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Many studies have been devoted to investigation of toxic benzo(a)pyrene (BaP) compound, but studies involving changes at the cellular level are insufficient to understand the mechanisms of polycyclic aromatic hydrocarbons (PAHs) effect on plants. To study the toxicity of BaP, a model vegetation experiment was conducted on cultivation of spring barley (Hordeum sativum distichum) on artificially polluted BaP soil at different concentrations. The article discusses the intake of BaP from the soil into the plant and its effect on the organismic and cellular levels of plant organization. The BaP content in the organs of spring barley was determined by the method of saponification. With an increase in the concentration of BaP in the soil, its content in plants also rises, which leads to inhibition of growth processes. The BaP content in the green part of Hordeum sativum increased from 0.3 µg kg -1 in control soil up to 2.6 µg kg -1 and 16.8 µg kg -1 under 20 and 400 ng/g BaP applying in soil, as well as in roots: 0.9 µg kg -1 , 7.7 µg kg -1 , 42.8 µg kg -1 , respectively. Using light and electron microscopy, changes in the tissues and cells of plants were found and it was established that accumulation of BaP in plant tissues caused varying degrees of ultrastructural damage depending on the concentration of pollutant. BaP had the greatest effect on the root, significant changes were found in it both at histological and cytological levels, while changes in the leaves were observed only at the cytological level. The results provide significant information about the mechanism of action of BaP on agricultural plants.

Nowadays, nanotechnology is one of the most dynamically developing and most promising technologies. However, the safety issues of using metal nanoparticles, their environmental impact on soil and plants are poorly understood. These studies are especially important in terms of copper-based nanomaterials because they are widely used in agriculture. Concerning that, it is important to study the mechanism behind the mode of CuO nanoparticles action at the ultrastructural intracellular level. It is established that the contamination with CuO has had a negative influence on the development of spring barley. A greater toxic effect has been exerted by the introduction of CuO nanoparticles as compared to the macrodispersed form. A comparative analysis of the toxic effects of copper oxides and nano-oxides on plants has shown changes in the tissue and intracellular levels in the barley roots. However, qualitative changes in plant leaves have not practically been observed. In general, conclusions can be made that copper oxide in nano-dispersed form penetrates better from the soil into the plant and can accumulate in large quantities in it.

Naked barley grains were germinated at 25 °C for 12, 24, and 36 h, followed by hot air and infrared drying. Changes in structural, physicochemical, and functional properties of naked barley flour and starch after germination and drying were evaluated. Germinated and dried flour showed loose structure in starch granules surface. Germination and drying enhanced bulk density and water solubility of flour, while oil absorption capacity decreased. Treated barley starch showed greater water holding capacity, viscosity, and gelatinization temperature than those of native ones, and the maximum increase in water holding capacity of germinated barley was 20.25% of the native ones. Moreover, molecular weight, amylopectin long chains, and crystallinity of treated starch decreased, and the maximum decrease in the crystallinity of germinated barley was 78.36% of the native ones. Generally, germination for 24 and 36 h by infrared drying induced significant changes of flour and starch compared with hot air drying ones. Therefore, the combination of germination and infrared drying can be suggested as a promising method for modifying the properties of naked barley flour and starch to promote their application in the food industry., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β-glucan depositions on the inside. During germination, arabinoxylan and β-glucan are hydrolysed, but unlike β-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Effects of Cu toxicity from contaminated soil were analysed in spring barley (Hordeum sativum distichum), a widely cultivated species in South Russia. In this study, H. sativum was planted outdoors in one of the most fertile soils-Haplic Chernozem spiked with high concentration of Cu and examined between the boot and head emergence phase of growth. Copper toxicity was observed to cause slow ontogenetic development of plants, changing their morphometric parameters (shape, size, colour). To the best of our knowledge, the ultrastructural changes in roots, stems and leaves of H. sativum induced by excess Cu were fully characterized for the first time using transmission electron microscopy. The plant roots were the most effected, showing degradation of the epidermis, reduced number of parenchyma cells, as well as a significant decrease in the diameter of the stele and a disruption and modification to its cell structure. The comparative analysis of the ultrastructure of control plants and plants exposed to the toxic effects of Cu has made it possible to reveal significant disruption of the integrity of the cell wall and cytoplasmic membranes in the root with deposition of electron-dense material. The changes in the ultrastructure of the main cytoplasmic organelles-endoplasmic reticulum, mitochondria, chloroplasts and peroxisomes-in the stem and leaves were found. The cellular Cu deposition, anatomical and ultrastructural modifications could mainly account for the primary impact points of metal toxicity. Therefore, this work extends the available knowledge of the mechanisms of the Cu effect tolerance of barley.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid β-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley ( Hordeum vulgare ) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7 ) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvCMF7 is a homolog of the Arabidopsis ( Arabidopsis thaliana ) CONSTANS, CO-like, and TOC1 ( CCT ) Motif transcription factor gene CHLOROPLAST IMPORT APPARATUS2 , which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization., (© 2019 American Society of Plant Biologists. All rights reserved.)

