Exogenous Melatonin Enhances the Low Phosphorus Tolerance of Barley Roots of Different Genotypes.

Melatonin (N-acetyl-5-methoxytryptamine) plays an important role in plant growth and development, and in the response to various abiotic stresses. However, its role in the responses of barley to low phosphorus (LP) stress remains largely unknown. In the present study, we investigated the root phenotypes and metabolic patterns of LP-tolerant (GN121) and LP-sensitive (GN42) barley genotypes under normal P, LP, and LP with exogenous melatonin (30 µM) conditions. We found that melatonin improved barley tolerance to LP mainly by increasing root length. Untargeted metabolomic analysis showed that metabolites such as carboxylic acids and derivatives, fatty acyls, organooxygen compounds, benzene and substituted derivatives were involved in the LP stress response of barley roots, while melatonin mainly regulated indoles and derivatives, organooxygen compounds, and glycerophospholipids to alleviate LP stress. Interestingly, exogenous melatonin showed different metabolic patterns in different genotypes of barley in response to LP stress. In GN42, exogenous melatonin mainly promotes hormone-mediated root growth and increases antioxidant capacity to cope with LP damage, while in GN121, it mainly promotes the P remobilization to supplement phosphate in roots. Our study revealed the protective mechanisms of exogenous MT in alleviating LP stress of different genotypes of barley, which can be used in the production of phosphorus-deficient crops.

Gold Nanoparticles-Induced Modifications in Cell Wall Composition in Barley Roots.

The increased use of nanoparticles (NP) in different industries inevitably results in their release into the environment. In such conditions, plants come into direct contact with NP. Knowledge about the uptake of NP by plants and their effect on different developmental processes is still insufficient. Our studies concerned analyses of the changes in the chemical components of the cell walls of Hordeum vulgare L. roots that were grown in the presence of gold nanoparticles (AuNP). The analyses were performed using the immunohistological method and fluorescence microscopy. The obtained results indicate that AuNP with different surface charges affects the presence and distribution of selected pectic and arabinogalactan protein (AGP) epitopes in the walls of root cells.

Barley Isolated Microspore Culture.

The production of doubled haploids (DHs) has proved to be a highly valuable tool to obtain new cultivars. Among the cereals, barley (Hordeum vulgare L.) is the most successful species in large-scale haploid production. Techniques employed for this purpose are based on either the gynogenetic or the androgenetic pathway. Interspecific cross with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther culture (AC), and isolated microspore culture (IMC) are the most used methods. Among all of them, IMC is regarded as a particularly effective system owing to the great increase in green plant numbers per spike and also the higher induction of chromosome doubling when compared with other methods. Thus, IMC provides the best way to mass scale production of new varieties.

Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

Complete Substitution of Clover Hay and its Impact on Performance of Barki Sheep in Arid and Semi-arid Regions.

BACKGROUND AND OBJECTIVE: Barley straw is a farm by product that may be useful in animal feeding but its use is limited due to its low nutritional value and high fiber content. Therefore, this study aimed to improving the nutritive value of barley straw by bacterial treatment with Condensed Molasses Soluble (CMS) in the ration of Barki lambs. MATERIALS AND METHODS: In Experiment 1, 12 Barki rams, which were divided into 3 equal groups (4 each), were given rations which consisted of 50% CFM+50%, either clover hay (C) or bacterial treated barley straw (T1) or T1+ CMS (T2). In Experiment 2, 10 Barki lambs were divided into 2 groups, one (C) and T2 as descripted in Experiment 1. Data analyzed by using one way ANOVA model. RESULTS: Digestibility of Dry Matter (DM), Organic Matter (OM), Crude Fiber (CF) and Ether Extract (EE) were insignificantly affected among all rations, while Digestible Crude Protein (DCP) recorded a higher value in T2 compared to T1. Changes in Total Digestible Nutrients (TDN) values were insignificant. Values of DCP were higher for T2 compared to C and T1. The Body Weight Gain (BWG) and Feed Conversion Ratio (FCR) of growing lambs didn't statistically differ among the groups. CONCLUSION: Finally, complete replacement of clover hay by treated barley straw plus CMS may be considered good feeding strategies in the feeding of Barki lambs in Arid and semi-arid regions.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture.

