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Background: The amino acid profile of plants is an important parameter in assessments of their growth potential, resource-use efficiency and/or quality as food and feed. Screening studies may involve large number of samples but the classical amino acid analysis is limited by the fact that it is very time consuming with typical chromatographic run times of 70 min or more. Results: We have here developed a high-throughput method for analysis of amino acid profiles in plant materials. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection by a single quadrupole (QDa) mass spectrometer. The chromatographic run time is reduced to 10 min and the precision, accuracy and sensitivity of the method are in line with other recent methods utilizing advanced and more expensive mass spectrometers. The sensitivity of the method is at least a factor 10 better than that of methods relying on detection by fluorescence or UV. It is possible to downscale sample size to 20 mg without compromising reproducibility, which makes the method ideal for analysis of very small sample amounts. Conclusion: The developed method allows high-throughput analysis of amino acid profiles in plant materials. The analysis is robust and accurate as well as compatible with both free amino acids and protein hydrolysates. The QDa detector offers high sensitivity and accuracy, while at the same time being relatively simple to operate and cheap to purchase, thus significantly reducing the overall analytical costs compared to methods based on more advanced mass spectrometers.

Silicon (Si) has many beneficial effects in plants, especially for the survival from biotic and abiotic stresses. However, Si may negatively affect the quality of lignocellulosic biomass for bioenergy purposes. Despite many studies, the regulation of Si distribution and deposition in plants remains to be fully understood. Here, we have identified the Brachypodium distachyon mutant low-silicon 1 (Bdlsi1-1), with impaired channeling function of the Si influx transporter BdLSI1, resulting in a substantial reduction of Si in shoots. Bioimaging by laser ablation-inductively coupled plasma-mass spectrometry showed that the wild-type plants deposited Si mainly in the bracts, awns and leaf macrohairs. The Bdlsi1-1 mutants showed substantial (>90%) reduction of Si in the mature shoots. The Bdlsi1-1 leaves had fewer, shorter macrohairs, but the overall pattern of Si distribution in bracts and leaf tissues was similar to that in the wild-type. The Bdlsi1-1 plants supplied with Si had significantly lower seed weights, compared to the wild-type. In low-Si media, the seed weight of wild-type plants was similar to that of Bdlsi1-1 mutants supplied with Si, while the Bdlsi1-1 seed weight decreased further. We conclude that Si deficiency results in widespread alterations in leaf surface morphology and seed formation in Brachypodium, showing the importance of Si for successful development in grasses.

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.

A novel zinc (Zn) fertilizer concept based on Zn doped layered double hydroxides (Zn-doped Mg-Fe-LDHs) has been investigated. Zn-doped Mg-Fe-LDHs were synthetized, their chemical composition was analyzed and their nutrient release was studied in buffered solutions with different pH values. Uptake of Zn by barley (Hordeum vulgare cv. Antonia) was evaluated in short- (8 weeks), medium- (11 weeks) and long-term (28 weeks) experiments in quartz sand and in a calcareous soil enriched with Zn-doped Mg-Fe-LDHs. The Zn release rate of the Zn-doped Mg-Fe-LDHs was described by a first-order kinetics equation showing maximum release at pH 5.2, reaching approximately 45% of the total Zn content. The Zn concentrations in the plants receiving the LDHs were between 2- and 9.5-fold higher than those in plants without Zn addition. A positive effect of the LDHs was also found in soil. This work documents the long-term Zn release capacity of LDHs complying with a release-on-demand behavior and serves as proof-of-concept that Zn-doped Mg-Fe-LDHs can be used as Zn fertilizers.

Basic leucine zipper (bZIP) transcription factors control important developmental and physiological processes in plants. In Arabidopsis thaliana, the three gene F-bZIP subfamily has been associated with zinc deficiency and salt stress response. Benefiting from the present abundance of plant genomic data, we performed an evolutionary and structural characterization of plant F-bZIPs. We observed divergence during seed plant evolution, into two groups and inferred different selective pressures for each. Group 1 contains AtbZIP19 and AtbZIP23 and appears more conserved, whereas Group 2, containing AtbZIP24, is more prone to gene loss and expansion events. Transcriptomic and experimental data reinforced AtbZIP19/23 as pivotal regulators of the zinc deficiency response, mostly via the activation of genes from the ZIP metal transporter family, and revealed that they are the main regulatory switch of AtZIP4. A survey of AtZIP4 orthologs promoters across different plant taxa revealed an enrichment of the Zinc Deficiency Response Element (ZDRE) to which both AtbZIP19/23 bind. Overall, our results indicate that while the AtbZIP24 function in the regulation of the salt stress response may be the result of neo-functionalization, the AtbZIP19/23 function in the regulation of the zinc deficiency response may be conserved in land plants (Embryophytes).