Aluminum (Al) is one of the most important crust elements causing reduced plant production in acidic soils. Barley ( Hordeum vulgare L.) is considered to be one of the crops that is most sensitive to Al, and the root cell wall is the primary target of Al toxicity. In this study, we evaluate the possible involvement of specific pectic epitopes in the cells of barley roots in response to aluminum exposure. We targeted four different pectic epitopes recognized by LM5, LM6, LM19, and LM20 antibodies using an immunocytochemical approach. Since Al becomes available and toxic to plants in acidic soils, we performed our analyses on barley roots that had been grown in acidic conditions (pH 4.0) with and without Al and in control conditions (pH 6.0). Differences connected with the presence and distribution of the pectic epitopes between the control and Al-treated roots were observed. In the Al-treated roots, pectins with galactan sidechains were detected with a visually lower fluorescence intensity than in the control roots while pectins with arabinan sidechains were abundantly present. Furthermore, esterified homogalacturonans (HGs) were present with a visually higher fluorescence intensity compared to the control, while methyl-esterified HGs were present in a similar amount. Based on the presented results, it was concluded that methyl-esterified HG can be a marker for newly arising cell walls. Additionally, histological changes were detected in the roots grown under Al exposure. Among them, an increase in root diameter, shortening of root cap, and increase in the size of rhizodermal cells and divisions of exodermal and cortex cells were observed. The presented data extend upon the knowledge on the chemical composition of the cell wall of barley root cells under stress conditions. The response of cells to Al can be expressed by the specific distribution of pectins in the cell wall and, thus, enables the knowledge on Al toxicity to be extended by explaining the mechanism by which Al inhibits root elongation.

Asexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.

Hordeum species develop a central spikelet flanked by two lateral spikelets at each inflorescence node. In 'two-rowed' spikes, the central spikelet alone is fertile and sets grain, while in 'six-rowed' spikes, lateral spikelets can also produce grain. Induced loss-of-function alleles of any of five Six-rowed spike (VRS) genes (VRS1-5) cause complete to intermediate gains of lateral spikelet fertility. Current six-row cultivars contain natural defective vrs1 and vrs5 alleles. Little information is known about VRS mechanism(s). We used comparative developmental, expression and genetic analyses on single and double vrs mutants to learn more about how VRS genes control development and assess their agronomic potential. We show that all VRS genes repress fertility at carpel and awn emergence in developing lateral spikelets. VRS4, VRS3 and VRS5 work through VRS1 to suppress fertility, probably by inducing VRS1 expression. Pairing vrs3, vrs4 or vrs5 alleles increased lateral spikelet fertility, despite the presence of a functional VRS1 allele. The vrs3 allele caused loss of spikelet identity and determinacy, improved grain homogeneity and increased tillering in a vrs4 background, while with vrs5, decreased tiller number and increased grain weight. Interactions amongst VRS genes control spikelet infertility, determinacy and outgrowth, and novel routes to improving six-row grain., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful information for our understanding of this detailed structure. It is evident that new technologies together with improved sample preparations and image analyses should be adequately combined. This mini review highlights 3D imaging used for chromosome analysis so far with future imaging directions also highlighted.

Microscopic investigations of biological objects are an integral part in plant research and most fields of life sciences. They allow the description of morphological, histological, and structural aspects of individual cells or tissues. Based on various cell biological tools and methods it is possible to characterize different plant genotypes or study their adaptation to changing environmental conditions. In combination with antibodies raised against specific antigens and epitopes light and electron microscopy enable investigation of the function of single genes/proteins in plant growth and development or their role related to abiotic or biotic stresses.Here, we describe sample preparation of barley roots for cell biological investigations using light and electron microscopy, to characterize morphological, structural, and functional aspects on root sections and the root surface.

The main objective of this study was to assess the uptake and translocation of MnFe 2 O 4 magnetic nanoparticles (MNPs) in hydroponically grown barley (Hordeum vulgare L.). Hydrothermally synthesized and well characterized MNPs (average crystallite size of 14.5 ± 0.5 nm) with varied doses (62.5, 125, 250, 500, and 1000 mg L -1 ) were subjected to the plants at germination and early growing stages (three weeks). The tissues analyzed by vibrating-sample magnetometer (VSM) and transmission electron microscopy (TEM) revealed the uptake and translocation of MNPs, as well as their internalization in the leaf cells. Also, elemental analysis proved that manganese (Mn) and iron (Fe) contents were ∼7-9 times and ∼4-7 times higher in the leaves of MNPs-treated plants than the ones for non-treated control, respectively. 250 mg L -1 of MNPs significantly (at least p < 0.05) promoted the fresh weight (FW, %10.25). However, higher concentrations (500 and 1000 mg L -1 ) remarkably reduced the increase to %8 and %5, respectively, possibly due to the restricted water uptake. Also, catalase activity was increased from 91 (μM H 2 O 2 min -1 mg -1 ) to 138 in leaves, and decreased to 66 in roots upon 1000 mg L -1 of MNPs application. Chlorophyll and carotenoid contents were not significantly changed, except chlorophyll a (%6 increase at 1000 mg L -1 , p < 0.05). Overall, MnFe 2 O 4 NPs were up-taken from the roots and migrated to the leaves which promoted the growth parameters of barley. Hence, MNPs can be suggested for barley breeding programs and can be proposed as effective delivery system for agrochemicals. However, the possible negative effect of MNPs due to its potential horizontal transfer from plants to animals via the food chain must be also considered., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH 4 + remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles-nucleus and mitochondria -became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes., (© 2018 American Society of Plant Biologists. All rights reserved.)