BACKGROUND: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. RESULTS: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, 'Jersey' and 'Mercada' that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. 'Mercada' that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating 'Mercada' embryos contained a low number of plastome copies whose replication was not always completed. Contrary to 'Mercada', cv. 'Jersey' that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in 'Jersey' regenerants. CONCLUSIONS: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Secondary metabolites changes in germinated barley and its relationship to anti-wrinkle activity.

The purpose of this research was to identify metabolite change during barley (Hordeum vulgare) germination and reveal active principles for the anti-wrinkle activity. Barley was germinated with deionized water (DW) and mineral-rich water (MRW) for the comparison of the effect of mineral contents on the metabolites changes during germination. The effects of germinated barley extracts (GBEs) on collagen production and collagenase inhibition were evaluated in vitro using human dermal fibroblasts (HDFs). A pronounced anti-wrinkle activity was observed in the test group treated with the MRW-GBEs. In order to find out the active components related to the anti-wrinkle activity, an orthogonal projection to latent structure-discriminant analysis (OPLS-DA) was performed, using the data from secondary metabolites profiling conducted by UPLC-PDA-ESI-MS. The anti-wrinkle activity of MRW-GBEs was revealed to be associated with the increase of oligomeric compounds of procyanidin and prodelphinidin, indicating that it can be used as an active ingredient for anti-wrinkle agents.

Timing-dependent effects of salicylic acid treatment on phytohormonal changes, ROS regulation, and antioxidant defense in salinized barley (Hordeum vulgare L.).

Cross-talk between exogenous salicylic acid (SA) and endogenous phytohormone pathways affects the antioxidant defense system and its response to salt stress. The study presented here investigated the effects of SA treatment before and during salt stress on the levels of endogenous plant growth regulators in three barley cultivars with different salinity tolerances: Hordeum vulgare L. cvs. Akhisar (sensitive), Erginel (moderate), and Kalayci (tolerant). The cultivars' relative leaf water contents, growth parameters, proline contents, chlorophyll a/b ratios, and lipid peroxidation levels were measured, along with the activities of enzymes involved in detoxifying reactive oxygen species (ROS) including superoxide-dismutase, peroxidase, catalase, ascorbate-peroxidase, and glutathione-reductase. In addition, levels of several endogenous phytohormones (indole-3-acetic-acid, cytokinins, abscisic acid, jasmonic acid, and ethylene) were measured. Barley is known to be more salt tolerant than related plant species. Accordingly, none of the studied cultivars exhibited changes in membrane lipid peroxidation under salt stress. However, they responded differently to salt-stress with respect to their accumulation of phytohormones and antioxidant enzyme activity. The strongest and weakest increases in ABA and proline accumulation were observed in Kalayci and Akhisar, respectively, suggesting that salt-stress was more effectively managed in Kalayci. The effects of exogenous SA treatment depended on both the timing of the treatment and the cultivar to which it was applied. In general, however, where SA helped mitigate salt stress, it appeared to do so by increasing ROS scavenging capacity and antioxidant enzyme activity. SA treatment also induced changes in phytohormone levels, presumably as a consequence of SA-phytohormone salt-stress cross-talk.

Effects of Growth Conditions and Cultivar on the Content and Physiochemical Properties of Arabinoxylan in Barley.

