An optimized method for extraction and purification of inorganic phosphate from plant material for oxygen isotope ratio analysis.

Compound-specific stable isotope ratio analysis of oxygen isotopes in inorganic phosphate can be used to study biological phosphorus cycling and the transformation processes controlling the fate of phosphorus. However, methods for extraction of inorganic phosphate from plant tissue for oxygen isotope ratio analysis are not consistent. Further, the purification into solid silver phosphate can be challenging and laborious. In this work, a detailed and optimized method to provide a more consistent, easily implementable and reproducible extraction using trichloroacetic acid and subsequent purification of inorganic phosphate from plant material for oxygen isotope ratio analysis is presented. Key focus points were: uniform extraction of inorganic phosphate from barley leaves, removal of dissolved organic material, flexibility in regards to the amount of inorganic phosphate extracted for the purification into silver phosphate, reduced use of chemicals and, removal of co-precipitated oxygen-bearing compounds before analysis. Most notable optimizations to the method and associated effects were:•Drying of plant material before inorganic phosphate extraction increases the method applicability to a broader range of plant sample types.•Removal of dissolved organic matter improves inorganic phosphate purification.•Sample volume adjustment according to inorganic phosphate content is vital for effective and quantitative precipitations.

Stable Isotope Fractionation of Metals and Metalloids in Plants: A Review.

This work critically reviews stable isotope fractionation of essential (B, Mg, K, Ca, Fe, Ni, Cu, Zn, Mo), beneficial (Si), and non-essential (Cd, Tl) metals and metalloids in plants. The review (i) provides basic principles and methodologies for non-traditional isotope analyses, (ii) compiles isotope fractionation for uptake and translocation for each element and connects them to physiological processes, and (iii) interlinks knowledge from different elements to identify common and contrasting drivers of isotope fractionation. Different biological and physico-chemical processes drive isotope fractionation in plants. During uptake, Ca and Mg fractionate through root apoplast adsorption, Si through diffusion during membrane passage, Fe and Cu through reduction prior to membrane transport in strategy I plants, and Zn, Cu, and Cd through membrane transport. During translocation and utilization, isotopes fractionate through precipitation into insoluble forms, such as phytoliths (Si) or oxalate (Ca), structural binding to cell walls (Ca), and membrane transport and binding to soluble organic ligands (Zn, Cd). These processes can lead to similar (Cu, Fe) and opposing (Ca vs. Mg, Zn vs. Cd) isotope fractionation patterns of chemically similar elements in plants. Isotope fractionation in plants is influenced by biotic factors, such as phenological stages and plant genetics, as well as abiotic factors. Different nutrient supply induced shifts in isotope fractionation patterns for Mg, Cu, and Zn, suggesting that isotope process tracing can be used as a tool to detect and quantify different uptake pathways in response to abiotic stresses. However, the interpretation of isotope fractionation in plants is challenging because many isotope fractionation factors associated with specific processes are unknown and experiments are often exploratory. To overcome these limitations, fundamental geochemical research should expand the database of isotope fractionation factors and disentangle kinetic and equilibrium fractionation. In addition, plant growth studies should further shift toward hypothesis-driven experiments, for example, by integrating contrasting nutrient supplies, using established model plants, genetic approaches, and by combining isotope analyses with complementary speciation techniques. To fully exploit the potential of isotope process tracing in plants, the interdisciplinary expertise of plant and isotope geochemical scientists is required.

Fertilizer Type Affects Stable Isotope Ratios of Nitrogen in Human Blood Plasma─Results from Two-Year Controlled Agricultural Field Trials and a Randomized Crossover Dietary Intervention Study.

The stable nitrogen isotope ratio δ15N is used as a marker of dietary protein sources in blood. Crop fertilization strategies affect δ15N in plant foods. In a double-blinded randomized cross-over dietary intervention trial with 33 participants, we quantified the effect of fertilizer type (conventional: synthetic fertilizer and organic: animal or green manure) on δ15N in blood plasma. At study baseline, plasma δ15N was +9.34 ± 0.29‰ (mean ± standard deviation). After 12 days intervention with a diet based on crops fertilized with animal manure, plasma δ15N was shifted by +0.27 ± 0.04‰ (mean ± standard error) compared to synthetic fertilization and by +0.22 ± 0.04‰ compared to fertilization with green manure (both p < 0.0001). Accordingly, differences in the δ15N values between fertilizers are propagated to the blood plasma of human consumers. The results indicate a need to consider agricultural practices when using δ15N as a dietary biomarker.

