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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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wiggenhauser, Moore, Wang, Bienert, Laursen and Blotevogel.)

The stable nitrogen isotope ratio δ 15 N is used as a marker of dietary protein sources in blood. Crop fertilization strategies affect δ 15 N 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 δ 15 N in blood plasma. At study baseline, plasma δ 15 N was +9.34 ± 0.29‰ (mean ± standard deviation). After 12 days intervention with a diet based on crops fertilized with animal manure, plasma δ 15 N 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 δ 15 N values between fertilizers are propagated to the blood plasma of human consumers. The results indicate a need to consider agricultural practices when using δ 15 N as a dietary biomarker.

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 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., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The oxygen isotope signature of sulphate (δ 18 O sulphate ) is increasingly used to study nutritional fluxes and sulphur transformation processes in a variety of natural environments. However, mechanisms controlling the δ 18 O sulphate signature in soil-plant systems are largely unknown. The objective of this study was to determine key factors, which affect δ 18 O sulphate 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, δ 18 O sulphate values of synthetic fertilizer were not affected by soil water. Correlations between soil and plant δ 18 O sulphate values were controlled by water δ 18 O values and fertilizer treatments. Additionally, plant δ 34 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., (© 2020 John Wiley & Sons Ltd.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The variability of stable isotope ratios (δ(2) H, δ(13) C, δ(15) N, δ(18) O and δ(34) S) along the production chain of pasta (durum wheat, flour and pasta) produced by using both conventional and organic farming systems in four Italian regions in 2 years was investigated. The aim was to evaluate if and how the farming system and geographical origin affect stable isotope ratios determined along the production chain. Irrespective of the processing technology, 65% of the samples were correctly classified according to the farming system and 98% were correctly classified regarding the geographical region. When considering both farming system and geographical region simultaneously, 80% of the samples were correctly classified. The measured isotope parameters were thus primarily affected by the geographical origin. In conclusion, it is expected that the use of these parameters will allow the development of analytical control procedures that can be used to check the geographical origin of Italian organic and conventional pasta and its raw materials. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background and Aims: Retranslocation of iron (Fe) from source tissues enhances plant tolerance to Fe deficiency. Previous work has shown that silicon (Si) can alleviate Fe deficiency by enhancing acquisition and root to shoot translocation of Fe. Here the role of Si in Fe mobilization in older leaves and the subsequent retranslocation of Fe to young leaves of cucumber (Cucumis sativus) plants growing under Fe-limiting conditions was investigated., Methods: Iron ((57)Fe or naturally occurring isotopes) was measured in leaves at different positions on plants hydroponically growing with or without Si supply. In parallel, the concentration of the Fe chelator nicotianamine (NA) along with the expression of nicotianamine synthase (NAS) involved in its biosynthesis and the expression of yellow stripe-like (YSL) transcripts mediating Fe-NA transport were also determined., Key Results: In plants not receiving Si, approximately half of the total Fe content remained in the oldest leaf. In contrast, Si-treated plants showed an almost even Fe distribution among leaves with four different developmental stages, thus providing evidence of enhanced Fe remobilization from source leaves. This Si-stimulated Fe export was paralleled by an increased NA accumulation and expression of the YSL1 transporter for phloem loading/unloading of the Fe-NA complex., Conclusions: The results suggest that Si enhances remobilization of Fe from older to younger leaves by a more efficient NA-mediated Fe transport via the phloem. In addition, from this and previous work, a model is proposed of how Si acts to improve Fe homeostasis under Fe deficiency in cucumber., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

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., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

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.

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.

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.

The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10 d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification., (© 2013 John Wiley & Sons Ltd.)

Novel procedures for analytical authentication of organic plant products are urgently needed. Here we present the first study encompassing stable isotopes of hydrogen, carbon, nitrogen, oxygen, magnesium and sulphur as well as compound-specific nitrogen and oxygen isotope analysis of nitrate for discrimination of organically and conventionally grown plants. The study was based on wheat, barley, faba bean and potato produced in rigorously controlled long-term field trials comprising 144 experimental plots. Nitrogen isotope analysis revealed the use of animal manure, but was unable to discriminate between plants that were fertilised with synthetic nitrogen fertilisers or green manures from atmospheric nitrogen fixing legumes. This limitation was bypassed using oxygen isotope analysis of nitrate in potato tubers, while hydrogen isotope analysis allowed complete discrimination of organic and conventional wheat and barley grains. It is concluded, that multi-isotopic analysis has the potential to disclose fraudulent substitutions of organic with conventionally cultivated plants., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

· Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast. · A combined approach was performed including analyses of apoplastic Fe, reduction-based Fe acquisition and Fe-mobilizing compounds in roots along with the expression of related genes. · Si-treated plants accumulated higher concentrations of root apoplastic Fe, which rapidly decreased when Fe was withheld from the nutrient solution. Under Fe-deficient conditions, Si also increased the accumulation of Fe-mobilizing compounds in roots. Si supply stimulated root activity of Fe acquisition at the early stage of Fe deficiency stress through regulation of gene expression levels of proteins involved in Fe acquisition. However, when the period of Fe deprivation was extended, these reactions further decreased as a consequence of Si-induced enhancement of the Fe status of the plants. · This work provides new evidence for the beneficial role of Si in plant nutrition and clearly indicates that Si-mediated alleviation of Fe deficiency includes an increase of the apoplastic Fe pool in roots and an enhancement of Fe acquisition., (© 2013 The Authors New Phytologist © 2013 New Phytologist Trust.)

Agricultural methods may affect the nutritional composition of plants and cause complex changes in the food matrix. Whether this affects the dietary absorption of minerals that are important for maintaining health thorough life remains unclear. We compared the effects of organic and conventional diets on intake and absorption of zinc and copper in men. Two double-blinded, cross-over, intervention trials (3 dietary periods of 12 days with 2-week-long wash-out) were performed in 2008 (n = 17) and 2009 (n = 16) in young men. The diets were based on 9 crops grown in rigidly controlled organic and conventional systems in 2 replications over 2 years. The primary outcomes were intake and absorption of zinc and copper. The absorption was determined by faecal excretion of stable enriched isotopes extrinsically added to the entire menu. Within each year, the intake and absorption of zinc (overall mean ± SD; 12.35 ± 0.47 mg per 10 MJ and 44.6% ± 12.1, respectively) and copper (overall mean ± SD; 2.12 ± 0.28 mg per 10 MJ and 41.2% ± 13.2, respectively) were not different between the organic and conventional diets. The growing season had no effect on zinc intake and absorption, but the copper intake was higher (P = 0.01) and absorption lower (P < 0.005) in 2008 compared with 2009 (overall mean absorption ± SD; 35.3% ± 13.5 in 2008 and 54.0% ± 10.7 in 2009). In conclusion, organic agriculture does not affect the intake and absorption of copper and zinc in men. Consequently, it does not seem to promote the health beneficial role of these minerals in vivo.

Plant tissue analysis is a valuable tool for evaluating the nutritional status and quality of crops and is widely used for scientific and commercial purposes. The majority of plant analyzes are now performed by techniques based on ICP spectrometry such as inductively coupled plasma-optical emission spectroscopy (ICP-OES) or ICP-mass spectrometry (ICP-MS). These techniques enable fast and accurate measurements of multielement profiles when combined with appropriate methods for sample preparation and digestion. This chapter presents state-of-the-art methods for digestion of plant tissues and subsequent analysis of their multielement composition by ICP spectrometry. Details on upcoming techniques, expected to gain importance within the field of multielement plant tissue analysis over the coming years, are also provided. Finally, attention is given to laser ablation ICP-MS (LA-ICP-MS) for multielement bioimaging of plant tissues. The presentation of the methods covers instructions on all steps from sampling and sample preparation to data interpretation.

Trace elements are unevenly distributed and speciated throughout the cereal grain. The germ and the outer layers of the grain have the highest concentrations of trace elements. A large fraction of the trace elements is therefore lost during the milling process. The bioavailability of the remaining trace elements is very low. This is usually ascribed to the formation of poorly soluble complexes with the phosphorus storage compound phytic acid. Hence, analysis of the total concentration of trace elements in grain tissues must be combined with a speciation analysis in order to assess their contribution to human nutrition. This chapter deals with the fractionation of anatomically very different cereal tissues. Procedures for microscaling of digestion procedures are outlined together with requirements for the use of certified reference materials in elemental profiling of grain tissue fractions. Methods for extraction and analysis of complexes containing trace elements in the grain tissue fractions are described. Finally, the chapter concludes with criteria for choice of chromatographic methods and setting of ICP-MS instrument parameters.