Wheat is one of the most important crops for food and feed and its straw is a potential feedstock for biorefinery purposes. Nitrogen (N) is an essential input factor in wheat agriculture but no information is available on how it affects straw composition during maturation and at harvest. To investigate this, we conducted a large scale field experiment in which wheat plants were cultivated at three levels of externally applied N. The plants were harvested at different stages of maturation, spanning green straw at heading (ear emergence) to fully yellow straw at final maturity. Defined parts of the straw were analyzed for cell wall characteristics relevant for further biomass processing. The straw N concentration corroborated with the level of N input, but the yield of straw biomass was not largely affected. High N treatment modified cell wall composition, namely increased abundance of arabinogalactan proteins (AGPs) and lignin in the mature straw. A general decrease in pectin methylesterification as well as in ferulate linkages was also observed. Importantly, no significant changes in crystalline cellulose and silicon concentration or in saccharification efficiency were observed among the different N treatments. Nitrogen fertilization partially alters the cell wall composition in wheat straw but is not a limiting factor in wheat biomass refinery.

A catalytic manganese (Mn) cluster is required for the oxidation of water in the oxygen-evolving complex (OEC) of photosystem II (PSII) in plants. Despite this essential role of Mn in generating the electrons driving photosynthesis, limited information is available on how Mn deficiency affects PSII functionality. We have here used parameters derived from measurements of fluorescence induction kinetics (OJIP transients), non-photochemical quenching (NPQ) and PSII subunit composition to investigate how latent Mn deficiency changes the photochemistry in two barley genotypes differing in Mn efficiency. Mn deficiency caused dramatic reductions in the quantum yield of PSII and led to the appearance of two new inflection points, the K step and the D dip, in the OJIP fluorescence transients, indicating severe damage to the OEC. In addition, Mn deficiency decreased the ability to induce NPQ in the light, rendering the plants incapable of dissipating excess energy in a controlled way. Thus, the Mn deficient plants became severely affected in their ability to recover from high light-induced photoinhibition, especially under strong Mn deficiency. Interestingly, the Mn-efficient genotype was able to maintain a higher NPQ than the Mn-inefficient genotype when exposed to mild Mn deficiency. However, during severe Mn deficiency, there were no differences between the two genotypes, suggesting a general loss of the ability to disassemble and repair PSII. The pronounced defects of PSII activity were supported by a dramatic decrease in the abundance of the OEC protein subunits, PsbP and PsbQ in response to Mn deficiency for both genotypes. We conclude that regulation of photosynthetic performance by means of maintaining and inducing NPQ mechanisms contribute to genotypic differences in the Mn efficiency of barley genotypes growing under conditions with mild Mn deficiency.

Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence.

Low concentration of zinc (Zn) in the endosperm of cereals is a major factor contributing to Zn deficiency in human populations. We have investigated how combined Zn and nitrogen (N) fertilization affects the speciation and localization of Zn in durum wheat (Triticum durum). Zn-binding proteins were analysed with liquid chromatography ICP-MS and Orbitrap MS(2) , respectively. Laser ablation ICP-MS with simultaneous Zn, sulphur (S) and phosphorus (P) detection was used for bioimaging of Zn and its potential ligands. Increasing the Zn and N supply had a major impact on the Zn concentration in the endosperm, reaching concentrations higher than current breeding targets. The S concentration also increased, but S was only partly co-localized with Zn. The mutual Zn and S enrichment was reflected in substantially more Zn bound to small cysteine-rich proteins (apparent size 10-30 kDa), whereas the response of larger proteins (apparent size > 50 kDa) was only modest. Most of the Zn-responsive proteins were associated with redox- and stress-related processes. This study offers a methodological platform to deepen the understanding of processes behind endosperm Zn enrichment. Novel information is provided on how the localization and speciation of Zn is modified during Zn biofortification of grains.

Better understanding of root function is central for the development of plants with more efficient nutrient uptake and translocation. We here present a method for multielement bioimaging at the cellular level in roots of the genetic model system Arabidopsis (Arabidopsis thaliana). Using conventional protocols for microscopy, we observed that diffusible ions such as potassium and sodium were lost during sample dehydration. Thus, we developed a protocol that preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, cryo-sectioned, and freeze dried. Samples are finally analyzed by laser ablation-inductively coupled plasma-mass spectrometry, 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 Arabidopsis unable to synthesize the metal chelator nicotianamine. The mutant accumulated substantially more zinc and manganese than the wild type in the tissues surrounding the vascular cylinder. For iron, the images looked completely different, with iron bound mainly in the epidermis of the wild-type plants but confined to the cortical cell walls of the mutant. The method offers the power of inductively coupled plasma-mass spectrometry to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to Arabidopsis, 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.