Lectin Activity in Commonly Consumed Plant-Based Foods: Calling for Method Harmonization and Risk Assessment.

Lectins are ubiquitous proteins characterized through their ability to bind different types of carbohydrates. It is well known that active lectins from insufficiently prepared legumes can cause adverse human health effects. The objective of this study was to determine the activity of lectins in samples across plant families representing commercially available edible plants, and the feasibility of inactivating lectins through soaking and boiling. Lectins were extracted from the plant families Adoxaceae, Amaranthaceae, Cannabaceae, Fabaceae, Gramineae, Lamiaceae, Linaceae, Pedaliaceae, and Solanaceae. A hemagglutination assay based on non-treated or trypsin treated rabbit erythrocytes was used to measure the lectin activity. The results showed the highest lectin activity in species from the Fabaceae family and demonstrated that soaking and boiling have an effect on the levels of active lectins. This is the first large study that combines lectin activity obtained from two different assays with raw and processed edible plants. In addition, we examined the current risk assessment, and regulations necessary for an adequate official reporting of results. We encourage the scientific community to further explore this field and agree on harmonized methods for analysis and interpretation, and hope that our methodology can initiate this development.

The oxygen isotopic signature of soil- and plant-derived sulphate is controlled by fertilizer type and water source.

The oxygen isotope signature of sulphate (δ18 Osulphate ) is increasingly used to study nutritional fluxes and sulphur transformation processes in a variety of natural environments. However, mechanisms controlling the δ18 Osulphate signature in soil-plant systems are largely unknown. The objective of this study was to determine key factors, which affect δ18 Osulphate values in soil and plants. The impact of an 18 O-water isotopic gradient and different types of fertilizers was investigated in a soil incubation study and a radish (Raphanus sativus L.) greenhouse growth experiment. Water provided 31-64% of oxygen atoms in soil sulphate formed via mineralization of organic residues (green and chicken manures) while 49% of oxygen atoms were derived from water during oxidation of elemental sulphur. In contrast, δ18 Osulphate values of synthetic fertilizer were not affected by soil water. Correlations between soil and plant δ18 Osulphate values were controlled by water δ18 O values and fertilizer treatments. Additionally, plant δ34 S data showed that the sulphate isotopic composition of plants is a function of S assimilation. This study documents the potential of using compound-specific isotope ratio analysis for investigating and tracing fertilization strategies in agricultural and environmental studies.

The molecular-physiological functions of mineral macronutrients and their consequences for deficiency symptoms in plants.

The visual deficiency symptoms developing on plants constitute the ultimate manifestation of suboptimal nutrient supply. In classical plant nutrition, these symptoms have been extensively used as a tool to characterise the nutritional status of plants and to optimise fertilisation. Here we expand this concept by bridging the typical deficiency symptoms for each of the six essential macronutrients to their molecular and physiological functionalities in higher plants. We focus on the most recent insights obtained during the last decade, which now allow us to better understand the links between symptom and function for each element. A deep understanding of the mechanisms underlying the visual deficiency symptoms enables us to thoroughly understand how plants react to nutrient limitations and how these disturbances may affect the productivity and biodiversity of terrestrial ecosystems. A proper interpretation of visual deficiency symptoms will support the potential for sustainable crop intensification through the development of new technologies that facilitate automatised management practices based on imaging technologies, remote sensing and in-field sensors, thereby providing the basis for timely application of nutrients via smart and more efficient fertilisation.

Authenticity testing of organically grown vegetables by stable isotope ratio analysis of oxygen in plant-derived sulphate.