The present study aims to evaluate the effect of the growth conditions and the cultivar on the total and water-extractable (W-E) arabinoxylan (AX) in barley. For this purpose, nine barley varieties from two different years were analyzed. The total AX content ranged from 5.97 to 8.98 wt % d.m., while the W-E AX ranged from 0.06 to 0.35 wt % d.m. The W-E AX molecular properties were characterized by high-pressure size exclusion chromatography (HPSEC)-triple detector array (TDA). The molecular weight was between 2.3 × 105 and 12.6 × 105 Da, the polydispersity was between moderate and broad (1.1 < Mw/Mn < 4.3), and the conformation was a stiff semiflexible coil (0.5 < α < 1.3). The results indicate that the year influences the content of total AX and W-E AX and some molecular characteristics of W-E AX, such as its polydispersity and its conformation. Finally, the results demonstrated that the W-E AX can be used as an index of the malting attitude of barley because it positively correlates with germinative energy and kernel dimension.

Phosphorus-acquisition strategies of canola, wheat and barley in soil amended with sewage sludges.

Crops have different strategies to acquire poorly-available soil phosphorus (P) which are dependent on their architectural, morphological, and physiological root traits, but their capacity to enhance P acquisition varies with the type of fertilizer applied. The objective of this study was to examine how P-acquisition strategies of three main crops are affected by the application of sewage sludges, compared with a mineral P fertilizer. We carried out a 3-months greenhouse pot experiment and compared the response of P-acquisition traits among wheat, barley and canola in a soil amended with three sludges or a mineral P fertilizer. Results showed that the P-acquisition strategy differed among crops. Compared with canola, wheat and barley had a higher specific root length and a greater root carboxylate release and they acquired as much P from sludge as from mineral P. By contrast, canola shoot P content was greater with sludge than with mineral P. This was attributed to a higher root-released acid phosphatase activity which promoted the mineralization of sludge-derived P-organic. This study showed that contrasted P-acquisition strategies of crops allows increased use of renewable P resources by optimizing combinations of crop and the type of P fertilizer applied within the cropping system.

Root plasticity and Pi recycling within plants contribute to low-P tolerance in Tibetan wild barley.

BACKGROUND: Barley is a low phosphorus (P) demand cereal crop. Tibetan wild barley, as a progenitor of cultivated barley, has revealed outstanding ability of tolerance to low-P stress. However, the underlying mechanisms of low-P adaption and the relevant genetic controlling are still unclear. RESULTS: We identified low-P tolerant barley lines in a doubled-haploid (DH) population derived from an elite Tibetan wild barley accession and a high-yield cultivar. The tolerant lines revealed greater root plasticity in the terms of lateral root length, compared to low-P sensitive lines, in response to low-P stress. By integrating the QTLs associated with root length and root transcriptomic profiling, candidate genes encoding isoflavone reductase, nitrate reductase, nitrate transporter and transcriptional factor MYB were identified. The differentially expressed genes (DEGs) involved the growth of lateral root, Pi transport within cells as well as from roots to shoots contributed to the differences between low-P tolerant line L138 and low-P sensitive lines L73 in their ability of P acquisition and utilization. CONCLUSIONS: The plasticity of root system is an important trait for barley to tolerate low-P stress. The low-P tolerance in the elite DH line derived from a cross of Tibetan wild barley and cultivated barley is characterized by enhanced growth of lateral root and Pi recycling within plants under low-P stress.

Aluminum Alters the Histology and Pectin Cell Wall Composition of Barley Roots.

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.

Intensification in pastoralist cereal use coincides with the expansion of trans-regional networks in the Eurasian Steppe.

The pace of transmission of domesticated cereals, including millet from China as well as wheat and barley from southwest Asia, throughout the vast pastoralist landscapes of the Eurasian Steppe (ES) is unclear. The rich monumental record of the ES preserves abundant human remains that provide a temporally deep and spatially broad record of pastoralist dietary intake. Calibration of human δ13C and δ15N values against isotope ratios derived from co-occurring livestock distinguish pastoralist consumption of millet from the products of livestock and, in some regions, identify a considerable reliance by pastoralists on C3 crops. We suggest that the adoption of millet was initially sporadic and consumed at low intensities during the Bronze Age, with the low-level consumption of millet possibly taking place in the Minusinsk Basin perhaps as early as the late third millennium cal BC. Starting in the mid-second millennium cal BC, millet consumption intensified dramatically throughout the ES with the exception of both the Mongolian steppe where millet uptake was strongly delayed until the end of first millennium cal BC and the Trans-Urals where instead barley or wheat gained dietary prominence. The emergence of complex, trans-regional political networks likely facilitated the rapid transfer of cultivars across the steppe during the transition to the Iron Age.

