Leaf-Atmosphere NH3 Exchange in Barley Mutants with Reduced Activities of Glutamine Synthetase

Mutants of barley (Hordeum vulgare L. cv Maris Mink) with 47 or 66% of the glutamine synthetase (GS) activity of the wild type were used for studies of NH3 exchange with the atmosphere. Under normal light and temperature conditions, tissue NH4+ concentrations were higher in the two mutants compared with wild-type plants, and this was accompanied by higher NH3 emission from the leaves. The emission of NH3 increased with increasing leaf temperatures in both wild-type and mutant plants, but the increase was much more pronounced in the mutants. Similar results were found when the light intensity (photosynthetic photon flux density) was increased. Compensation points for NH3 were estimated by exposing intact shoots to 10 nmol NH3 mol-1 air under conditions with increasing temperatures until the plants started to emit NH3. Referenced to 25[deg]C, the compensation points were 5.0 nmol mol-1 for wild-type plants, 8.3 nmol mol-1 for 47% GS mutants, and 11.8 nmol mol-1 for 66% GS mutants. Compensation points for NH3 in single, nonsenescent leaves were estimated on the basis of apoplastic pH and NH4+ concentrations. These values were 0.75, 3.46, and 7.72 nmol mol-1 for wild type, 47% GS mutants, and 66% GS mutants, respectively. The 66% GS mutant always showed higher tissue NH4+ concentrations, NH3 emission rates, and NH3 compensation points compared with the 47% GS mutant, indicating that NH4+ release was curtailed by some kind of compensatory mechanism in plants with only 47% GS activit

Interactions between uptake of amino acids and inorganic nitrogen in wheat plants

Soil-borne amino acids may constitute a source of nitrogen (N) for plants in various terrestrial ecosystems but their importance for total N nutrition is unclear, particularly in nutrient-rich arable soils. One reason for this uncertainty is lack of information on how the absorption of amino acids by plant roots is affected by the simultaneous presence of inorganic N forms. The objective of the present study was to study absorption of glycine (Gly) and glutamine (Gln) by wheat roots and their interactions with nitrate (NO<sub>3</sub><sup>−</sup>) and ammonium (NH<sub>4</sub><sup>+</sup>) during uptake. The underlying hypothesis was that amino acids, when present in nutrient solution together with inorganic N, may lead to down-regulation of the inorganic N uptake, thereby resulting in similar total N uptake rates. Amino acids were enriched with double-labelled <sup>15</sup>N and <sup>13</sup>C, while NO<sub>3</sub><sup>−</sup> and NH<sub>4</sub><sup>+</sup> acquisition was determined by their rate of removal from the nutrient solution surrounding the roots. The uptake rates of NO<sub>3</sub><sup>−</sup> and NH<sub>4</sub><sup>+</sup> did not differ from each other and were generally about twice as high as the uptake rate of organic N when the different N forms were supplied separately in concentrations of 2 mM. Nevertheless, replacement of 50% of the inorganic N with organic N was able to restore the N uptake to the same level as that in the presence of only inorganic N. Co-provision of NO<sub>3</sub><sup>−</sup> did not affect glycine uptake, while the presence of glycine down-regulated NO<sub>3</sub><sup>−</sup> uptake. The ratio between <sup>13</sup>C and <sup>15</sup>N were lower in shoots than in roots and also lower than the theoretical values, reflecting higher C losses via respiratory processes compared to N losses. It is concluded that organic N can constitute a significant N-source for wheat plants and that there is an interaction between the uptake of inorganic and organic N

