with excess Mg2+ that are frequently encountered in the field and aimed to find better supply conditions. For tomato plants cultivated in plastic pots using a plastic film house soil, the fertilizer supply varied in either the nitrogen (N) or potassium (K) concentration, which were either 0.1 (lowest) or 0.75 times (lower) than the standard fertilizer concentrations. Water was supplied either at 30 (sufficient) or 80 kPa (limited) of the soil water potential. Lycopene content on a dry-weight basis (mg/kg) was enhanced by the combination of lowest N supply and sufficient water supply. However, this enhancement was not occurred by the combination of the lowest N supply and limited water supply. Sugars and organic acids were decreased by limiting the water supply. Therefore, we carefully suggest that an adjustment of nitrogen with sufficient watering could be one of strategies to enhance fruit quality in excess Mg2+ soils.

Metabolomic response of Perilla frutescens leaves, an edible-medicinal herb, to acclimatize magnesium oversupply.

High salt accumulation, resulting from the rampant use of chemical fertilizers in greenhouse cultivation, has deleterious effects on plant growth and crop yield. Herein, we delineated the effects of magnesium (Mg) oversupply on Perilla frutescens leaves, a traditional edible and medicinal herb used in East-Asian countries. Mg oversupply resulted in significantly higher chlorophyll content coupled with lower antioxidant activities and growth, suggesting a direct effect on subtle metabolomes. The relative abundance of bioactive phytochemicals, such as triterpenoids, flavonoids, and cinnamic acids, was lower in the Mg-oversupplied plants than in the control. Correlation analysis between plant phenotypes (plant height, total fresh weight of the shoot, leaf chlorophyll content, and leaf antioxidant content) and the altered metabolomes in P. frutescens leaves suggested an acclimatization mechanism to Mg oversupply. In conclusion, P. frutescens preferentially accumulated compatible solutes, i.e., carbohydrates and amino acids, to cope with higher environmental Mg levels, instead of employing secondary and antioxidative metabolism.

Comparative Metabolomics Unravel the Effect of Magnesium Oversupply on Tomato Fruit Quality and Associated Plant Metabolism.

In general, greenhouse cultivation involves the rampant application of chemical fertilizers, with the aim of achieving high yields. Oversaturation with mineral nutrients that aid plant growth, development, and yield may lead to abiotic stress conditions. We explore the effects of excess magnesium on tomato plant metabolism, as well as tomato fruit quality using non-targeted mass spectrometry (MS)-based metabolomic approaches. Tomato plants were subjected to three different experiments, including high magnesium stress (MgH), extremely high magnesium stress (MgEH), and a control with optimal nutrient levels. Leaves, roots, and fruits were harvested at 16 weeks following the treatment. A metabolic pathway analysis showed that the metabolism induced by Mg oversupply was remarkably different between the leaf and root. Tomato plants allocated more resources to roots by upregulating carbohydrate and polyamine metabolism, while these pathways were downregulated in leaves. Mg oversupply affects the fruit metabolome in plants. In particular, the relative abundance of threonic acid, xylose, fucose, glucose, fumaric acid, malic acid, citric acid, oxoglutaric acid, threonine, glutamic acid, phenylalanine, and asparagine responsible for the flavor of tomato fruits was significantly decreased in the presence of Mg oversupply. Altogether, we concluded that Mg oversupply leads to drastically higher metabolite transport from sources (fully expanded leaves) to sinks (young leaves and roots), and thus, produces unfavorable outcomes in fruit quality and development.

The Relationship between Turgor Pressure Change and Cell Hydraulics of Midrib Parenchyma Cells in the Leaves of Zea mays.