Tritrophic interactions involving a biocontrol agent, a pathogen and a plant have been analyzed predominantly from the perspective of the biocontrol agent. We have conducted the first comprehensive transcriptomic analysis of all three organisms in an effort to understand the elusive properties of Pseudozyma flocculosa in the context of its biocontrol activity against Blumeria graminis f.sp. hordei as it parasitizes Hordeum vulgare. After inoculation of P. flocculosa, the tripartite interaction was monitored over time and samples collected for scanning electron microscopy and RNA sequencing. Based on our observations, P. flocculosa indirectly parasitizes barley, albeit transiently, by diverting nutrients extracted by B. graminis from barley leaves through a process involving unique effectors. This brings novel evidence that such molecules can also influence fungal-fungal interactions. Their release is synchronized with a higher expression of powdery mildew haustorial effectors, a sharp decline in the photosynthetic machinery of barley and a developmental peak in P. flocculosa. The interaction culminates with a collapse of B. graminis haustoria, thereby stopping P. flocculosa growth, as barley plants show higher metabolic activity. To conclude, our study has uncovered a complex and intricate phenomenon, described here as hyperbiotrophy, only achievable through the conjugated action of the three protagonists., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: While most water loss from leaf surfaces occurs via stomata, part of this loss also occurs through the leaf cuticle, even when the stomata are fully closed. This component, termed residual transpiration, dominates during the night and also becomes critical under stress conditions such as drought or salinity. Reducing residual transpiration might therefore be a potentially useful mechanism for improving plant performance when water availability is reduced (e.g. under saline or drought stress conditions). One way of reducing residual transpiration may be via increased accumulation of waxes on the surface of leaf. Residual transpiration and wax constituents may vary with leaf age and position as well as between genotypes. This study used barley genotypes contrasting in salinity stress tolerance to evaluate the contribution of residual transpiration to the overall salt tolerance, and also investigated what role cuticular waxes play in this process. Leaves of three different positions (old, intermediate and young) were used., Results: Our results show that residual transpiration was higher in old leaves than the young flag leaves, correlated negatively with the osmolality, and was positively associated with the osmotic and leaf water potentials. Salt tolerant varieties transpired more water than the sensitive variety under normal growth conditions. Cuticular waxes on barley leaves were dominated by primary alcohols (84.7-86.9%) and also included aldehydes (8.90-10.1%), n-alkanes (1.31-1.77%), benzoate esters (0.44-0.52%), phytol related compounds (0.22-0.53%), fatty acid methyl esters (0.14-0.33%), β-diketones (0.07-0.23%) and alkylresorcinols (1.65-3.58%). A significant negative correlation was found between residual transpiration and total wax content, and residual transpiration correlated significantly with the amount of primary alcohols., Conclusions: Both leaf osmolality and the amount of total cuticular wax are involved in controlling cuticular water loss from barley leaves under well irrigated conditions. A significant and negative relationship between the amount of primary alcohols and a residual transpiration implies that some cuticular wax constituents act as a water barrier on plant leaf surface and thus contribute to salinity stress tolerance. It is suggested that residual transpiration could be a fundamental mechanism by which plants optimize water use efficiency under stress conditions.

Fusarium crown rot (FCR), caused by various Fusarium species, is a chronic disease of cereals in many semi-arid regions worldwide. To clarify what effects drought-stress may have on FCR development, visual assessment, histological analysis and quantitative PCR were used to analyse the infection process of F. pseudograminearum in barley. This study observed for the first time that the severity of FCR symptom reflects the quantity of pathogens in infected tissues of barley under both drought-stressed and well-watered conditions. Drought-stress prolongs the initial infection phase but enhances the proliferation and spread of Fusarium pathogens after the initial infection phase. Under drought-stressed conditions, the invading hyphae were frequently observed to re-emerge from stomata and invade again the surrounding epidermis cells. Under the well-watered conditions, however, very few hyphae re-emerged from stomata and most infection was caused by hyphae intracellularly grown. It was also observed that drought-stress increased the length and density of trichomes dramatically especially in the susceptible genotypes, and that the length and density of trichomes were positively related to fungal biomass of F. pseudograminearum in plants., Competing Interests: The authors have declared that no competing interests exist.