Elevated Phosphorus Impedes Manganese Acquisition by Barley Plants

The occurrence of manganese (Mn) deficiency in cereal crops has increased in recent years. This coincides with increasing phosphorus (P) status of many soils due to application of high levels of animal manure and P-fertilizers. In order to test the hypothesis that elevated P my lead to Mn deficiency we have here conducted a series of hydroponics and soil experiments examining how the P supply affects the Mn nutrition of barley. Evidence for a direct negative interaction between P and Mn during root uptake was obtained by on-line inductively coupled plasma mass spectrometry (ICP-MS). Addition of a pulse of KH2PO4 rapidly and significantly reduced root Mn uptake, while a similar concentration of KCl had no effect. Addition of a P pulse to the same nutrient solution without plants did not affect the concentration of Mn, revealing that no precipitation of Mn–P species was occurring. Barley plants growing at a high P supply in hydroponics with continuous replenishment of Mn2+ had up to 50% lower Mn concentration in the youngest leaves than P limited plants. This P-induced depression of foliar Mn accelerated the development of Mn deficiency as evidenced by a marked change in the fluorescence induction kinetics of chlorophyll a. Also plants growing in soil exhibited lower leaf Mn concentrations in response to elevated P. In contrast, leaf concentrations of Fe, Cu, and N increased with the P supply, supporting that the negative effect of P on Mn acquisition was specific rather than due to a general dilution effect. It is concluded that elevated P supply directly interferes with Mn uptake in barley roots and that this negative interaction can induce Mn deficiency in the shoot. This finding has major implications in commercial plant production where many soils have high P levels

Multi-elemental speciation analysis of barley genotypes diering in tolerance to cadmium toxicity using SEC-ICP-MS and ESI-TOF-MS

Plants respond to Cd exposure by synthesizing heavy-metal-binding oligopeptides, called phytochelatins (PCs). These peptides reduce the activity of Cd2+ ions in the plant tissues by forming Cd chelates. The main objective of the present work was to develop an analytical technique, which allowed identication of the most prominent Cd species in plant tissue by SEC-ICP-MS and ESI-TOF-MS. An integrated part of the method development was to test the hypothesis that dierential Cd tolerance between two barley genotypes was linked to dierences in Cd speciation. Only one fraction of Cd species, ranging from 7001800 Da, was detected in the shoots of both genotypes. In the roots, two additional fractions ranging from 29004600 and 670015 000 Da were found. The Cd-rich SEC fractions were heart-cut, de-salted and demetallized using reversed-phase chromatography (RPC), followed by ESI-MS-TOF to identify the ligands. Three dierent families of PCs, viz. (gGlu-Cys)n-Gly (PCn), (gGlu-Cys)n-Ser (iso-PCn) and Cys-(gGlu-Cys)n-Gly (des-gGlu-PCn), the last lacking the N-terminal amino acid, were identied. The PCs induced by Cd toxicity also bound several essential trace elements in plants, including Zn, Cu, and Ni, whereas no Mn species were detected. Zn, Cu and Ni-species were distributed between the 7001800 Da and 670015 000 Da fractions, whereas only Cd species were found in the 29004600 Da fraction dominated by PC3 ligands. Although the total tissue concentration of Cd was similar for the two species, the tolerant barley genotype synthesized signicantly more CdPC3 species with a high Cd specicity than the intolerant genotype, clearly indicating a correlation between Cd tolerance and the CdPC speciation

Multi-element bioimaging of Arabidopsis thaliana roots

Better understanding of root function is central for development of plants with more efficient nutrient uptake and translocation. We here present a method for multi-element bioimaging at the cellular level in roots of the genetic model system Arabidopsis thaliana. Using conventional protocols for microscopy we observed that diffusible ions such as potassium (K+) and sodium (Na+) were lost during sample dehydration. Thus, we developed a protocol which preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, then cryo-sectioned and freeze dried. Samples are finally analyzed by Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS), utilizing a specially designed internal standard procedure. The method can be further developed to maintain the native composition of proteins, enzymes, RNA and DNA, making it attractive in combination with other omics techniques. To demonstrate the potential of the method we analyzed a mutant of A. thaliana unable to synthesize the metal chelator nicotianamine (NA). The mutant accumulated substantially more zinc (Zn) and manganese (Mn) than the wild type in the tissues surrounding the vascular cylinder. For iron (Fe) the images looked completely different, with Fe bound mainly in the epidermis of the WT plants, but confined to the cortical cell walls of the mutant. The method offers the power of ICP-MS to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to A. thaliana, the molecular and genetic approaches available in this system can now be fully exploited in order to gain a better mechanistic understanding of these processes

Micro-scaled high-throughput digestion of plant tissue samples for multi-elemental analysis

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