The multielemental composition of organic and conventional winter wheat, spring barley, faba bean, and potato was analyzed with inductively coupled plasma-optical emission spectrometry (ICP-OES) and -mass spectrometry (ICP-MS). The crops were cultivated in two years at three geographically different field locations, each accommodating one conventional and two organic cropping systems. The conventional system produced the highest harvest yields for all crops except the nitrogen-fixing faba bean, whereas the dry matter content of each crop was similar across systems. No systematic differences between organic and conventional crops were found in the content of essential plant nutrients when statistically analyzed individually. However, chemometric analysis of multielemental fingerprints comprising up to 14 elements allowed discrimination. The discrimination power was further enhanced by analysis of up to 25 elements derived from semiquantitative ICP-MS. It is concluded that multielemental fingerprinting with semiquantitative ICP-MS and chemometrics has the potential to enable authentication of organic crops.

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.

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.

Background: Quantitative multi-elemental analysis by inductively coupled plasma (ICP) spectrometry depends on a complete digestion of solid samples. However, fast and thorough sample digestion is a challenging analytical task which constitutes a bottleneck in modern multi-elemental analysis. Additional obstacles may be that sample quantities are limited and elemental concentrations low. In such cases, digestion in small volumes with minimum dilution and contamination is required in order to obtain high accuracy data., Results: We have developed a micro-scaled microwave digestion procedure and optimized it for accurate elemental profiling of plant materials (1-20 mg dry weight). A commercially available 64-position rotor with 5 ml disposable glass vials, originally designed for microwave-based parallel organic synthesis, was used as a platform for the digestion. The novel micro-scaled method was successfully validated by the use of various certified reference materials (CRM) with matrices rich in starch, lipid or protein. When the micro-scaled digestion procedure was applied on single rice grains or small batches of Arabidopsis seeds (1 mg, corresponding to approximately 50 seeds), the obtained elemental profiles closely matched those obtained by conventional analysis using digestion in large volume vessels. Accumulated elemental contents derived from separate analyses of rice grain fractions (aleurone, embryo and endosperm) closely matched the total content obtained by analysis of the whole rice grain., Conclusion: A high-throughput micro-scaled method has been developed which enables digestion of small quantities of plant samples for subsequent elemental profiling by ICP-spectrometry. The method constitutes a valuable tool for screening of mutants and transformants. In addition, the method facilitates studies of the distribution of essential trace elements between and within plant organs which is relevant for, e.g., breeding programmes aiming at improvement of the micronutrient density in edible plant parts. Compared to existing vial-in-vial systems, the new method developed here represents a significant methodological advancement in terms of higher capacity, reduced labour consumption, lower material costs, less contamination and, as a consequence, improved analytical accuracy following micro-scaled digestion of plant samples.

The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as (48)SO(+). Simultaneously, Fe was measured as (72)FeO(+), P as (47)PO(+), and Zn as (66)Zn(+), enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP(6)) was the main Fe binding ligand, with the stoichiometry Fe(4)(IP(6))(18). When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.

Barley (Hordeum vulgare) genotypes display a marked difference in their ability to tolerate growth at low manganese (Mn) concentrations, a phenomenon designated as differential Mn efficiency. Induction of Mn deficiency in two genotypes differing in Mn efficiency led to a decline in the quantum yield efficiency for both, although faster in the Mn-inefficient genotype. Leaf tissue and thylakoid Mn concentrations were reduced under Mn deficiency, but no difference between genotypes was observed and no visual Mn deficiency symptoms were developed. Analysis of the fluorescence induction kinetics revealed that in addition to the usual O-J-I-P steps, clear K and D steps were developed in the Mn-inefficient genotype under Mn deficiency. These marked changes indicated damages to photosystem II (PSII). This was further substantiated by state transition measurements, indicating that the ability of plants to redistribute excitation energy was reduced. The percentage change in state transitions for control plants with normal Mn supply of both genotypes was 9% to 11%. However, in Mn-deficient leaves of the Mn-inefficient genotypes, state transitions were reduced to less than 1%, whereas no change was observed for the Mn-efficient genotypes. Immunoblotting and the chlorophyll a/b ratio confirmed that Mn deficiency in general resulted in a significant reduction in abundance of PSII reaction centers relative to the peripheral antenna. In addition, PSII appeared to be significantly more affected by Mn limitation than PSI. However, the striking genotypic differences observed in Mn-deficient plants, when analyzing state transitions and fluorescence induction kinetics, could not be correlated with specific changes in photosystem proteins. Thus, there is no simple linkage between protein expression and the differential reduction in state transition and fluorescence induction kinetics observed for the genotypes under Mn deficiency.

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.

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.