Rice false smut, caused by the fungal pathogen Villosiclava virens, is one of the most important rice diseases in the world. Previous studies reported that the pathogen has less number of cell wall-degraded genes and attacks dominantly rice stamen filaments and extends intercellularly. To reveal why the fungus infects plant stamen filaments, inoculation test on barley was carried out with the similar protocol to rice. The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly, usually resulting in severe damage of the stamen filament tissues. It also attacked young barley lodicules and grew intercellularly by chance. The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps. Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent. The cell walls were very soft and flexible, and often folded. However, V. virens extended dominantly in the noncellulose regions and seemed never to degrade microfibrils in barley and rice cell walls. This suggested that the unique structures of rice and barley stamen filaments should be fit for their function of elongation in anthesis, and also endow with the susceptibility to the fungus, V. virens., (© 2016 Wiley Periodicals, Inc.)

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. 'Compositum-Barley' and tetraploid 'Miracle-Wheat' (T. turgidum convar. compositum (L.f.) Filat.) display noncanonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation 'Miracle-Wheat' produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in 'Compositum-Barley,' i.e., compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched head(t) (bh(t)) locus regulating spike branching in tetraploid 'Miracle-Wheat.' Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bh(t) locus in a collection of mutant and wild-type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of 'Miracle-Wheat.' mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative downstream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat's yield potential., (Copyright © 2015 by the Genetics Society of America.)

The aim of this study was to determine the impact of lead (Pb) stress as 0.6mM Pb(NO3)2 on the content of free, thylakoid- and chromatin-bound polyamines (PAs) and diamine oxidase (DAO) activity in detached greening barley leaves. Additionally, photosynthetic-related parameters, generation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content and ultrastructural changes under Pb-stress were studied. The level of putrescine (Put) was reduced progressively to 56% at 24h of Pb stress, and it was correlated with 38% increase of DAO activity. Spermidine (Spd) content was not affected by Pb-stress, while the free spermine (Spm) level significantly increased by about 83% at 6h, and in that time the lowest level of H2O2 was observed. The exogenous applied Spm to Pb-treated leaves caused a decrease in the content of H2O2. In greening leaves exposed to Pb an accumulation of chlorophylls a and b was inhibited by about 39 and 47%, respectively, and photosynthetic parameters of efficiency of electron transport and photochemical reaction in chloroplasts as ΦPSII, ETR and RFd were lowered by about 23-32%. The level of thylakoid-bound Put decreased by about 22%. Moreover, thylakoids isolated from chloroplasts of Pb-treated leaves were characterized with lower Put/Spm ratio as compared to control leaves. In the presence of Pb the significant decrease in the number of thylakoids per granum and cap-shape invaginations of cytoplasmic material were noticed. In Pb-stressed leaves the level of chromatin-bound Spm increased by about 48% and sometimes condensed chromatin in nuclei was observed. We conclude that in greening barley leaves exposed to Pb-stress changes in free, thylakoid- and chromatin-bound PAs play some role in the functioning of leaves or plants in heavy metal stress conditions., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD)., Results: Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers., Conclusions: Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA., (© 2014 Society of Chemical Industry.)

Programmed cell death is a key feature of epidermal plant immunity, which is particularly effective against biotrophic microbes that depend on living host tissue. The covered smut fungus Ustilago hordei establishes a compatible biotrophic interaction with its host plant barley. The maize smut U. maydis triggers a nonhost response in barley, which results in epidermal cell death. Similarly, Ustilago mutants being deleted for pep1, a gene encoding a secreted effector, are blocked upon host penetration. We studied the epidermal responses of barley to incompatible Ustilago strains. Molecular and cellular analyses were used to test the impact of Bax inhibitor-1 (BI-1), a suppressor of programmed cell death, on the barley nonhost resistance to U. maydis as well as Ustilago Δpep1 mutants. Overexpression of BI-1 resulted in partial break of barley nonhost resistance to U. maydis. By contrast, the epidermal cell death response triggered by pep1 deletion mutants was not impaired by BI-1. Hypersensitive-response-like cell death caused by U. maydis wild-type infection showed features of necrotic cell death, while Δpep1 mutant-induced host responses involved hallmarks of autophagy. Therefore, we propose that the mechanisms of epidermal cell death in response to different types of incompatible pathogens depend on spatial and temporal appearance of cell-death-triggering stimuli.

Key Message: The recessive labile locus mapped on chromosome 5HL causes irregular spikelet fertility and controls floret development as well as row-type in barley. The labile-barley displays a variable number of fertile spikelets at each rachis internode (0-3 fertile spikelets/rachis internode) which is intermediate between that observed in two- or six-rowed types. Previous re-sequencing of Vrs1 in 219 labile-barley (Hordeum vulgare L. convar. labile) accessions showed that all carried a six-rowed specific allele. We therefore hypothesized that this seemingly random reduction in spikelet fertility is most likely caused by the labile (lab) locus, which we aimed to phenotypically and genetically define. Here, we report a detailed phenotypic analysis of spikelet fertility in labile-barleys in comparison to two- and six-rowed genotypes using scanning electron microscopy analysis. We found that the first visible morphological deviation occurred during the stamen primordium stage, when we regularly observed the appearance of arrested central floral primordia in labile but not in two- or six-rowed barleys. At late stamen and early awn primordium stages, lateral florets in two-rowed and only some in labile-barley showed retarded development and reduction in size compared with fully fertile lateral florets in six-rowed barley. We used two F2 mapping populations to generate whole genome genetic linkage maps and ultimately locate the lab locus as a recessive Mendelian trait to a 4.5-5.8 cM interval at approximately 80 cM on chromosome 5HL. Our results will help identifying the role of the lab gene in relation to other spikelet fertility factors in barley.