Analytical methods for authenticity testing of organically grown vegetables are urgently needed. Here we present a novel method for organic authentication based on stable isotope ratio analysis of oxygen in plant-derived sulphate. We combined this method with stable isotope ratio analysis of bulk plant tissue and plant-derived nitrate to discriminate organic and conventional potato, carrot, and cabbage from rigidly controlled long-term field trials and from a case study using retail potatoes. It was shown that oxygen isotope ratios of sulphate from organic vegetables were significantly lower compared to their conventional counterparts and the values were directly linked to the fertilisation strategy. The classification power of sulphate isotope analysis was superior compared to known bulk tissue isotope markers and nitrate isotope values. In conclusion, oxygen isotope analysis of plant-derived sulphate represents a promising new method for authentication of organic vegetables.

Sensitive Detection of Phosphorus Deficiency in Plants Using Chlorophyll a Fluorescence.

Phosphorus (P) is a finite natural resource and an essential plant macronutrient with major impact on crop productivity and global food security. Here, we demonstrate that time-resolved chlorophyll a fluorescence is a unique tool to monitor bioactive P in plants and can be used to detect latent P deficiency. When plants suffer from P deficiency, the shape of the time-dependent fluorescence transients is altered distinctively, as the so-called I step gradually straightens and eventually disappears. This effect is shown to be fully reversible, as P resupply leads to a rapid restoration of the I step. The fading I step suggests that the electron transport at photosystem I (PSI) is affected in P-deficient plants. This is corroborated by the observation that differences at the I step in chlorophyll a fluorescence transients from healthy and P-deficient plants can be completely eliminated through prior reduction of PSI by far-red illumination. Moreover, it is observed that the barley (Hordeum vulgare) mutant Viridis-zb(63), which is devoid of PSI activity, similarly does not display the I step. Among the essential plant nutrients, the effect of P deficiency is shown to be specific and sufficiently sensitive to enable rapid in situ determination of latent P deficiency across different plant species, thereby providing a unique tool for timely remediation of P deficiency in agriculture.

Yield, Quality, and Nutrient Concentrations of Strawberry (Fragaria ×ananassa Duch. cv. 'Sonata') Grown with Different Organic Fertilizer Strategies.

Four combinations of two solid organic fertilizers (Monterra Malt and chicken manure) applied before planting and two liquid organic fertilizers (broad bean and Pioner Hi-Fruit/K-Max) given through drip irrigation (fertigation) were compared with inorganic fertilization regarding growth, yield, nutrient concentration, and fruit quality of strawberries. Broad bean fertigation combined with Monterra Malt resulted in a similar fruit yield as inorganic fertilizer and a higher yield than Monterra Malt combined with Pioner; however, total soluble solids, firmness, and titratable acid were improved with Pioner fertigation, although these parameters were more affected by harvest time than the applied fertilizers. The concentrations of most nutrients in fruits and leaves were higher in inorganically fertigated plants. The reductions in fruit yield in three of four treatments and fruit weight in all organic treatments may be due to a combination of the following conditions in the root zone: (1) high pH and high NH4(+)/NO3(-) ratio; (2) high EC and/or high NaCl concentration; (3) cation imbalance; and (4) nutrient deficiency.

Compound-Specific δ¹5N and δ¹³C Analyses of Amino Acids for Potential Discrimination between Organically and Conventionally Grown Wheat.

We present a study deploying compound-specific nitrogen and carbon isotope analysis of amino acids to discriminate between organically and conventionally grown plants. We focused on grain samples of common wheat and durum wheat grown using synthetic nitrogen fertilizers, animal manures, or green manures from nitrogen-fixing legumes. The measurement of amino acid δ(15)N and δ(13)C values, after protein hydrolysis and derivatization, was carried out using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Our results demonstrated that δ(13)C of glutamic acid and glutamine in particular, but also the combination of δ(15)N and δ(13)C of 10 amino acids, can improve the discrimination between conventional and organic wheat compared to stable isotope bulk tissue analysis. We concluded that compound-specific stable isotope analysis of amino acids represents a novel analytical tool with the potential to support and improve the certification and control procedures in the organic sector.

