Wild-type Barley Research Articles

Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley

Key messageThe hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm.The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Heat Pre-Treatment Modified Host and Non-Host Interactions of Powdery Mildew with Barley Brassinosteroid Mutants and Wild Types.

High temperatures associated with climate change may increase the severity of plant diseases. This study investigated the effect of heat shock treatment on host and non-host barley powdery mildew interactions using brassinosteroid (BR) mutants of barley. Brassinosteroids are plant steroid hormones, but so far little is known about their role in plant-fungal interactions. Wild type barley cultivar Bowman and its near-isogenic lines with disturbances in BR biosynthesis or signalling showed high compatibility to barley powdery mildew race A6, while cultivar Delisa and its BR-deficient mutants 522DK and 527DK were fully incompatible with this pathogen (host plant-pathogen interactions). On the other hand, Bowman and its mutants were highly resistant to wheat powdery mildew, representing non-host plant-pathogen interactions. Heat pre-treatment induced shifts in these plant-pathogen interactions towards higher susceptibility. In agreement with the more severe disease symptoms, light microscopy showed a decrease in papillae formation and hypersensitive response, characteristic of incompatible interactions, when heat pre-treatment was applied. Mutant 527DK, but not 522DK, maintained high resistance to barley powdery mildew race A6 despite heat pre-treatment. By 10 days after heat treatment and infection, a noticeable shift became apparent in the chlorophyll a fluorescence and in various leaf reflectance parameters at all genotypes.

Comparative Analysis of Root Transcriptome of High-NUE Mutant and Wild-Type Barley under Low-Nitrogen Conditions

Although nitrogen (N) deficiency greatly affects N absorption and metabolism in barley, the global transcriptomic changes in morphological and physiological adaptation to altered N availability remains largely unclear. We conducted a comparative transcriptome analysis of roots in A9-29 (low N tolerant line of barley) and Hua 30 (low N-sensitive variety of barley) under low N conditions to elucidate the responses and the underlying molecular mechanism. The results demonstrated that the root architecture was strongly influenced and that the root morphological indexes (total root length, total root area surface, and root volume) were remarkably promoted in A9-29 compared to Hua30 under low N stress. The transcriptome analysis of roots identified 1779 upregulated differentially expressed genes (DEGs) and 1487 downregulated DEGs specifically expressed in A9-29 under low N stress. Specific DEGs in A9-29 were largely enriched in energy metabolism, lipid metabolism, and the metabolism of other amino acids. In addition, transcription factor genes ERFs and IAA-related genes were specifically expressed in A9-29. To conclude, this study could provide a foundation for improving low N tolerance in barley.

Over-expression of the barley Phytoglobin 1 (HvPgb1) evokes leaf-specific transcriptional responses during root waterlogging

Oxygen deprivation (hypoxia) in the root due to waterlogging causes profound metabolic changes in the aerial organs depressing growth and limiting plant productivity in barley (Hordeum vulgare L.). Genome-wide analyses in waterlogged wild type (WT) barley (cv. Golden Promise) plants and plants over-expressing the phytoglobin 1 HvPgb1 [HvPgb1(OE)] were performed to determine leaf specific transcriptional responses during waterlogging. Normoxic WT plants outperformed their HvPgb1(OE) counterparts for dry weight biomass, chlorophyll content, photosynthetic rate, stomatal conductance, and transpiration. Root waterlogging severely depressed all these parameters in WT plants but not in HvPgb1(OE) plants, which exhibited an increase in photosynthetic rate. In leaftissue, root waterlogging repressed genes encoding photosynthetic components and chlorophyll biosynthetic enzymes, while induced those of reactive oxygen species (ROS)-generating enzymes. This repression was alleviated in HvPgb1(OE) leaves which also exhibited an induction of enzymes participating in antioxidant responses. In the same leaves, the transcript levels of several genes participating in nitrogen metabolism were also higher relative to WT leaves. Ethylene levels were diminished by root waterlogging in leaves of WT plants, but not in HvPgb1(OE), which were enriched in transcripts of ethylene biosynthetic enzymes and ethylene response factors. Pharmacological treatments increasing the level or action of ethylene further suggested the requirement of ethylene in plant response to root waterlogging. In natural germplasm an elevation in foliar HvPgb1 between 16h and 24h of waterlogging occurred in tolerant genotypes but not in susceptible ones. By integrating morpho-physiological parameters with transcriptome data, this study provides a framework defining leaf responses to root waterlogging and indicates that the induction of HvPgb1 may be used as a selection tool to enhance resilience to excess moisture.