Aluminum (Al) toxicity is a major limiting factor for plant production in acid soils. Wild barley germplasm is a treasure trove of useful genes and offers rich sources of genetic variation for crop improvement. Al-stress-hydroponic-experiments were performed, and the physiochemical characteristic of two contrasting Tibetan wild barley genotypes (Al-resistant XZ16 and Al-sensitive XZ61) and Al-resistant cv. Dayton were compared. Ultrastructure of chloroplasts and root cells in XZ16 was less injured than that in Dayton and XZ61. Moreover, XZ16 secreted significantly more malate besides citrate and exhibited less Al uptake and distribution than both of XZ61 and Dayton in response to Al stress, simultaneously maintained higher H⁺-, Ca²⁺Mg²⁺- and total-ATPase activities over XZ61. The protein synthesis inhibitor cycloheximide reduced citrate secretion from XZ16, but not from Dayton. In Tibetan wild barley, our findings highlight the significant correlations between Al tolerance, ATPase activity and citrate secretion, providing some insights into the physiological basis for Al-detoxification., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Breeding for resistance is a key task to control Fusarium head blight (FHB), a devastating disease of small cereals leading to economic losses and grain contamination with mycotoxins harmful for humans and animals. In the present work, FHB resistance of the six-rowed spring barley 'Chevron' to FHB in Germany was compared with those of adapted German spring barley cultivars. Both under natural infection conditions and after spray inoculation with conidia of Fusarium culmorum, F. sporotrichioides, and F. avenaceum under field conditions, Chevron showed a high level of quantitative resistance to the infection and contamination of grain with diverse mycotoxins. This indicates that Chevron is not only a little susceptible to deoxynivalenol-producing Fusarium spp. but also to Fusarium spp. producing type A trichothecenes and enniatins. Monitoring the initial infection course of F. culmorum on barley lemma tissue by confocal laser-scanning microscopy provided evidence that FHB resistance of Chevron is partially mediated by a preformed penetration resistance, because direct penetration of floral tissue by F. culmorum was observed rarely on Chevron but was common on susceptible genotypes. Alternatively, F. culmorum penetrated Chevron lemma tissue via stomata, which was unusual for susceptible genotypes. We generated double-haploid barley populations segregating for the major FHB resistance quantitative trait loci (QTL) Qrgz-2H-8 of Chevron. Subsequently, we characterized these populations by spray inoculation with conidia of F. culmorum and F. sporotrichioides. This suggested that Qrgz-2H-8 was functional in the genetic background of European elite barley cultivars. However, the degree of achieved resistance was very low when compared with quantitative resistance of the QTL donor Chevron, and the introgression of Qrgz-2H-8 was not sufficient to mediate the cellular resistance phenotype of Chevron in the European backgrounds.

A hydroponic experiment was carried out to examine the effect of hydrogen sulfide (H2 S) in alleviating chromium (Cr) stress in barley. A 2-factorial design with 6 replications was selected, including 3 levels of NaHS (0 μM, 100 μM, and 200 μM) and 2 levels of Cr (0 μM and 100 μM) as treatments. The results showed that NaHS addition enhances plant growth and photosynthesis slightly compared with the control. Moreover, NaHS alleviated the inhibition in plant growth and photosynthesis by Cr stress. Higher levels of NaHS exhibited more pronounced effects in reducing Cr concentrations in roots, shoots, and leaves. Ultrastructural examination of plant cells supported the facts by indication of visible alleviation of cell disorders in both root and leaf with exogenous application of NaHS. An increased number of plastoglobuli, disintegration, and disappearance of thylakoid membranes and starch granules were visualized inside the chloroplast of Cr-stressed plants. Starch accumulation in the chloroplasts was also noticed in the Cr-treated cells, with the effect being much less in Cr + NaHS-treated plants. Hence, it is concluded that H2 S produced from NaHS can improve plant tolerance under Cr stress., (© 2013 SETAC.)

Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spike1 (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1), a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.

