Pai Pedas

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.

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.

Competition between uptake of ammonium and potassium in barley and Arabidopsis roots: molecular mechanisms and physiological consequences.

Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1–3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.

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

BackgroundQuantitative 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.ResultsWe 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.ConclusionA 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.

Genotypic differences in manganese efficiency: field experiments with␣winter barley (Hordeum vulgare L.)

Eight different winter barley genotypes were grown in a plot experiment in northern Denmark, on soil where Mn deficiency had been previously demonstrated, to assess field performance and grain yield under two treatments: with foliar Mn fertilisation and without foliar Mn fertilisation. A high Mn efficiency was demonstrated for two of the genotypes and a low Mn efficiency was observed for one of the genotypes in three consecutive years; highly variable results were obtained for other genotypes. Mn efficiency based on grain yields could not be related to whole shoot Mn concentration, biomass or Mn uptake at various vegetative growth stages. Chlorophyll a fluorescence analysis was able to differentiate between the two Mn treatments up to 7 weeks after the last Mn application, whereas Mn concentrations, shoot biomass and Mn accumulation could not. It was not possible to fully alleviate Mn deficiency by repeated foliar spraying on the control plots. Given the growing conditions at the trial site and the low Mn in plant tissue, it is possible that Mn deficiency was extreme enough that Mn efficiency mechanisms broke down.

Differential Capacity for High-Affinity Manganese Uptake Contributes to Differences between Barley Genotypes in Tolerance to Low Manganese Availability

There is considerable variability among barley (Hordeum vulgare) genotypes in their ability to grow in soils containing a low level of plant available manganese (Mn). The physiological basis for the tolerance to low Mn availability is unknown. In this work, Mn2+ influx and compartmentation in roots of the Mn-efficient genotype Vanessa and the Mn-inefficient genotype Antonia were investigated. Two separate Mn transport systems, mediating high-affinity Mn2+ influx at concentrations up to 130 nm and low-affinity Mn2+ influx at higher concentrations, were identified in both genotypes. The two genotypes differed only in high-affinity kinetics with the Mn-efficient genotype Vanessa having almost 4 times higher Vmax than the inefficient Antonia, but similar Km values. Online inductively coupled plasma-mass spectrometry measurements verified that the observed differences in high-affinity influx resulted in a higher Mn net uptake of Vanessa compared to Antonia. Further evidence for the importance of the differences in high-affinity uptake kinetics for Mn acquisition was obtained in a hydroponic system with mixed cultivation of the two genotypes at a continuously low Mn concentration (10–50 nm) similar to that occurring in soil solution. Under these conditions, Vanessa had a competitive advantage and contained 55% to 75% more Mn in the shoots than did Antonia. Subcellular compartmentation analysis of roots based on 54Mn2+ efflux established that up to 93% and 83% of all Mn was present in the vacuole in Vanessa and Antonia, respectively. It is concluded that differential capacity for high-affinity Mn influx contributes to differences between barley genotypes in Mn efficiency.

Review: The role of atomic spectrometry in plant science

Inductively coupled plasma-mass spectrometry (ICP-MS) is the state-of-the-art technique for multi-elemental analysis of plant tissue. It provides a powerful tool in functional genomics, linking altered elemental profiles of mutants with gene expression and function. In addition, with its unmatched sensitivity, ICP-MS enables characterization of the substrate specificity and regulation of membrane transport proteins. Digestion of plant tissue has traditionally represented a bottleneck due to the low capacity of commercially available equipment. However, recent developments in micro-scaled digestion, combined with semi-quantitative analysis and chemometrics, have enabled high-throughput multi-elemental profiling and multivariate classification of large sample sets, thereby supporting a range of new applications in molecular breeding, quality assessment and authenticity testing of plants. Novel hyphenated techniques based on liquid chromatography and ICP-MS (LC-ICP-MS) have significantly improved the understanding of elemental species and their importance for e.g. the bioactivity of metals in plants. Development of procedures for sample pre-treatment, extraction and multi-dimensional separation now allows characterization of important metallo-biomolecules in plants, such as the coordination complexes of phytochelatins, metallothioneins, nicotianamine and inositol phosphates. These are key ligands involved in ion homeostasis, translocation and long-term storage of elements. Much emphasis has also been given to studies of covalently bound Se and As species, primarily due to their impact on human health. LC-ICP-MS has extensively been complemented by molecular mass spectrometry for structural information of biologically relevant species. This review covers the most recent developments in multi-elemental analysis (Part A) and speciation analysis (Part B) in plant science. A number of relevant cases are presented in order to demonstrate how the analytical developments have unravelled the functional roles of elements in plants science. These cases show that ICP-MS is an essential technology in plant metallomic platforms.

Manganese deficiency leads to genotype-specific changes in fluorescence induction kinetics and state transitions.

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.

Responses of barley root and shoot proteomes to long-term nitrogen deficiency, short-term nitrogen starvation and ammonium.

Cereals are major crops worldwide, and improvement of their nitrogen use efficiency is a crucial challenge. In this study proteins responding to N supply in barley roots and shoots were analysed using a proteomics approach, to provide insight into mechanisms of N uptake and assimilation. Control plants grown hydroponically for 33 d with 5 mm nitrate, plants grown under N deficiency (0.5 mm nitrate, 33 d) or short-term N starvation (28 d with 5 mm nitrate followed by 5 d with no N source) were compared. N deficiency caused changes in C and N metabolism and ascorbate-glutathione cycle enzymes in shoots and roots. N starvation altered proteins of amino acid metabolism in roots. Both treatments caused proteome changes in roots that could affect growth. Shoots of plants grown with ammonium as N source (28 d with 5 mm nitrate followed by 5 d with 5 mm ammonium) showed responses similar to N deficient shoots, characterized by turnover of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and increases in proteins of the chloroplastic transcription and translation machinery. Identified proteins in 67 and 49 varying spots in roots and shoots respectively, corresponded to 62 functions and over 80 gene products, considerably advancing knowledge of N responses in barley.

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.