Altered properties and structures of root exudate polysaccharides in a root hairless mutant of barley.

Root exudates and rhizosheaths of attached soil are important features of growing roots. To elucidate factors involved in rhizosheath formation, wild-type (WT) barley (Hordeum vulgare L. cv. Pallas) and a root hairless mutant, bald root barley (brb), were investigated with a combination of physiological, biochemical, and immunochemical assays. When grown in soil, WT barley roots bound ∼5-fold more soil than brb per unit root length. High molecular weight (HMW) polysaccharide exudates of brb roots had less soil-binding capacity than those of WT root exudates. Carbohydrate and glycan monoclonal antibody analyses of HMW polysaccharide exudates indicated differing glycan profiles. Relative to WT plants, root exudates of brb had reduced signals for arabinogalactan-protein (AGP), extensin, and heteroxylan epitopes. In contrast, the root exudate of 2-week-old brb plants contained ∼25-fold more detectable xyloglucan epitope relative to WT. Root system immunoprints confirmed the higher levels of release of the xyloglucan epitope from brb root apices and root axes relative to WT. Epitope detection with anion-exchange chromatography indicated that the increased detection of xyloglucan in brb exudates was due to enhanced abundance of a neutral polymer. Conversely, brb root exudates contained decreased amounts of an acidic polymer, with soil-binding properties, containing the xyloglucan epitope and glycoprotein and heteroxylan epitopes relative to WT. We, therefore, propose that, in addition to physically structuring soil particles, root hairs facilitate rhizosheath formation by releasing a soil-binding polysaccharide complex.

The barley mutant multiflorus2.b reveals quantitative genetic variation for new spikelet architecture

Key messageSpikelet indeterminacy and supernumerary spikelet phenotypes in barley multiflorus2.b mutant show polygenic inheritance. Genetic analysis of multiflorus2.b revealed major QTLs for spikelet determinacy and supernumerary spikelet phenotypes on 2H and 6H chromosomes.Understanding the genetic basis of yield forming factors in small grain cereals is of extreme importance, especially in the wake of stagnation of further yield gains in these crops. One such yield forming factor in these cereals is the number of grain-bearing florets produced per spikelet. Wild-type barley (Hordeum vulgare L.) spikelets are determinate structures, and the spikelet axis (rachilla) degenerates after producing single floret. In contrast, the rachilla of wheat (Triticum ssp.) spikelets, which are indeterminate, elongates to produce up to 12 florets. In our study, we characterized the barley spikelet determinacy mutant multiflorus2.b (mul2.b) that produced up to three fertile florets on elongated rachillae of lateral spikelets. Apart from the lateral spikelet indeterminacy (LS-IN), we also characterized the supernumerary spikelet phenotype in the central spikelets (CS-SS) of mul2.b. Through our phenotypic and genetic analyses, we identified two major QTLs on chromosomes 2H and 6H, and two minor QTLs on 3H for the LS-IN phenotype. For, the CS-SS phenotype, we identified one major QTL on 6H, and a minor QTL on 5H chromosomes. Notably, the 6H QTLs for CS-SS and LS-IN phenotypes co-located with each other, potentially indicating that a single genetic factor might regulate both phenotypes. Thus, our in-depth phenotyping combined with genetic analyses revealed the quantitative nature of the LS-IN and CS-SS phenotypes in mul2.b, paving the way for cloning the genes underlying these QTLs in the future.