Silicon (Si) is generally considered as a benefic element for higher plants, especially for those grown under abiotic stressed environments. Current study is carried out in a hydroponic experiment to analyze the effect of Si application on barley growth, photosynthesis and ultra-structure under chromium (Cr) stress. The treatments consisted of three Si (0, 1 and 2mM) and two Cr (0 and 100 μM) levels. The results showed that Si application at both levels enhanced plant growth relative to the control, and alleviated Cr toxicity, reflected by significant increase in growth and photosynthetic parameters, such as SPAD value, net photosynthetic rate (P(n)), cellular CO(2) concentration (C(i)), stomatal conductance (G(s)) and transpiration rate (T(r)), and chlorophyll fluorescence efficiency (Fv/Fm), with 2mM Si having greater effect than 1mM Si. Cr stress caused ultra-structural disorders in leaves, such as uneven swelling of chloroplast, increased amount of plastoglobuli, disintegrated and disappeared thylakoid membranes, increased size and number of starch granules in leaves, and root ultra-structural modification, including increased vacuolar size, presence of Cr metal in cell walls and vacuoles, disruption and disappearance of nucleus. Exogenous Si alleviated these ultra-structural disorders both in roots and leaves. Apparently, Si and Cr behaved antagonistically, indicating that Si could be a candidate for Cr detoxification in crops under Cr-contaminated soil., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

A typical barley (Hordeum vulgare) floret consists of reproductive organs three stamens and a pistil, and non-reproductive organs-lodicules and two floral bracts, abaxial called 'lemma' and adaxial 'palea'. The floret is subtended by two additional bracts called outer or empty glumes. Together these organs form the basic structural unit of the grass inflorescence, a spikelet. There are commonly three spikelets at each rachis (floral stem of the barley spike) node, one central and two lateral spikelets. Rare naturally occurring or induced phenotypic variants that contain a third bract subtending the central spikelets have been described in barley. The gene responsible for this phenotype was called the THIRD OUTER GLUME1 (Trd1). The Trd1 mutants fail to suppress bract growth and as a result produce leaf-like structures that subtend each rachis node in the basal portion of the spike. Also, floral development at the collar is not always suppressed. In rice and maize, recessive mutations in NECK LEAF1 (Nl1) and TASSEL SHEATH1 (Tsh1) genes, respectively, have been shown to be responsible for orthologous phenotypes. Fine mapping of the trd1 phenotype in an F(3) recombinant population enabled us to position Trd1 on the long arm of chromosome 1H to a 10 cM region. We anchored this to a conserved syntenic region on rice chromosome Os05 and selected a set of candidate genes for validation by resequencing PCR amplicons from a series of independent mutant alleles. This analysis revealed that a GATA transcription factor, recently proposed to be Trd1, contained mutations in 10 out of 14 independent trd1 mutant alleles that would generate non-functional TRD1 proteins. Together with genetic linkage data, we confirm the identity of Trd1 as the GATA transcription factor ortholog of rice Nl1 and maize Tsh1 genes.

• Owing to its evolutional plasticity and adaptability, barley (Hordeum vulgare) is one of the most widespread crops in the world. Despite this evolutionary success, sexual reproduction of small grain cereals is poorly investigated, making discovery of novel genes and functions a challenging priority. Barley gene Jekyll appears to be a key player in grain development; however, its role in flowers has remained unknown. • Here, we studied RNAi lines of barley, where Jekyll expression was repressed to different extents. The impact of Jekyll on flower development was evaluated based on differential gene expression analysis applied to anthers and gynoecia of wildtype and transgenic plants, as well as using isotope labeling experiments, hormone analysis, immunogold- and TUNEL-assays and in situ hybridization. • Jekyll is expressed in nurse tissues mediating gametophyte-sporophyte interaction in anthers and gynoecia, where JEKYLL was found within the intracellular membranes. The repression of Jekyll impaired pollen maturation, anther dehiscence and induced a significant loss of fertility. The presence of JEKYLL on the pollen surface also hints at possible involvement in the fertilization process. • We conclude that the role of Jekyll in cereal sexual reproduction is clearly much broader than has been hitherto realized., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

Greenhouse hydroponic experiments were conducted using Cd-sensitive (Dong 17) and tolerant (Weisuobuzhi) barley genotypes to evaluate genotypic differences in response of photosynthesis and ultrastructure to Cd toxicity in the presence of exogenous glutathione (GSH). Addition of 20 mg L(-1) GSH in 5 μM Cd culture medium (Cd + GSH) significantly alleviated Cd-induced growth inhibition and reduced Cd concentration in leaves and roots especially in the sensitive genotype Dong 17. Exogenous GSH greatly ameliorated Cd-induced damages on leaf/root ultrastructure, e.g., compared with Cd alone treatment, chloroplasts in plants treated with Cd + GSH become better or in relatively normal shape with well-structured thylakoid membranes and parallel pattern of lamellae and unfolded more starch grains but less osmiophilic plastoglobuli; nuclei of root cells were better formed and chromatin distributed more uniformly in both genotypes, and number of plastids and mitochondria cristae in Dong 17 resumed to control level. The examination of photosynthetic performance revealed GSH dramatically increased net photosynthetic rate (P(n)), stomatal conductance (G(s)), and transpiration rate (T(r)) in the both genotypes and strongly stimulated Cd-induced decrease in the maximal photochemical efficiency (F(v)/F(m)) especially in the sensitive genotype.

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation., (© 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.)

Large data sets are generated from plants by the various 'omics platforms. Currently, a limiting step in data analysis is the assessment of protein function and its translation into a biological context. The lack of robust high-throughput transformation systems for monocotyledonous plants, to which the vast majority of crop plants belong, is a major restriction and impedes exploitation of novel traits in agriculture. Here we present a stable root transformation system for barley, termed STARTS, that allows assessment of gene function in root tissues within 6 weeks. The system is based on the finding that a callus, produced on root induction medium from the scutellum of the immature embryo, is able to regenerate roots from single transformed cells by concomitant suppression of shoot development. Using Agrobacterium tumefaciens-mediated transfer of genes involved in root development and pathogenesis, we show that those calli regenerate large amounts of uniformly transformed roots for in situ functional analysis of newly expressed proteins., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Genes encoding two new isoforms of sucrose synthase from barley, HvSs3 and HvSs4, have been characterised and their expression patterns compared with those previously described for HvSs1 and HvSs2, in different organs and during seed maturation and germination. Their response to several abiotic stimuli has also been investigated in leaves: HvSs1 is up-regulated by anoxia and HvSs3 by water deprivation while no response is observed to 150 mM NaCl treatment; HvSs1 and HvSs3 are also induced by cold temperatures. Using translational fusions and transient expression analyses, the four isozymes have been localised not only to the cytoplasm but also along several cytoplasmic tracks and at the inner side of the cell membrane; besides, HvSS1 is also associated with mitochondria, a localisation that has been predicted in silico with the TargetP and Predotar programmes. These data suggest distinct although partially overlapping roles, for the four barley sucrose synthase isoforms, in the channelling of carbon towards different metabolic pathways within the cell.

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses., (Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd. No claim to original US government works.)

Seeds are generally viewed in the context of plant reproduction and the supply of food and feed, but only seldom as a site of photosynthesis. However, the seeds of many plant species are green, at least during their early development, which raises the issue of the significance of this greening for seed development. Here we describe the two contrasting modes of photosynthesis in the developing seed. The dicotyledonous pea seed has a green embryo, while the monocotyledonous barley caryopsis has a chlorenchymatic layer surrounding its non-green endosperm (storage organ). We have employed pulse-amplitude-modulated fluorescence and oxygen-sensitive microsensors to localize and describe gradient distributions of photosynthetic activity across the seed/caryopsis, and have discussed its role in maintaining the endogenous O₂ balance. We also report the lack of photosynthetic activity in the stay-green embryo axis of the sacred lotus (Nelumbo nucifera) seed following imbibition. The observations are discussed with respect to in vivo light supply and contrasted with the characteristics of leaf photosynthesis., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We investigated by fluorescence in situ hybridization (FISH) the synteny between Brachypodium distachyon with a small genome (1C = 320 Mb) and barley with a large genome (1C = 5,100 Mb) at the chromosome level. Reciprocal genomic in situ hybridization (GISH) between B. distachyon and barley labeled mainly 45S ribosomal DNA loci, indicating that most high copy DNA is weakly conserved between both grasses. Of 13 BAC clones with inserts from different B. distachyon chromosomes, only two belonging to chromosome 1 yielded hybridization signals on a barley metaphase chromosome (on 7HS and 7HL, respectively), confirming synteny between both chromosomes. FISH experiments to characterize the synteny of single-copy loci were performed. Two of four Brachypodium sylvaticum BACs spanning a 223-kb interval homologous to the region of barley that harbors a gibberellic-acid-insensitive semi-dwarfing gene, sdw3, hybridized specifically to a central position of B. distachyon chromosome 1 short arm but not to the homologous region of the barley genome. Repeat-free sequences PCR amplified from four non-overlapping barley BACs linked to the core of Sdw3 region yielded signals at distinct positions in the middle of barley chromosome arm 2HS. Together, these results (1) confirmed the synteny between B. distachyon chromosome 1 and barley chromosomes 2H and 7H at the cytological level, (2) indicated mid-arm position for the Sdw3 locus genetically mapped at the centromere of barley chromosome 2H, and (3) proved that the sdw3 core interval of < 100 kb in B. distachyon corresponds to a megabase-sized syntenic region in barley.

Shown antimicrotrubules activity of new 2,6-dinitroaniline compounds. Investigated their ability on apoptotic processes in a plant cell when used as a composition with inhibitors of acetyl-CoA carboxylase.

Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.

Hypersensitive response (HR) against Blumeria graminis f. sp. hordei infection in barley (Hordeum vulgare) was associated with stomata "lock-up" leading to increased leaf water conductance (g(l)). Unique spatio-temporal patterns of HR formation occurred in barley with Mla1, Mla3, or MlLa R genes challenged with B. graminis f. sp. hordei. With Mla1, a rapid HR, limited to epidermal cells, arrested fungal growth before colonies initiated secondary attacks. With Mla3, mesophyll HR preceded that in epidermal cells whose initial survival supported secondary infections. With MlLa, mesophyll survived and not all attacked epidermal cells died immediately, allowing colony growth and secondary infection until arrested. Isolines with Mla1, Mla3, or MlLa genes inoculated with B. graminis f. sp. hordei ranging from 1 to 100 conidia mm(2) showed abnormally high g(l) during dark periods whose timing and extent correlated with those of each HR. Each isoline showed increased dark g(l) with the nonpathogen B. graminis f. sp. avenae which caused a single epidermal cell HR. Guard cell autofluorescence was seen only after drying of epidermal strips and closure of stomata suggesting that locked open stomata were viable. The data link stomatal lock-up to HR associated cell death and has implications for strategies for selecting disease resistant genotypes.

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.

The CELLULOSE SYNTHASE-LIKE C (CSLC) family is an ancient lineage within the CELLULOSE SYNTHASE/CELLULOSE SYNTHASE-LIKE (CESA/CSL) polysaccharide synthase superfamily that is thought to have arisen before the divergence of mosses and vascular plants. As studies in the flowering plant Arabidopsis have suggested synthesis of the (1,4)-beta-glucan backbone of xyloglucan (XyG), a wall polysaccharide that tethers adjacent cellulose microfibrils to each other, as a probable function for the CSLCs, CSLC function was investigated in barley (Hordeum vulgare L.), a species with low amounts of XyG in its walls. Four barley CSLC genes were identified (designated HvCSLC1-4). Phylogenetic analysis reveals three well supported clades of CSLCs in flowering plants, with barley having representatives in two of these clades. The four barley CSLCs were expressed in various tissues, with in situ PCR detecting transcripts in all cell types of the coleoptile and root, including cells with primary and secondary cell walls. Co-expression analysis showed that HvCSLC3 was coordinately expressed with putative XyG xylosyltransferase genes. Both immuno-EM and membrane fractionation showed that HvCSLC2 was located in the plasma membrane of barley suspension-cultured cells and was not in internal membranes such as endoplasmic reticulum or Golgi apparatus. Based on our current knowledge of the sub-cellular locations of polysaccharide synthesis, we conclude that the CSLC family probably contains more than one type of polysaccharide synthase.

Ramularia collo-cygni causes leaf spots on barley (Hordeum vulgare), a disease of growing economical importance. Scanning electron microscopy was used to study the life cycle of the fungus on barley during the vegetation period and in winter. The infectious stage started with conidium germination on the surface and the penetration into the leaf via the stomatal pore where the hyphae grew within the cells that became necrotic. The conidiophores emerged through the stomatal pore. On older leaves, however, they frequently emerged apart from it and the results suggested a pushing apart of adjacent cell walls of the epidermal cells. An assessment of the amount of conidium formation of one heavily infested barley plant resulted in 4.05 x 10(6) conidia per plant. For the first time, conidiophores, conidium production and germination of conidia were also observed in winter on barley and on maize leaves.

The decay of seed dormancy during after-ripening is not well understood, but elucidation of the mechanisms involved may be important for developing strategies for modifying dormancy in crop species and, for example, addressing the problem of preharvest sprouting in cereals. We have studied the germination characteristics of barley (Hordeum vulgare 'Betzes') embryos, including a description of anatomical changes in the coleorhiza and the enclosed seminal roots. The changes that occur correlate with abscisic acid (ABA) contents of embryo tissues. To understand the molecular mechanisms involved in dormancy loss, we compared the transcriptome of dormant and after-ripened barley embryos using a tissue-specific microarray approach. Our results indicate that in the coleorhiza, ABA catabolism is promoted and ABA sensitivity is reduced and that this is associated with differential regulation by after-ripening of ABA 8'-hydroxylase and of the LIPID PHOSPHATE PHOSPHATASE gene family and ABI3-INTERACTING PROTEIN2, respectively. We also identified other processes, including jasmonate responses, cell wall modification, nitrate and nitrite reduction, mRNA stability, and blue light sensitivity, that were affected by after-ripening in the coleorhiza that may be downstream of ABA signaling. Based on these results, we propose that the coleorhiza plays a major role in causing dormancy by acting as a barrier to root emergence and that after-ripening potentiates molecular changes related to ABA metabolism and sensitivity that ultimately lead to degradation of the coleorhiza, root emergence, and germination.

Objective: To establish the standard for quality control of Fructus Hordei germinatus, Fructus Oryzae germinatus and Fructus Setariae germinatus., Methods: The digital microscope and infrared spectroscopy were used in the pharmacognostical study., Results: Distinguished differences were found on morphological and microscopical features of these three crude drugs. Whereas, their infrared spectrums were basically all the same., Conclusion: The study provides a convenient, effect method for the identification of these three medicinal materials.

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast-enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO(2)) affinity chromatography and were subsequently analysed using LC-ESI-MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na(+)/H(+) antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the toxicological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 microg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression--alteration that can be caused by cytoskeleton disorder. Using antibodies to alpha-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of microtubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.