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.

Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of the high-affinity low-capacity isoform Gln-1;1 was reduced. The expression of Gln-1;3 did not respond to ammonium treatment while Gln-1;4 and Gln-1;5 isogenes in all cases were expressed at a very low level. Gln-2 was highly expressed in shoots but only at a very low level in roots. To investigate the specific functions of the two isogenes Gln-1;1 and Gln-1;2 in shoots for ammonium detoxification, single and double knock-out mutants were grown under standard N supply or with high ammonium provision. Phenotypes of the single mutant gln1;1 were similar to the wild type, while growth of the gln1;2 single mutant and the gln1;1:gln1;2 double mutant was significantly impaired irrespective of N regime. GS1 activity was significantly reduced in both gln1;2 and gln1;1:gln1;2 Along with this, the ammonium content increased while that of Gln decreased, showing that Gln-1;2 was essential for ammonium assimilation and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots.

The aim of the current paper is to discuss the sustainability aspect of biorefinery systems with focus on biomass supply chains, processing of biomass feedstocks in biorefinery platforms and sustainability assessment methodologies. From the stand point of sustainability, it is important to optimize the agricultural production system and minimize the related environmental impacts at the farming system level. These impacts are primarily related to agri-chemical inputs and the related undesired environmental emissions and to the repercussions from biomass production. At the same time, the biorefineries need a year-round supply of biomass and about 40-60% of the total operating cost of a typical biore finery is related to the feedstocks chosen, and thus highlights on the careful prioritization of feedstocks mainly based on their economic and environmental loadings. Regarding the processing in biorefinery platforms, chemical composition of biomasses is important. Biomasses with higher concentrations of cellulose and hemicelluloses compared to lignin are preferred for bioethanol production in the lignocellulosic biorefinery, since the biodegradability of cellulose is higher than lignin. A green biorefinery platform enables the extraction of protein from grasses, producing an important alternative to importing protein sources for food products and animal feed, while also allowing processing of residues to deliver bioethanol. Currently, there are several approaches to integrate biorefinery platforms, which are aimed to enhance their economic and environmental sustainability. Regarding sustainability assessment, the complexities related to the material flows in a biorefinery and the delivery of alternative biobased products means dealing with multiple indicators in the decision-making process to enable comparisons of alternatives. Life Cycle Assessment is regarded as one of the most relevant tools to assess the environmental hotspots in the biomass supply chains, at proce

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.

With more than 60% of the land farmed, with vulnerable freshwater and marine environments, and with one of the most intensive, export-oriented livestock sectors in the world, the nitrogen (N) pollution pressure from Danish agriculture is severe. Consequently, a series of policy action plans have been implemented since the mid 1980s with significant effects on the surplus, efficiency and environmental loadings of N. This paper reviews the policies and actions taken and their ability to mitigate effects of reactive N (Nr) while maintaining agricultural production. In summary, the average N-surplus has been reduced from approximately 170 kg N ha-1 yr-1 to below 100 kg N ha-1 yr-1 during the past 30 yrs, while the overall N-efficiency for the agricultural sector (crop + livestock farming) has increased from around 20-30% to 40-45%, the N-leaching from the field root zone has been halved, and N losses to the aquatic and atmospheric environment have been significantly reduced. This has been achieved through a combination of approaches and measures (ranging from command and control legislation, over market-based regulation and governmental expenditure to information and voluntary action), with specific measures addressing the whole N cascade, in order to improve the quality of ground- and surface waters, and to reduce the deposition to terrestrial natural ecosystems. However, there is still a major challenge in complying with the EU Water Framework and Habitats Directives, calling for new approaches, measures and technologies to mitigate agricultural N losses and control N flows.

Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude that GS1 activity may be downregulated via a chain of processes elicited by metabolic imbalances and environmental constraints. We suggest that a pivotal role of GS1 may be related to the maintenance of essential nitrogen (N) flows and internal N sensing during critical stages of plant development. A number of more refined overexpression strategies exploiting gene stacking combined with tissue and cell specific targeting to overcome metabolic bottlenecks are considered along with their potential in relation to new N management strategies.

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., 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.