Alternate partial root-zone N-fertigation increases water use efficiency and N uptake of barley at elevated CO2

Elevated atmospheric CO2 concentration (e[CO2]) increases water use efficiency (WUE) while reducing nitrogen (N) concentration of crops particularly under drought conditions; yet the combined effects of e[CO2] and different N-fertigation regimes on WUE and crop N nutrition remain largely elusive. In this experiment, the growth and physiological responses of two barley genotypes, wild type barley Steptoe (WT) and its correspondent ABA-deficient mutant barley Az34, to three N-fertigation regimes at ambient CO2 (a[CO2]) (400 ppm) and e[CO2] (800 ppm) were investigated. From tillering to grain filling stage, the plants were subjected to three N-fertigation regimes: 1) N-fertigation at full irrigation volume (FIN); 2) N-fertigation at reduced irrigation volume (DIN); 3) alternate N-fertigation at reduced irrigation volume (PRDN). Although e[CO2] had little effect on gs, Tr and plant water use of WT, especially under DIN and PRDN, it increased An, resulting in an increased WUE at stomatal, leaf and whole plant levels. For Az34, the positive effect of e[CO2] on WUE was attributed to both significantly enhanced An and lowered gs and Tr. For both genotypes, e[CO2] increased 100-grain weight and shoot dry biomass but didn’t affect grain yield and WUE for grain production (WUEg). PRDN increased grain yield, HI and WUEg of both genotypes regardless of [CO2], compared to FIN. DIN and PRDN increased N uptake of both genotypes at e[CO2] compared to FIN. Compared to a[CO2], e[CO2] increased 15N uptake and 15N recovery rate of both genotypes by enhancing plant biomass. In addition, both genotypes grown under DIN and PRDN allocated more N to the grain compared to the FIN plants. Collectively, N-fertigation at reduced irrigation volume promoted N allocation to the grain and increased WUE, particularly under e[CO2]. Such information is conductive for optimizing WUE and N nutrition of crops in a future water-limited and CO2-enriched environment.

Enhancement of root architecture and nitrate transporter gene expression improves plant growth and nitrogen uptake under long-term low-nitrogen stress in barley (Hordeum vulgare L.) seedlings

Over application of nitrogen (N) fertilizers to crops ultimately causes N pollution in the ecosphere. Studying the response of plant growth and N uptake to low-N stress may aid in elucidating the mechanism of low N tolerance in plants and developing crop cultivars with high nitrogen use efficiency (NUE). In this study, a high-NUE mutant line A9-29 and the wild-type barley cultivar Hua30 were subjected to hydroponic culture with high and low N supply, and the dry weight, N accumulation, root morphology, and expression levels of the potential genes involved in nitrate uptake and assimilation were measured at seedling stage. The results showed that under low-N conditions, A9-29 had a higher dry weight, N content, N influx rate and larger root uptake area than did Hua30. Under long-term low-N stress, compared with Hua30, A9-29 demonstrated higher expression of the HvNRT2/3 genes, especially HvNRT2.1, HvNRT2.5, and HvNRT3.3. Similarly, the expression levels of N assimilation genes including HvNIA1, HvNIR1, HvGS1_1, HvGS1_3, and HvGLU2 increased significantly in A9-29. Taken together, our results suggested that the larger root area and the upregulation of nitrate transporter and assimilation genes may contribute to stronger N uptake capacity for plant growth and N accumulation in responding to long-term low-N stress. These findings may aid in understanding the mechanism of low N tolerance and developing barley cultivars with high-NUE.

Root hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu).

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.

In situ determination of guard cell ion flux underpins the mechanism of ABA-mediated stomatal closure in barley plants exposed to PEG-induced drought stress

