Søren Husted

Iron fortification of rice seeds through activation of the nicotianamine synthase gene

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression.

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganese-hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11–EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.

Elevated Nicotianamine Levels in Arabidopsis halleri Roots Play a Key Role in Zinc Hyperaccumulation

The hyperaccumulation of micronutrients and toxic metals (such as zinc and cadmium, respectively) represents an extreme trait of metallophytes adapted to metal-rich environments. This work demonstrates that elevated production of the metal chelator nicotianamine, specifically in roots of the metallophyte Arabidopsis halleri, is important for efficient root-to-shoot translocation of zinc. Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.

Manganese Efficiency in Barley: Identification and Characterization of the Metal Ion Transporter HvIRT1

Manganese (Mn) deficiency is an important plant nutritional disorder in many parts of the world. Barley (Hordeum vulgare) genotypes differ considerably in their ability to grow in soils with low Mn2+ availability. Differential genotypic Mn efficiency can be attributed to differences in Mn2+ uptake kinetics in the low nanomolar concentration range. However, the molecular basis for these differences has not yet been clarified. We present here the identification and characterization of the first barley gene encoding a plasma membrane-localized metal transport protein able to transport Mn2+. The gene is designated HvIRT1 (for IRON-REGULATED TRANSPORTER1) because it belongs to the ZIP gene family and has a high similarity to rice (Oryza sativa) OsIRT1. A novel yeast uptake assay based on inductively coupled plasma-mass spectrometry analysis of 31 different metal and metalloid ions showed that the HvIRT1 protein, in addition to Mn2+, also transported Fe2+/Fe3+, Zn2+, and Cd2+. Both Mn and iron deficiency induced an up-regulation of HvIRT1 in two barley genotypes differing in Mn efficiency, but the expression levels in all cases were highest (up to 40%) in the Mn-efficient genotype. The higher expression of HvIRT1 correlated with an increased Mn2+ uptake rate. We conclude that HvIRT1 is an important component controlling Mn2+ uptake in barley roots and contributes to genotypic differences in Mn2+ uptake kinetics.

Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

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.

Resistance modelling of ammonia exchange over oilseed rape

Ammonia (NH3) surface/atmosphere exchange is bi-directional and as such resistance models must include canopy concentrations. An existing single layer model that describes the exchange in terms of adsorption to leaf cuticles and bi-directional transport through leaf stomata, which is governed by a stomatal compensation point ( s), is applied here to NH3 exchange over oilseed rape and compared with measured fluxes. For the first time the model is tested using values of s based on the apoplastic ratio [NH4 C ]=pH ( s) measured directly in the field. Strong NH 3 emission from decomposing leaf litter at the ground and the likelihood of high [NH4 C ] in the siliques complicate the exchange pattern with oilseed rape and limit the application of the original model. This is therefore extended by: (a) the inclusion of a litter layer (2-layer model), with an emission potential ( l), (b) additionally dividing the plant canopy into a foliage- and a silique-layer (3-layer model) and (c) considering the relative humidity (h) dependency of l. The 2-layer model is able to predict night-time emission, but daytime emission is estimated to originate from the litter layer, which is in contradiction to the NH 3 sources and sinks derived for this canopy. The 3-layer model using a constant value of l requires an emission potential for the siliques of about 1300, which is consistent with bioassay estimates. Together with a parameterization of l that increases with h this model indicates that during daytime emission originates from the siliques, in agreement with the source/sink analysis. It is concluded that the leaf stomata were an effective NH3 sink, whereas the leaf litter dominates night-time emissions and the silique-layer (probably) daytime emissions. Although the 2-layer model reproduces the net exchange, the 3-layer model appears to be the mechanistically more accurate description.

Megapixel imaging of (micro)nutrients in mature barley grains

Understanding the accumulation and distribution of essential nutrients in cereals is of primary importance for improving the nutritional quality of this staple food. While recent studies have improved the understanding of micronutrient loading into the barley grain, a detailed characterization of the distribution of micronutrients within the grain is still lacking. High-definition synchrotron X-ray fluorescence was used to investigate the distribution and association of essential elements in barley grain at the micro scale. Micronutrient distribution within the scutellum and the embryo was shown to be highly variable between elements in relation to various morphological features. In the rest of the grain, the distribution of some elements such as Cu and Zn was not limited to the aleurone layer but extended into the endosperm. This pattern of distribution was less marked in the case of Fe and, in particular, Mn. A significant difference in element distribution was also found between the ventral and dorsal part of the grains. The correlation between the elements was not consistent between and within tissues, indicating that the transport and storage of elements is highly regulated. The complexity of the spatial distribution and associations has important implications for improving the nutritional content of cereal crops such as barley.

Ammonia Flux between Oilseed Rape Plants and the Atmosphere in Response to Changes in Leaf Temperature, Light Intensity, and Air Humidity (Interactions with Leaf Conductance and Apoplastic NH4+ and H+ Concentrations)

NH3 exchange between oilseed rape (Brassica napus) plants and the atmosphere was examined at realistic ambient NH3 levels under controlled environmental conditions. Different leaf conductances to NH3 diffusion were obtained by changing leaf temperature (10 to 40[deg]C), light intensity (0 to 600 [mu]mol m-2 s-1), and air humidity (20 to 80%), respectively. NH3 adsorption to the cuticle with subsequent NH3 transport through the epidermis had no significant effect on the uptake of atmospheric NH3, even at 80% relative air humidity. NH3 fluxes increased linearly with leaf conductance when light intensities were increased from 0 to 600 [mu]mol m-2 s-1. Increasing leaf temperatures from 10 to 35[deg]C caused an exponential increase in NH3 emission from plants exposed to low ambient NH3 concentrations, indicating that leaf conductance was not the only factor responding to the temperature increase. The exponential relationship between NH3 emission and temperature was closely matched by the temperature dependence of the mole fraction of gaseous NH3 above the leaf apoplast (NH3 compensation point), as calculated on the basis of NH4+ and H+ concentrations in the leaf apoplast at the different leaf temperatures. NH3 fumigation experiments showed that an increase in leaf temperature may cause a plant to switch from being a strong sink for atmospheric NH3 to being a significant NH3 source. In addition to leaf temperature, the size of the NH3 compensation point depended on plant N status and was related to plant ontogeny.

Sources and sinks of ammonia within an oilseed rape canopy.

Within-canopy profiles of ammonia (NH 3) and measurements of the canopy turbulence characteristics were used to calculate the vertical source/sink density profile of NH 3 and sensible heat in a mature oilseed rape (Brassica napus) canopy. For the analysis, the inverse Lagrangian technique (ILT) based on localized near-field theory was used. Turbulence was measured with a standard ultrasonic anemometer, which, although not ideal for in-canopy work, is estimated to lead to a parameterization of the normalized standard deviation of the vertical wind component .w=u/, which is 11% accurate for heights >0.16 m during the day. The NH3 profiles in the canopy consistently show largest concentrations at the ground caused by NH 3 release from decomposing litter leaves on the ground surface with values of up to 150 ng m 2 s 1 predicted by the ILT. The inverse Lagrangian source/sink analysis performs well for both sensible heat and NH3, although it proves to be sensitive to the choice of the source/sink layers and becomes uncertain at the ground. Despite the large estimated ground level emission (26 gN H 3-N ha 1 per day), the analysis indicates that for the runs considered all NH 3 is recaptured by the lowest 0.7 m of the 1.38 m tall canopy, and that the bi-directional net exchange with the atmosphere is governed by the top 0.5 m, leading to a net emission from the canopy of 12 g NH3-N ha 1 per day. Since measurements of apoplastic [NH4 C ] and pH indicate that no significant stomatal emission from foliage should have occurred, this suggests that the siliques were a further source of NH3.

INFLUENCE OF NITROGEN NUTRITION AND METABOLISM ON AMMONIA VOLATILIZATION IN PLANTS

Barley (Hordeum vulgare L. cv. Golf) was grown in solution culture with controlled nitrogen availability in order to study the influence of nitrogen nutrition on ammonia emission from the leaves. Ammonia emission measured in cuvettes connected to an automatic NH3 monitor was close to zero for nitrate grown plants but increased to 0.9–1.3 nmol NH3 m-2 leaf area s-1 after 3–5 days of ammonium nutrition. Increasing concentrations from 0.5 to 10 mM NH4+ in the root medium increased NH3 emission from the shoots, root glutamine synthetase activity and NH4+ concentrations in apoplast, xylem sap and bulk tissue, while apoplastic pH values decreased.Inhibition of glutamine synthetase in nitrate grown barley plants by addition of 1 mM methionine sulfoximine (MSO) to the root medium caused ammonia emission to increase 5 to 10-fold after 2–3 hours. At the same time shoot tissue ammonium concentrations started to increase. Addition of an inhibitor of photorespiration, 1 mM pyrid-2-yl hydroxymethane sulfonate (HPMS) reduced this increase in ammonia emission showing a relation between NH3 emission and photorespiration.Oil seed rape (Brassica napus L. cv. Global) plants grown at 3 different nitogen levels (2N, 4N and 7N) in a sand/soil mixture showed increasing NH3 compensation points with increasing N level. This increase was highly correlated with increasing NH4+ concentrations in the leaf apoplast and total leaf tissue. The NH3 compensation points could be succesfully predicted on basis of the pH and NH4+ concentration in the leaf apoplast.