Leaf dehydration decreases water potential and cell turgor pressure. Therefore, changes in cell turgor pressure may regulate water transport across plant cell membranes. Using a cell pressure probe, the hydraulic properties of parenchyma cells in the midrib of maize (Zea mays L.) leaves were measured (half time of water exchange in cells as a measure of hydraulic conductivity Lp). Using intact plants with root systems encased in a pressure chamber, the root systems were pressurized and the turgor pressure in leaf cells increased by increments up to 0.3 MPa. However, the increase in the cell turgor did not increase but stabilized values. Increased water potential in leaf cells seemed to have stabilizing effects on the probably due to enhanced water availability. When the cell turgor decreased by 0.1 MPa to 0.3 MPa with releasing the pressure in the pressure chamber, was temporarily increased to a large degree,a factor of up to 13 within 30 min.

Diurnal changes in CN metabolism and response of rice seedlings to UV-B radiation.

Plants regulate a number of primary metabolites, including carbohydrates, organic acids, and amino acids, in response to UV-B radiation. Therefore, it is essential to understand the time-dependent response of rice plants to UV-B stress. This study focused on the response of plants to UV-B at different leaf developmental phases (emerging, growing, and maturing) in an attempt to fully comprehend the metabolic shift. We analyzed the expression levels of genes related to starch/sucrose metabolism in the leaf blades of rice seedlings (Oryza sativa L. "Saechuchenog") exposed to UV-B irradiation for short (1 day) and long terms (5 days) using quantitative real-time polymerase chain reaction. We also examined the diurnal variations in the contents of primary metabolites using an established GCTOF-MS (gas chromatography time of flight-mass spectrometry) method. The results showed that the levels of primary metabolites were largely dependent upon the diurnal rhythm and leaf developmental phase. The young leaves (sink) produced and accumulated starch rather than sucrose. The short-term (4 h, 1 day) UV-B exposure inhibited sucrose synthesis, which could be the first target of UV-B radiation. Following short- and long-term (5 days) exposure to UV-B radiation, the dynamic response of primary metabolites was evaluated. It was found that the content of carbohydrates decreased throughout the period of exposure to UV-B stress, especially in terms of sucrose concentration. However, the content of the majority of amino acids increased after an early decrease. Our data revealed that the metabolic response, as well as the gene expression, differed with the period (intensity) of exposure to UV-B radiation and with the phase of leaf development. These findings provide new insights for a better understanding of the metabolic response of a variety of plant species exposed to a wide range of UV-B radiation.

Composite Transport Model and Water and Solute Transport across Plant Roots: An Update.

The present review examines recent experimental findings in root transport phenomena in terms of the composite transport model (CTM). It has been a well-accepted conceptual model to explain the complex water and solute flows across the root that has been related to the composite anatomical structure. There are three parallel pathways involved in the transport of water and solutes in roots - apoplast, symplast, and transcellular paths. The role of aquaporins (AQPs), which facilitate water flows through the transcellular path, and root apoplast is examined in terms of the CTM. The contribution of the plasma membrane bound AQPs for the overall water transport in the whole plant level was varying depending on the plant species, age of roots with varying developmental stages of apoplastic barriers, and driving forces (hydrostatic vs. osmotic). Many studies have demonstrated that the apoplastic barriers, such as Casparian bands in the primary anticlinal walls and suberin lamellae in the secondary cell walls, in the endo- and exodermis are not perfect barriers and unable to completely block the transport of water and some solute transport into the stele. Recent research on water and solute transport of roots with and without exodermis triggered the importance of the extension of conventional CTM adding resistances that arrange in series (epidermis, exodermis, mid-cortex, endodermis, and pericycle). The extension of the model may answer current questions about the applicability of CTM for composite water and solute transport of roots that contain complex anatomical structures with heterogeneous cell layers.

The composite water and solute transport of barley (Hordeum vulgare) roots: effect of suberized barriers.

Background and Aims: Roots have complex anatomical structures, and certain localized cell layers develop suberized apoplastic barriers. The size and tightness of these barriers depend on the growth conditions and on the age of the root. Such complex anatomical structures result in a composite water and solute transport in roots. Methods: Development of apoplastic barriers along barley seminal roots was detected using various staining methods, and the suberin amounts in the apical and basal zones were analysed using gas chromatography-mass spectometry (GC-MS). The hydraulic conductivity of roots ( Lp r ) and of cortical cells ( Lp c ) was measured using root and cell pressure probes. Key Results: When grown in hydroponics, barley roots did not form an exodermis, even at their basal zones. However, they developed an endodermis. Endodermal Casparian bands first appeared as 'dots' as early as at 20 mm from the apex, whereas a patchy suberin lamellae appeared at 60 mm. The endodermal suberin accounted for the total suberin of the roots. The absolute amount in the basal zone was significantly higher than in the apical zone, which was inversely proportional to the Lp r . Comparison of Lp r and Lp c suggested that cell to cell pathways dominate for water transport in roots. However, the calculation of Lp r from Lp c showed that at least 26 % of water transport occurs through the apoplast. Roots had different solute permeabilities ( P sr ) and reflection coefficients ( σ sr ) for the solutes used. The σ sr was below unity for the solutes, which have virtually zero permeability for semi-permeable membranes. Conclusions: Suberized endodermis significantly reduces Lp r of seminal roots. The water and solute transport across barley roots is composite in nature and they do not behave like ideal osmometers. The composite transport model should be extended by adding components arranged in series (cortex, endodermis) in addition to the currently included components arranged in parallel (apoplastic, cell to cell pathways).

Direct observation of local xylem embolisms induced by soil drying in intact Zea mays leaves.

The vulnerability of vascular plants to xylem embolism is closely related to their stable long-distance water transport, growth, and survival. Direct measurements of xylem embolism are required to understand what causes embolism and what strategies plants employ against it. In this study, synchrotron X-ray microscopy was used to non-destructively investigate both the anatomical structures of xylem vessels and embolism occurrence in the leaves of intact Zea mays (maize) plants. Xylem embolism was induced by water stress at various soil drying periods and soil water contents. X-ray images of dehydrated maize leaves showed that the ratio of gas-filled vessels to all xylem vessels increased with decreased soil water content and reached approximately 30% under severe water stress. Embolism occurred in some but not all vessels. Embolism in maize leaves was not strongly correlated with xylem diameter but was more likely to occur in the peripheral veins. The rate of embolism formation in metaxylem vessels was higher than in protoxylem vessels. This work has demonstrated that xylem embolism remains low in maize leaves under water stress and that there xylem has characteristic spatial traits of vulnerability to embolism.

Where do roots take up water? Neutron radiography of water flow into the roots of transpiring plants growing in soil.

Where and how fast does water flow from soil into roots? The answer to this question requires direct and in situ measurement of local flow of water into roots of transpiring plants growing in soil. We used neutron radiography to trace the transport of deuterated water (D2O) in lupin (Lupinus albus) roots. Lupins were grown in aluminum containers (30 × 25 × 1 cm) filled with sandy soil. D2O was injected in different soil regions and its transport in soil and roots was monitored by neutron radiography. The transport of water into roots was then quantified using a convection-diffusion model of D2O transport into roots. The results showed that water uptake was not uniform along roots. Water uptake was higher in the upper soil layers than in the lower ones. Along an individual root, the radial flux was higher in the proximal segments than in the distal segments. In lupins, most of the water uptake occurred in lateral roots. The function of the taproot was to collect water from laterals and transport it to the shoot. This function is ensured by a low radial conductivity and a high axial conductivity. Lupin root architecture seems well designed to take up water from deep soil layers.

Gating of aqùaporins by light and reactive oxygen species in leaf parenchyma cells of the midrib of Zea mays.