ABA regulates stomatal movement by affecting ion transport in guard cells; yet in situ measurement of ABA-mediated dynamics of guard cell ion transport and the involvement of other phytohormones in regulating stomatal aperture under drought stress are still lacking. In this study, hydroponically grown plants of wild type barley Steptoe (WT) and its correspondent ABA-deficient barley mutant Az34 were treated with 10% polyethylene glycol (PEG) 6000 for 0, 2, 4, and 24 h or 9 d to mimic short- and long-term drought stress. The K+, H+ and Ca2+ fluxes in the guard cell were monitored in situ by noninvasive micro-test technology. Upon 10% PEG treatment, leaf ABA concentration ([ABA]leaf) of both barley genotypes increased dramatically after 2 h and reached the highest level after the 24 h. Compared to the control, a significant increase in Ca2+ influx in both genotypes was observed after 2 h exposure to PEG, and reached the largest value after 4 h in WT. The increase of [ABA]leaf coincided with the increase of K+ efflux and Ca2+ influx and the decrease of stomatal conductance in WT under short-term drought stress, though the concentrations of IAA, GA3 and ZR in WT were all increased at 4 h. K+ efflux of guard cells was significantly greater in WT than in Az34 at 24 h after PEG treatment. The results elucidate the role of ABA in mediating ion transports in guard cells, hereby regulating the stomatal movement in barley exposed to drought stress.

Effects of exogenous nitric oxide on wild barley (Hordeum brevisubulatum) under salt stress

Soil salinization has become more and more serious. Exogenous nitric oxide (NO) could act in alleviating toxicity in plants. In this study, we aimed to explore the growth, photosynthetic pigments, water-solubles content, proteins, antioxidant enzyme parameters, and element contents of two types of wild barley (Hordeum brevisubulatum (Trin.) Link.), wild-type barley and Saertu wild barley cultivars, in response to salt stress. Both cultivars were first treated with 300 µmol·L −1 NaCl (increased in 25 µmol·L −1 increments to 300 µmol·L −1 for 7 days). Next, plants were sprayed with different NO concentrations on days 1, 5, 10, 15 and 20 after the NaCl concentration reached 300 µmol·L −1. The results showed that salt stress alone decreased all growth parameters [plant height (HT), fresh weight (FW), total dry weight (DW)], the photosynthetic pigment and protein contents, and the activity of the antioxidant enzymes [ascorbic acid peroxidase (APX), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD)], and significantly increased the K+ and Na+ elements contents, water solubles and malondialdehyde (MDA) compared with the controls. However, the inhibitory effects of salt stress in both wild barley cultivars were alleviated by exogenous NO treatment, and 200 µmol·L −1 NO was the most effective concentration to improve plant growth, ion element levels and the physiology of wild barley under high salt stress. Altogether, this study demonstrated the beneficial role of exogenous NO donor, sodium nitroprusside, on wild barley under salinity stress and the underlying physiological and biochemical mechanisms.

Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures.

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 °C, whereas in the hydrophobic core, they were visible at both 20 and 5 °C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 °C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts.

Phosphorus Uptake and Growth of Wild-Type Barley and Its Root-Hairless Mutant Cultured in Buffered-and Non-Buffered-P Solutions

Root hairs play an important role in phosphorus (P) nutrition of plants. To better understand the relationship between root hairs and P acquisition efficiency (PAE) in barley, experiments were conducted with the wild-type barley (cv. ’Pallas’) and its root-hairless mutant (brb). A hydroponic split-root system was used to supply P as Ca3(PO4)2 (tri-calcium phosphate, TCP) to one-half and other nutrients to the other half of the root system. Using TCP as a sole P source can simulate a soil solution with buffered low P concentration in hydroponics to induce prolific root hair growth. Root morphology, plant growth, and P uptake efficiency were measured with 50 mg L−1 TCP supplied to the roots in the split-root system and 0, 35, or 1000 μM NaH2PO4 in a non-split-root hydroponic system. The wild-type plants developed root hairs, but they did not contribute to the significant genotypic differences in the P uptake rate when a soluble P source was supplied in the non-split root system, indicating that root hair formation does not contribute to P uptake in a non-split root solution. On the other hand, when grown in a split-root system with one-half of the roots supplied with TCP, the wild-type showed 1.25-fold greater P uptake than the root hairless mutant. This study provides evidence that root hairs play an essential role in plant P uptake when P bioavailability is limited in the root zone.

The difference in N metabolism between NAD(P)H-specific NR-deficient mutant and wild-type barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) is an important crop for cereal research. In this study, two barley genotypes the wild-type (Steptoe) and the mutant (Az12) were used. An experiment was conducted using 15N-tracing method to NADH-specific nitrate reductase (NR)-deficient mutant seedling of barley. The N-depleted seedlings were exposed to a nutrient solution containing nitrate and nitrite, and were labeled with 15N for 38 h under (14 L/10D) cycles. The two genotypes utilized 15NO3− and accumulated it as reduced 15N, predominately in the shoots. However, nitrate reduction in the Az12 shoots was 9% lower than that in the Steptoe shoots at 38 h. As a result, in the Az12, nitrate accumulation in shoots was 78% higher than that in the Steptoe. Accumulation of reduced 15N in the Az12 roots was nearly similar to that of the Steptoe roots, but 8% lower in the Az12 shoots than in the Steptoe shoots at the end of the experiment. Also for both genotypes, root contribution increased during L/D cycles and decreased during the subsequent light cycle. Upward transport of reduced 15N via the xylem in the Az12 was nearly two times higher than that in Steptoe during the second light period (24–38 h). In both genotypes, xylem transport of reduced 15N was far exceeded the downward phloem transport.AbbreviationsAnlaccumulation of reduced 15N from 15NO3− in non-labeled roots of split rootsAraccumulation in roots of reduced 15N from 15NO3−Asaccumulation in shoots of reduced 15N from 15NO3−Rr15NO3− reduction in rootsRs15NO3− reduction in shootsTptranslocation to root of shoot reduced 15N from 15NO3− in phloemTxtranslocation to shoot of root-reduced 15N from 15NO3− in xylemFWfresh weight

The rpl23 gene and pseudogene are hotspots of illegitimate recombination in barley chloroplast mutator seedlings

Previously, through a TILLING (Targeting Induced Local Lesions in Genomes) approach applied on barley chloroplast mutator (cpm) seedlings a high frequency of polymorphisms in the rpl23 gene was detected. All the polymorphisms corresponded to five differences already known to exist in nature between the rpl23 gene located in the inverted repeats (IRs) and the rpl23 pseudogene located in the large single copy region (LSC). In this investigation, polymorphisms in the rpl23 gene were verified and besides, a similar situation was found for the pseudogene in cpm seedlings. On the other hand, no polymorphisms were found in any of those loci in 40 wild type barley seedlings. Those facts and the independent occurrence of polymorphisms in the gene and pseudogene in individual seedlings suggest that the detected polymorphisms initially arose from gene conversion between gene and pseudogene. Moreover, an additional recombination process involving small recombinant segments seems to occur between the two gene copies as a consequence of their location in the IRs. These and previous results support the hypothesis that the CPM protein is a component of the plastome mismatch repair (MMR) system, whose failure of the anti-recombination activity results in increased illegitimate recombination between the rpl23 gene and pseudogene.

The barley transcription factor HvMYB1 is a positive regulator of drought tolerance

Transcription factors such as MYB have previously been associated with the plant response to drought. In this work, studies on the function of the barley (Hordeum vulgare L.) transcription factor HvMYB1 show that gene expression is upregulated in wildtype barley roots and leaves under drought and osmotic stress. Transgenic barley plants that overexpress HvMYB1 were found to be more resistant to drought, showing enhanced relative water content and reduced water loss rate and stomatal conductance as compared to control plants. Levels of the osmolyte proline were enhanced as was expression of dehydrin HvDNH6 in the transgenic lines under drought conditions. The levels of the reactive oxygen species H2O2 were enhanced in wildtype roots and leaves by drought, but less so in the HvMYB1 overexpressing lines. Enzyme activity of the low affinity H2O2 degrading enzyme catalase (EC 1.11.1.6) was also lower in droughted HvMYB1 overexpressing lines. Gene expression of the high affinity ROS scavengers ASCORBATE PEROXIDASE and GLUTATHIONE PEROXIDASE was found to be constitutively high in the overexpressing lines, whereas CATALASE gene expression was similar to the control plants. These results suggest a role for HvMYB1 in protecting plants against drought in the vegetative plant by acting as a mediator of abscisic acid action.

Interaction between root hairs and soil phosphorus on rhizosphere priming of soil organic matter

We hypothesized that the rhizosphere priming effect (RPE) of soil organic matter by mutant barley lacking root hairs is dependant on a large network of symbiotic arbuscular mycorrhizal fungi (AMF). We thus predicted that fertilizing with phosphate-P would reduce AMF abundance and, in turn, reduce RPE of mutant barley. We packed microcosms with a P-responsive soil in which we grew mutant barley lacking root hairs as well as wild type barley and narrowleaf plantain, each possessing root hairs. One set of microcosms was fertilized with phosphate-P while another set was not fertilized. The plants were grown in a labelling chamber with 13C-depleted CO2. Soil respiration and δ13C of headspace CO2 were measured after 3, 4 and 5 weeks and RPE was calculated using an isotope mass balance approach. Root hair length was measured and soils were analyzed for the 16:1ω5 neutral lipid fatty acid (i.e. AMF biomarker). AMF abundance was greater, whereas RPE was lower, in mutant barley soil under low-P than under high-P conditions. In the other two plant-types, P had no effect on AMF or on RPE. As our results contradict our prediction, we propose an alternative explanation based on plant N demand under high-P.

Recombinant expression of osmotin in barley improves stress resistance and food safety during adverse growing conditions.

BackgroundAlthough many genetic manipulations of crops providing biofortified or safer food have been done, the acceptance of biotechnology crops still remains limited. We report on a transgenic barley expressing the multi-functional protein osmotin that improves plant defense under stress conditions.MethodsAn Agrobacterium–mediated technique was used to transform immature embryos of the spring barley cultivar Golden Promise. Transgenic barley plants of the T0 and T1 generation were evaluated by molecular methods. Transgenic barley tolerance to stress was determined by chlorophyll, total protein, malondialdehyde and ascorbate peroxidase content. Methanol extracts of i) Fusarium oxysporum infected or ii) salt-stressed plants, were characterized by their acute toxicity effect on human dermal fibroblasts (HDF), genotoxicity and affection of biodiversity interactions, which was tested through monitoring barley natural virus pathogen–host interactions–the BYDV and WDV viruses transmitted to the plants by aphids and leafhoppers.ResultsTransgenic plants maintained the same level of chlorophyll and protein, which significantly declined in wild-type barley under the same stressful conditions. Salt stress evoked higher ascorbate peroxidase level and correspondingly less malondialdehyde. Osmotin expressing barley extracts exhibited a lower cytotoxicity effect of statistical significance than that of wild-type plants under both types of stress tested on human dermal fibroblasts. Extract of Fusarium oxysporum infected transgenic barley was not able to damage DNA in the Comet assay, which is in opposite to control plants. Moreover, this particular barley did not affect the local biodiversity.ConclusionOur findings provide a new perspective that could help to evaluate the safety of products from genetically modified crops.

Development of Decreased-Gluten Wheat Enabled by Determination of the Genetic Basis of lys3a Barley.

Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

A Novel (1,4)-β-Linked Glucoxylan Is Synthesized by Members of the Cellulose Synthase-Like F Gene Family in Land Plants.

As a significant component of monocot cell walls, (1,3;1,4)-β-glucan has conclusively been shown to be synthesized by the cellulose synthase-like F6 protein. In this study, we investigated the synthetic activity of other members of the barley (Hordeum vulgare) CslF gene family using heterologous expression. As expected, the majority of the genes encode proteins that are capable of synthesizing detectable levels of (1,3;1,4)-β-glucan. However, overexpression of HvCslF3 and HvCslF10 genes resulted in the synthesis of a novel linear glucoxylan that consists of (1,4)-β-linked glucose and xylose residues. To demonstrate that this product was not an aberration of the heterologous system, the characteristic (1,4)-β-linkage between glucose and xylose was confirmed to be present in wild type barley tissues known to contain HvCslF3 and HvCslF10 transcripts. This polysaccharide linkage has also been reported in species of Ulva, a marine green alga, and has significant implications for defining the specificity of the cell wall content of many crop species. This finding supports previous observations that members of a single CSL family may not possess the same carbohydrate synthetic activity, with the CSLF family now associated with the formation of not only (1,3)- and (1,4)-β-glucosidic linkages, but also (1,4)-β-glucosidic and (1,4)-β-xylosidic linkages.