Discrimination of conventional and organic white cabbage from a long-term field trial study using untargeted LC-MS-based metabolomics.

The influence of organic and conventional farming practices on the content of single nutrients in plants is disputed in the scientific literature. Here, large-scale untargeted LC-MS-based metabolomics was used to compare the composition of white cabbage from organic and conventional agriculture, measuring 1,600 compounds. Cabbage was sampled in 2 years from one conventional and two organic farming systems in a rigidly controlled long-term field trial in Denmark. Using Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA), we found that the production system leaves a significant (p = 0.013) imprint in the white cabbage metabolome that is retained between production years. We externally validated this finding by predicting the production system of samples from one year using a classification model built on samples from the other year, with a correct classification in 83 % of cases. Thus, it was concluded that the investigated conventional and organic management practices have a systematic impact on the metabolome of white cabbage. This emphasizes the potential of untargeted metabolomics for authenticity testing of organic plant products.

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato.

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.

Comparison of polyacetylene content in organically and conventionally grown carrots using a fast ultrasonic liquid extraction method.

A rapid and sensitive analytical method for quantification of polyacetylenes in carrot roots was developed. The traditional extraction method (stirring) was compared to a new ultrasonic liquid processor (ULP)-based methodology using high-performance liquid chromatography-ultraviolet (HPLC-UV) and mass spectrometry (MS) for identification and quantification of three polyacetylenes. ULP was superior because a significant reduction in extraction time and improved extraction efficiencies were obtained. After optimization, the ULP method showed good selectivity, precision [relative standard deviations (RSDs) of 2.3-3.6%], and recovery (93% of falcarindiol) of the polyacetylenes. The applicability of the method was documented by comparative analyses of carrots grown organically or conventionally in a 2 year field trial study. The average concentrations of falcarindiol, falcarindiol-3-acetate, and falcarinol in year 1 were 222, 30, and 94 mug of falcarindiol equiv/g of dry weight, respectively, and 3-15% lower in year 2. The concentrations were not significantly influenced by the growth system, but a significant year-year variation was observed for falcarindiol-3-acetate.

Latent manganese deficiency increases transpiration in barley (Hordeum vulgare).

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low nM range. The Mn-starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (F(v)/F(m)) was reduced from 0.83 in Mn-sufficient control plants to below 0.5 in Mn-starved plants. Leaf Mn concentrations declined from 30 to 7 microg Mn g(-1) dry weight in control and Mn-starved plants, respectively. Mn-starved plants had up to four-fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn-starved plants compared with the control. The observed increase in transpiration was substantiated by (13)C-isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn-starved plants. The extractable wax content of leaves of Mn-starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.

The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana.

Loss of aquaporin TIP1;1 in Arabidopsis has been suggested to result in early senescence and plant death. This was based on the fact that a partial reduction of TIP1;1 by RNA interference (RNAi) led to gradual phenotypes, ranging from indistinguishable from wild type to lethality, depending on the degree of downregulation of the target messenger, and displaying pleiotropic effects in primary metabolism and cell signalling. A hypothesis was put forward to suggest that TIP1;1, apart from its transport function, may play an essential role in vesicle routing. Here we identify an Arabidopsis transposon insertion line tip1;1-1 that is completely devoid of TIP1;1 protein, as demonstrated by western blotting and immunolocalization using an isoform-specific antibody. Strikingly, the complete absence of the protein did not result in any significant effect on metabolism or elemental composition of the plants. Microarray analysis did not indicate increased expression of other aquaporins to compensate for the lack of TIP1;1 in tip1;1-1. We further developed a double mutant of TIPs in Arabidopsis, lacking both TIP1;1 and its closest paralog TIP1;2. Arabidopsis mutants lacking both TIP1;1 and TIP1;2 showed a minor increase in anthocyanin content, and a reduction in catalase activity, but showed no changes in water status. In contrast to earlier reports, plants lacking TIP1;1 and TIP1;2 aquaporins are alive and thriving. We suggest that RNAi directed towards TIP1;1 may have resulted in off-target gene silencing, a notion that is potentially interesting for various studies analysing gene function by RNAi.