Changes of the water permeability aqùaporin (AQP) activity of leaf cells were investigated in response to different light regimes (low versus high). Using a cell pressure probe, hydraulic properties (half-time of water exchange, T(1/2) infinity 1/water permeability) of parenchyma cells in the midrib tissue of maize (Zea mays L.) leaves have been measured. A new perfusion technique was applied to excised leaves to keep turgor constant and to modify the environment around cells by perfusing solutions using a pressure chamber. In response to low light (LL) of 200 micromol m(-2) s(-1), T(1/2) decreased during the perfusion of a control solution of 0.5 mM CaCl(2) by a factor of two. This was in line with earlier results from leaf cells of intact maize plants at a constant turgor. In contrast, high light (HL) at intensities of 800 micromol m(-2) s(-1) and 1800 micromol m(-2) s(-1) increased the T(1/2) in two-thirds of cells by factors of 14 and 35, respectively. The effects of HL on T(1/2) were similar to those caused by H(2)O(2) treatment in the presence of Fe(2+), which produced *OH (Fenton reaction; reversible oxidative gating of aquaporins). Treatments with 20 mM H(2)O(2) following Fe(2+) pre-treatments increased the T(1/2) by a factor of 30. Those increased T(1/2) values could be partly recovered, either when the perfusion solution was changed back to the control solution or when LL was applied. 3mM of the antioxidant glutathione also reversed the effects of HL. The data suggest that HL could induce reactive oxygen species (ROS) such as *OH, and they affected water relations. The results provide evidence that the varying light climate adjusts water flow at the cell level; that is, water flow is maximized at a certain light intensity and then reduced again by HL. Light effects are discussed in terms of an oxidative gating of aquaporins by ROS.

Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays.

In response to light, water relation parameters (turgor, half-time of water exchange, T(1/2), and hydraulic conductivity, Lp; T(1/2) proportional 1/Lp) of individual cells of parenchyma sitting in the midrib of leaves of intact corn (Zea mays L.) plants were investigated using a cell pressure probe. Parenchyma cells were used as model cells for the leaf mesophyll, because they are close to photosynthetically active cells at the abaxial surface, and there are stomata at both the adaxial and abaxial sides. Turgor ranged from 0.2 to 1.0 MPa under laboratory light condition (40 micromol m(-2) s(-1) at the tissue level), and individual cells could be measured for up to 6 h avoiding the variability between cells. In accordance with earlier findings, there was a big variability in T(1/2)s measured ranging from 0.5 s to 100 s, but the action of light on T(1/2)s could nevertheless be worked out for cells having T(1/2)s greater than 2 s. Increasing light intensity ranging from 100 micromol m(-2) s(-1) to 650 micromol m(-2) s(-1) decreased T(1/2) by a factor up to five within 10 min and increased Lp (and aquaporin activity) by the same factor. In the presence of light, turgor decreased due to an increase in transpiration, and this tended to compensate or even overcompensated for the effect of light on T(1/2). For example, during prolonged illumination, cell turgor dropped from 0.2 to 1.0 MPa to -0.03 to 0.4 MPa, and this drop caused an increase of T(1/2) and a reduction of cell Lp, i.e. there was an effect of turgor on cell Lp besides that of light. To separate the two effects, cell turgor (water potential) was kept constant while changing light intensity by applying gas pressure to the roots using a pressure chamber. At a light intensity of 160 micromol m(-2) s(-1), there was a reduction of T(1/2) by a factor of 2.5 after 10-30 min, when turgor was constant within +/-0.05 MPa. Overall, the effects of light on T(1/2) (Lp) were overriding those of turgor only when decreases in turgor were less than about 0.2 MPa. Otherwise, turgor became the dominant factor. The results indicate that the hydraulic conductivity increased with increasing light intensity tending to improve the water status of the shoot. However, when transpiration induced by light tends to cause a low turgidity of the tissue, cell Lp was reduced. It is concluded that, when measuring the overall hydraulic conductivity of leaves, both the effects of light and turgor should be considered. Although the mechanism(s) of how light and turgor influence the cell Lp is still missing, it most likely involves the gating of aquaporins by both parameters.