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Barley produces several specialized metabolites, including five α-, β-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Transporters involved in manganese (Mn) uptake and intracellular Mn homeostasis in Arabidopsis and rice are well characterized, while much less is known for barley, which is particularly prone to Mn deficiency. In this study we have investigated the role of the iron-regulated transporter 1 (IRT1) for Mn uptake and translocation in barley plants. We employed an RNAi approach to reduce HvIRT1 expression to 5% of the wild-type level. This enabled characterization of the functional role of HvIRT1 by use of advanced imaging and phenotyping techniques applied to plants growing in hydroponics or soils with different Mn availability. Our results highlight the importance of HvIRT1 for the transport of Mn across the root endodermis into the stele. In the hvirt1-RNAi lines, a chlorotic phenotype with reduced shoot Mn concentration and impaired photosynthetic functionality was observed, especially under conditions with low Mn availability. We also document that HvIRT1 controlled the Mn distribution within the barley grain. Surprisingly, unlike other IRT1 orthologues, HvIRT1 played no significant role in iron uptake. We conclude that the barley IRT1 orthologue has a novel function with respect to ensuring sufficient shoot Mn concentrations. The preference of IRT1 for Mn instead of Fe is discussed in an evolutionary context., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: Manganese (Mn) has several essential functions in plants, including a role as cofactor in the oxygen evolving complex (OEC) of photosystem II (PSII). Manganese deficiency is a major plant nutritional disorder in winter cereals resulting in significant yield reductions and winter kill in more severe cases. Among the winter cereals, genotypes of winter barley are known to differ considerably in tolerance to Mn deficiency, but the genes controlling the Mn deficiency trait remains elusive., Results: Experiments were conducted using 248 barley varieties, cultivated in six distinct environments prone to induce Mn deficiency. High-throughput phenotyping for Mn deficiency was performed by chlorophyll a (Chl a) fluorescence analysis to quantify the quantum yield efficiency of PSII. High-throughput phenotyping in combination with ICP-OES based multi-element analyses allowed detection of marker-trait associations by genome wide association (GWA) mapping. Several key candidate genes were identified, including PSII subunit proteins, germin like proteins and Mn superoxide dismutase. The putative roles of the encoded proteins in Mn dependent metabolic processes are discussed., Conclusions: Fifty-four candidate genes were identified by Chl a fluorescence phenotyping and association genetics. Tolerance of plants to Mn deficiency, which is referred to as Mn efficiency, appeared to be a complex trait involving many genes. Moreover, the trait appeared to be highly dependent on the environmental conditions in field. This study provides the basis for an improved understanding of the parameters influencing Mn efficiency and is valuable in future plant breeding aiming at producing new varieties with improved tolerance to cultivation in soil prone to induce Mn deficiency.

Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

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.)

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals., (© 2013 Australian Centre for Plant Functional Genomics New Phytologist © 2013 New Phytologist Trust.)

The ammonia compensation point ( ) controls the direction and magnitude of NH3 exchange between plant leaves and the atmosphere. Very limited information is currently available on how responds to anticipated climate changes. Young barley plants were grown for 2 weeks at ambient (400 μmol mol(-1)) or elevated (800 μmol mol(-1)) CO2 concentration with or NH4NO3 as the nitrogen source. The concentrations of and H(+) in the leaf apoplastic solution were measured along with different foliar N pools and enzymes involved in N metabolism. Elevated CO2 caused a threefold decrease in the concentration in the apoplastic solution and slightly acidified it. This resulted in a decline of the from 2.25 and 2.95 nmol mol(-1) under ambient CO2 to 0.37 and 0.89 nmol mol(-1) at elevated CO2 in the and NH4NO3 treatments, respectively. The decrease in at elevated CO2 reflected a lower N concentration (-25%) in the shoot dry matter. The activity of nitrate reductase also declined (-45 to -60%), while that of glutamine synthetase was unaffected by elevated CO2. It is concluded that elevated CO2 increases the likelihood of plants being a sink for atmospheric NH3 and reduces episodes of NH3 emission from plants.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.

Heavy metal transporters belonging to the P(1B)-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P(1B)-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.

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., (© 2011 Blackwell Publishing Ltd.)

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+.

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.

Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn(2+) transport in crop species. To expand the knowledge on Zn(2+) transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Deltazrt1/Deltazrt2, defective in Zn(2+) uptake. This strategy resulted in isolation and identification of three new Zn(2+) transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Deltazrt1/Deltazrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe(2+) and Mn(2+) uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe(2+) and Mn(2+) limited media, respectively, indicating a specific role in Zn(2+) transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in -Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn(2+) transporters involved in Zn(2+) homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.

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.

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 Mn(2+) availability. Differential genotypic Mn efficiency can be attributed to differences in Mn(2+) 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 Mn(2+). 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 Mn(2+), also transported Fe(2+)/Fe(3+), Zn(2+), and Cd(2+). 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 Mn(2+) uptake rate. We conclude that HvIRT1 is an important component controlling Mn(2+) uptake in barley roots and contributes to genotypic differences in Mn(2+) uptake kinetics.

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 V(max) than the inefficient Antonia, but similar K(m) 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.