Manuring practices in the first millennium AD in southern Sweden inferred from isotopic analysis of crop remains.

This study uses crop stable nitrogen isotope analysis of charred grain to explore manuring practices in arable production at the affluent regional center Uppåkra and a set of smaller surrounding sites, dating to the first millennium AD in southern Sweden. The isotopic analysis focuses on hulled barley, the principle crop in the Scandinavian Iron Age, and the minor crops: bread wheat, emmer wheat, rye and oat, are included to compare manuring practices in cultivation of other crop species during this period. A field experiment was first conducted to establish relationships between manuring and δ15N values in modern grain from known growing conditions. The data formed an interpretive framework to reconstruct past agricultural practices and manuring intensity in the archaeological study area. Our results from the ancient grains have demonstrated that barley from the early phase in the study area (AD 0-200) varies widely in its δ15N values, reflecting mixed manuring regimes. In the following periods (AD 200-1000), isotopic values are relatively high overall, indicating systematic input of manure. In this paper, we explore whether the isotopic data that indicates sustained and high manuring levels could reflect the wealth of Uppåkra and its surrounding areas by showing prosperity also in its agricultural production, since intensive manuring would have required more resource and labor investments. The new crop nitrogen isotopic data shed light on the agricultural practices of a long-lived Iron Age center and its surrounding areas.

Evaluation of the effectiveness of in situ stabilization in the field aged arsenic-contaminated soil: Chemical extractability and biological response.

The effectiveness of in situ stabilization in the long-term As-contaminated soil was assessed. In situ stabilization of As was conducted through a Fe-based sorbent amendment. Chemical extractability of As was first determined by solubility/bioavailability research consortium extraction method and any change in human health risk through oral ingestion was characterized. Also, nonspecifically bound As in soil was determined by five-step sequential extraction. The results indicate that such extractable fractions of As decreased, and consequently risk through oral ingestion decreased probably due to hematite transformed from both the goethite in the original soil and the Fe-based sorbent, which was identified through the X-ray absorption spectroscopy. In ecotoxicity test with Hordeum vulgare, root and shoot elongation and germination rate decreased which was contrary to the chemical extraction data. Such increase in As toxicity is because of increased exchangeable Ca2+ concentration causing As accumulation in the membrane surface of H. vulgare. Also, adsorption of phosphorus onto the Fe-based sorbent decreased available phosphorus concentration causing phosphorus deficiency for growth. Our results demonstrate that the effectiveness of in situ stabilization should be evaluated by means of both chemical extractability and biological response, as chemical analysis alone may not be sufficient to assess the ecotoxicity.

Barley Anther Culture.

The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.

Isolated Microspore Culture in Barley.

Isolated microspore culture (IMC) is the most efficient way to produce large numbers of doubled-haploid (DH) barley plants in a short time. Yet, while IMC is more cost-efficient and less labor-intensive than anther culture, it is technically more complex and requires more experienced personnel if it is to yield its full potential. In part, this is because of multiple and important interactions that exist between factors at its many different phases, including genotype effects as well. When every phase is fine-tuned, the protocol that is presented below yields a useful number of DHs with almost all genotypes and can allow the production of up to 300 DH plants from a single F1 plant in just a few months.

Ethylene modulates root cortical senescence in barley.

Background and Aims: Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes. Methods: Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications. Key Results: The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD). Conclusions: The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue.