Stress In Barley Research Articles

Comparative proteomic analysis of β-aminobutyric acid-mediated alleviation of salt stress in barley

The non-protein amino acid β-aminobutyric acid (BABA) is known to induce plant resistance to a broad spectrum of biotic and abiotic stresses. This is the first study describing the effect of BABA seed priming on physiological and proteomic changes under salt stress conditions in barley (Hordeum vulgare). The aim of our study was to investigate the changes of fresh weight, dry weight and relative water content (RWC) as well as root proteome changes of two barley lines contrasting in salt tolerance (DH14, DH 187) in response to salt stress after seed priming in water or in 800 μM BABA.Seed priming with BABA significantly increased (p ≤ 0.05) RWC in both barley lines, which indicates considerably lower water loss in BABA-primed plants than in the non-primed control plants. Dry and fresh matter increased significantly in line DH 187, whereas no changes were detected in line DH14.BABA-primed plants of both lines showed different proteomic patterns than the non-primed control plants. The root proteins exhibiting significant abundance changes (1.75-fold change, p ≤ 0.05) were separated by two-dimensional polyacrylamide gel electrophoresis (2D- PAGE). Thirty-one spots, representing 24 proteins, were successfully identified by MALDI TOF/TOF mass spectrometry.The most prominent differences include the up-regulation of antioxidant enzymes (catalase, peroxidase and superoxide dismutase), PR proteins (chitinase, endo-1,3-β-glucosidase), and chaperones (cyclophilin, HSC 70). Our results indicate that BABA induces defence and detoxification processes which may enable faster and more effective responses to salt stress, increasing the chances of survival under adverse environmental conditions.

Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress.

Salinity is a major limiting factor for agricultural production worldwide. A better understanding of the mechanisms of salinity stress response will aid efforts to improve plant salt tolerance. In this study, a combination of small RNA and mRNA degradome sequencing was used to identify salinity responsive-miRNAs and their targets in barley. A total of 152 miRNAs belonging to 126 families were identified, of which 44 were found to be salinity responsive with 30 up-regulated and 25 down-regulated respectively. The majority of the salinity-responsive miRNAs were up-regulated at the 8h time point, while down-regulated at the 3h and 27h time points. The targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction. Finally, qRT-PCR was used to validate the identified miRNA and their targets. Our study systematically investigated the expression profile of miRNA and their targets in barley during salinity stress phase, which can contribute to understanding how miRNAs respond to salinity stress in barley and other cereal crops.

The Levels of Boron‐Uptake Proteins in Roots are Correlated with Tolerance to Boron Stress in Barley

ABSTRACTBoron (B) is an essential micronutrient required for plant growth and development. Recently, two major B‐uptake proteins, BOR1 and NIP5;1, have been identified and partially characterized. BOR1 is a high‐affinity B transporter involved in xylem loading in roots, and NIP5;1 is a major boric‐acid channel in the plasma membrane. The aim of the present study was to determine if plant tolerance to B stress (deficiency or toxicity) is correlated with natural levels of B‐uptake proteins in roots. We grew two barley (Hordeum vulgare L.) cultivars that differ in tolerance to low and high B (Schooner and Clipper, respectively) at three B levels (sub‐, near‐, and supraoptimal), and then we determined the concentration of BOR1 and NIP5;1 in roots using enzyme‐linked immunosorbent assay (ELISA) and protein‐specific antibodies that we developed. Shoot and root biomass in Schooner decreased at high B, while shoot mass in Clipper decreased in low B. Differences between cultivars in tolerance to B stress were unrelated to the concentration of B in plant tissue or to effects of B on root total protein concentration. BOR1 content per unit total protein and per gram of root was greater in Clipper than Schooner, while NIP5;1 content was greater in Schooner. The estimated activity of BOR1 (B uptake per BOR1) did not differ between cultivars. These results show that whole‐plant tolerance to B deficiency and toxicity is correlated with levels of the major B‐uptake proteins in roots (BOR1 and NIP5;1). Hence, BOR1 and NIP5;1 can potentially be used as biomarkers to identify plant genotypes with enhanced tolerance to B stress (e.g., for agriculture or phytoremediation).

Ultraviolet and photosynthetically active radiation can both induce photoprotective capacity allowing barley to overcome high radiation stress

The main objective of this study was to determine the effects of acclimation to ultraviolet (UV) and photosynthetically active radiation (PAR) on photoprotective mechanisms in barley leaves. Barley plants were acclimated for 7 days under three combinations of high or low UV and PAR treatments ([UV−PAR−], [UV−PAR+], [UV+PAR+]). Subsequently, plants were exposed to short-term high radiation stress (HRS; defined by high intensities of PAR – 1000 μmol m−2 s−1, UV-A – 10 W m−2 and UV-B 2 W m−2 for 4 h), to test their photoprotective capacity. The barley variety sensitive to photooxidative stress (Barke) had low constitutive flavonoid content compared to the resistant variety (Bonus) under low UV and PAR intensities. The accumulation of lutonarin and 3-feruloylquinic acid, but not of saponarin, was greatly enhanced by high PAR and further increased by UV exposure. Acclimation of plants to both high UV and PAR intensities also increased the total pool of xanthophyll-cycle pigments (VAZ). Subsequent exposure to HRS revealed that prior acclimation to UV and PAR was able to ameliorate the negative consequences of HRS on photosynthesis. Both total contents of epidermal flavonols and the total pool of VAZ were closely correlated with small reductions in light-saturated CO2 assimilation rate and maximum quantum yield of photosystem II photochemistry caused by HRS. Based on these results, we conclude that growth under high PAR can substantially increase the photoprotective capacity of barley plants compared with plants grown under low PAR. However, additional UV radiation is necessary to fully induce photoprotective mechanisms in the variety Barke. This study demonstrates that UV-exposure can lead to enhanced photoprotective capacity and can contribute to the induction of tolerance to high radiation stress in barley.

Cloning and characterization of up-regulated HbSINA4 gene induced by drought stress in Tibetan hulless barley.

Hulless barley is an important crop cereal in Tibetan, China. Drought is a major abiotic stress in barley production. In this study, we cloned the drought-related HbSINA4 gene from the variety 'Himalaya 10' and analyzed its expression patterns under different drought and rehydration conditions. The cDNA of HbSINA4 was 1052 bp long, including an open reading frame of 771 bp that encoded a protein of 256 amino acids. The molecular weight of HbSINA4 protein was predicted to be 29.53 kDa and the theoretical pI was 8.32. Bioinformatic analysis showed that the HbSINA4 gene contained a protein kinase domain profile family signature motif, with high similarity to that of Oryza sativa and Brachypodium distachyon. Real-time polymerase chain reaction (PCR) assays revealed that gene expression declined rapidly with increasing drought stress; in contrast, its expression increased after rehydration treatment. Therefore, the HbSINA4 gene responds to the drought stress and plays an important role in barely drought resistance. Furthermore, our results provide information which may be useful in other temperate crop studies and in aiding resistance to drought.

Structural and biochemical analysis of the Hordeum vulgare L. HvGR-RBP1 protein, a glycine-rich RNA-binding protein involved in the regulation of barley plant development and stress response.

The timing of whole-plant senescence influences important agricultural traits such as yield and grain protein content. Post-transcriptional regulation by plant RNA-binding proteins is essential for proper control of gene expression, development, and stress responses. Here, we report the three-dimensional solution NMR structure and nucleic acid-binding properties of the barley glycine-rich RNA-binding protein HvGR-RBP1, whose transcript has been identified as being >45-fold up-regulated in early—as compared to late—senescing near-isogenic barley germplasm. NMR analysis reveals that HvGR-RBP1 is a multidomain protein comprising a well-folded N-terminal RNA Recognition Motif (RRM) and a structurally disordered C-terminal glycine-rich domain. Chemical shift differences observed in 2D 1H–15N correlation (HSQC) NMR spectra of full-length HvGR-RBP1 and N-HvGR-RBP1 (RRM domain only) suggest that the two domains can interact both in-trans and intramolecularly, similar to what is observed in the tobacco NtGR-RBP1 protein. Further, we show that the RRM domain of HvGR-RBP1 binds single-stranded DNA nucleotide fragments containing the consensus nucleotide sequence 5′-TTCTGX-3′ with low micromolar affinity in vitro. We also demonstrate that the C-terminal glycine-rich (HvGR) domain of Hv-GR-RBP1 can interact nonspecifically with ssRNA in vitro. Structural similarities with other plant glycine-rich RNA-binding proteins suggest that HvGR-RBP1 may be multifunctional. Based on gene expression analysis following cold stress in barley and E. coli growth studies following cold shock treatment, we conclude that HvGR-RBP1 functions in a manner similar to cold-shock proteins and harbors RNA chaperone activity. HvGR-RBP1 is therefore not only involved in the regulation of barley development including senescence, but also functions in plant responses to environmental stress.

Automated analysis of barley organs using 3D laser scanning: an approach for high throughput phenotyping.

Due to the rise of laser scanning the 3D geometry of plant architecture is easy to acquire. Nevertheless, an automated interpretation and, finally, the segmentation into functional groups are still difficult to achieve. Two barley plants were scanned in a time course, and the organs were separated by applying a histogram-based classification algorithm. The leaf organs were represented by meshing algorithms, while the stem organs were parameterized by a least-squares cylinder approximation. We introduced surface feature histograms with an accuracy of 96% for the separation of the barley organs, leaf and stem. This enables growth monitoring in a time course for barley plants. Its reliability was demonstrated by a comparison with manually fitted parameters with a correlation R2 = 0.99 for the leaf area and R2 = 0.98 for the cumulated stem height. A proof of concept has been given for its applicability for the detection of water stress in barley, where the extension growth of an irrigated and a non-irrigated plant has been monitored.

Detection of early plant stress responses in hyperspectral images

Early stress detection in crop plants is highly relevant, but hard to achieve. We hypothesize that close range hyperspectral imaging is able to uncover stress related processes non-destructively in the early stages which are invisible to the human eye. We propose an approach which combines unsupervised and supervised methods in order to identify several stages of progressive stress development from series of hyperspectral images. Stress of an entire plant is detected by stress response levels at pixel scale. The focus is on drought stress in barley (Hordeum vulgare). Unsupervised learning is used to separate hyperspectral signatures into clusters related to different stages of stress response and progressive senescence. Whereas all such signatures may be found in both, well watered and drought stressed plants, their respective distributions differ. Ordinal classification with Support Vector Machines (SVM) is used to quantify and visualize the distribution of progressive stages of senescence and to separate well watered from drought stressed plants. For each senescence stage a distinctive set of most relevant Vegetation Indices (VIs) is identified. The method has been applied on two experiments involving potted barley plants under well watered and drought stress conditions in a greenhouse. Drought stress is detected up to ten days earlier than using NDVI. Furthermore, it is shown that some VIs have overall relevance, while others are specific to particular senescence stages. The transferability of the method to the field is illustrated by an experiment on maize (Zea mays).

Differential Responses of Antioxidative System to Soil Water Shortage in Barley (<i>Hordeum vulgare</i> L.) Genotypes

Drought is one of the major factors limiting the yield and quality of crops in the world. The activity of antioxidative system to tolerate the drought stress is significant in plants. In the present study, the activities and isoform profiles of catalase (CАТ), ascorbate peroxidase (APX), glutathione reductase (GR), and superoxide dismutase (SOD) were analyzed in four barley genotypes grown under soil water restriction. Drought stress caused increase in the activities of CАТ and SOD in all studied genotypes, while APX activity decreased. The total GR activity increased substantially in genotypes K 2778 and St.Garabag 7 and decreased in No. 77 local and St.Pallidum 596 genotypes under conditions of severe water stress. No detectable differences were observed in the isoenzyme pattern (the appearance of a new isoenzymes and disappearance of another one) between control plants and those subjected to soil drought. However, intensification of corresponding isoforms in electrophoretic spectra was observed in stressed barley leaves relative to watered ones. The obtained results possibly suggest that antioxidant protection in barley plants under drought conditions could be attributed mainly to SOD and CAT.

Yield increase induced by the fungal root endophyte Piriformospora indica in barley grown at low temperature is nutrient limited

The fungal root endophytes Chaetomium globosum, Epicoccum nigrum and Piriformospora indica have value as biocontrol and biofertilising organisms in barley, but have not been well tested at low temperatures. This study assessed the efficacy of the endophytes on barley varieties grown under low temperature stress with variable nutrient input. Seed from three cultivars of spring barley were inoculated with one of the three fungal root endophyte isolates – C. globosum, E. nigrum or P. indica - and grown in low temperature under higher and lower nutrient input regimes. Compared with the control, for P.indica-inoculated plants with the higher nutrient input, flowering was earlier and grain dry weight significantly greater for all barley varieties by a mean of 22 %. The nitrogen and carbon content of the grains did not differ significantly between treatments. Chaetomium globosum and Epicoccum nigrum conferred no significant benefits under either nutrient regime. Piriformospora indica is amenable to axenic culture, sporulates readily and can be multiplied rapidly, suggesting that it could be developed as an effective crop treatment in low temperature stressed barley and may have the potential to increase crop yield in colder growing conditions provided that adequate nutrients are supplied.

Alleviation of chromium toxicity by hydrogen sulfide in barley

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.

Leaf proteome alterations in the context of physiological and morphological responses to drought and heat stress in barley (Hordeum vulgare L.)

The objective of this study was to identify barley leaf proteins differentially regulated in response to drought and heat and the combined stresses in context of the morphological and physiological changes that also occur. The Syrian landrace Arta and the Australian cultivar Keel were subjected to drought, high temperature, or a combination of both treatments starting at heading. Changes in the leaf proteome were identified using differential gel electrophoresis and mass spectrometry. The drought treatment caused strong reductions of biomass and yield, while photosynthetic performance and the proteome were not significantly changed. In contrast, the heat treatment and the combination of heat and drought reduced photosynthetic performance and caused changes of the leaf proteome. The proteomic analysis identified 99 protein spots differentially regulated in response to heat treatment, 14 of which were regulated in a genotype-specific manner. Differentially regulated proteins predominantly had functions in photosynthesis, but also in detoxification, energy metabolism, and protein biosynthesis. The analysis indicated that de novo protein biosynthesis, protein quality control mediated by chaperones and proteases, and the use of alternative energy resources, i.e. glycolysis, play important roles in adaptation to heat stress. In addition, genetic variation identified in the proteome, in plant growth and photosynthetic performance in response to drought and heat represent stress adaption mechanisms to be exploited in future crop breeding efforts.

Mitigation of peroxidative stress for barley exposed to cadmium in the presence of water-extractable organic matter from compost-like materials

The effects of water-extractable organic matter (WEOM) from compost-like materials on peroxidative stress were investigated for hydroponic culture of barley exposed to Cd. In the presence of WEOM, lipoxygenase activity and malondialdehyde, indices of peroxidative stress in barley, were significantly reduced, compared to those with Cd alone (5μM) for a 30-d culture (p<0.05). In addition, Cd uptake in the presence of WEOM samples was significantly lower than that in their absence (p<0.05). These results indicate that the addition of WEOM can be effective in mitigating the peroxidative stress in barley exposed to Cd. Of the total Cd in the solution, 7–8% was complexed with WEOM, indicating that the complexation of Cd with WEOM is a minor factor in reducing Cd-induced stress in barley. The WEOM sample was purified by cation-exchange column and ultrafiltration to remove the nutrient minerals, such as Ca, Mg and Fe. When the purified WEOM was employed for hydroponic culture in the presence of Cd, significant decreases in peroxidative stress and Cd uptake were observed (p<0.05). These results show that the organic components in WEOM contribute to the mitigation of peroxidative stress in barley exposed to Cd.

Genetic variation in the root growth response of barley genotypes to salinity stress.

We aimed to identify genetic variation in root growth in the cereal crop barley (Hordeum vulgare L.) in response to the early phase of salinity stress. Seminal root elongation was examined at various concentrations of salinity in seedlings of eight barley genotypes consisting of a landrace, wild barley and cultivars. Salinity inhibited seminal root elongation in all genotypes, with considerable variation observed between genotypes. Relative root elongation rates were 60-90% and 30-70% of the control rates at 100 and 150mM NaCl, respectively. The screen identified the wild barley genotype CPI71284-48 as the most tolerant, maintaining root elongation and biomass in response to salinity. Root elongation was most significantly inhibited in the landrace Sahara. Root and shoot Na+ concentrations increased and K+ concentrations decreased in all genotypes in response to salinity. However, the root and shoot ion concentrations did not correlate with root elongation rates, suggesting that the Na+ and K+ concentrations were not directly influencing root growth, at least during the early phase of salt stress. The identification of genetic diversity in root growth responses to salt stress in barley provides important information for future genetic, physiological and biochemical characterisation of mechanisms of salinity tolerance.

Microelement Absorption and Metals Accumulation in Barley Seedlings under the Combined Toxicity of Cadmium and Aluminum

Hydroponic system was used to investigate the interaction between different levels of Cd and Al in barley seedlings. The results showed that stress exposure promoted Al or Cd accumulation in plants, leading to strongly inhibited growth. Concentration- and tissue-dependent responses to Al or Cd stress in barley leaves, stems, and roots were found in four nutrient concentrations. Thus, stress generally inhibited Mg, Zn, and Ca absorption, but increased Fe accumulation and Ca translocation from roots to aboveground parts. Binary metal combinations significantly increased Al levels in plant parts, and aggravated growth inhibition as well as nutrients absorption disorder caused by Al treatments, especially the combination of 90 µM Al+5 µM Cd. By contrast, combined treatments decreased Cd concentration in plants, and the combinations of 10 µM Cd+Al alleviated Cd-induced growth and metabolism anomaly.

Meta-analysis of QTL associated with tolerance to abiotic stresses in barley

A meta-analysis of quantitative trait loci (QTL) associated with tolerance to abiotic stresses in barley was carried out using results from 35 different experiments. “MetaQTL” software was used to project QTL positions on a reference map. Three hundred and thirty-seven QTL for traits associated with tolerance to abiotic stresses were included in this analysis which identified 79 metaQTL (MQTL) including 26 for drought, 11 for low temperature, 22 for salinity, 17 for water-logging, and 3 for mineral toxicity and deficiency. The distribution of MQTL was similar to that of the initial QTL. Many of these MQTL were located on chromosomes 2H (mainly for water-logging and drought) and 5H (mainly for salinity and low temperature). It inferred that chromosomes 2H and 5H were important for researches on barley abiotic tolerance, and the genes associated with abiotic stresses were concentrated relatively. As expected from trait correlations, 22.8 % of these MQTL displayed overlapping CIs. These overlapping regions were mainly on chromosomes 1H, 2H and 4H. The results indicated that the tolerance to diverse abiotic stresses were associated with each other in barley. Additionally, 67 candidate genes responsive to abiotic stresses were co-located with the abiotic-stress MQTL. Of them, a total of 55 had different conserved motifs. It inferred that the tolerance to abiotic stresses was contributed by multi-genes with diverse functions, though there might be some important genes associated with the tolerance to abiotic stresses in barley. Additionally, of these candidate genes, scsnp02622, scsnp01644 and scsnp19641 could be better for further studies of abiotic stresses tolerance.

Fluorescence parameters as early indicators of light stress in barley

Photosynthetic efficiency of two Syrian barley landraces Arabi Aswad and Arabi Abiad grown under different light intensities were studied by the application of qualitative and quantitative analysis of chlorophyll a fluorescence. Different values of fluorescence parameters, quantum efficiencies, specific and phenomenological energy fluxes were obtained for each cultivar. Both low and high light stresses decreased photosystem II (PSII) activity in barley seedlings depending on the stress type and its duration. Cultivar Arabi Aswad was more tolerant to high light while Arabi Abiad was more tolerant to low light stress. The results allowed us to select chlorophyll a fluorescence parameters related to energy flux within PSII which were specifically affected under low or high light stress. We found that the performance index parameter is a sensitive indicator to explore the effect of light changes on PSII activity immediately after stress application, while maximal quantum yield of PSII and phenomenological parameters were only modified after a long period of stress application indicating PSII damage. Thus, we recommend the former parameter for early detection of light stress.

Alleviation of aluminum toxicity by hydrogen sulfide is related to elevated ATPase, and suppressed aluminum uptake and oxidative stress in barley

Greenhouse hydroponic experiments were performed to evaluate potential role of H2S on Al toxicity in barley seedlings. Seedlings pretreated with 200μM NaHS as a donor of H2S for 24h and subsequently exposed to 100μM AlCl3 for 24h had significantly longer roots than those without NaHS. The promoted root elongation was correlated with a substantial decrease in Al-induced overproduction of lipid peroxidation, electrolyte leakage and Al accumulation in roots, and a marked increase in Al-induced depress activities of Na+K+-ATPase and H+-ATPase. The alleviating role of H2S on Al-induced toxicity was also found in a time- and dose-dependent experiment. Addition of 200 and 400μM NaHS to 100μM AlCl3 effectively alleviated Al-toxicity, markedly diminished Al-induced MDA accumulation, and increased chlorophyll content, net photosynthetic rate (Pn) and maximal photochemical efficiency (Fv/Fm) compared with Al alone. Exogenous H2S significantly elevated depressed CAT activities, and further improved root POD activity. Moreover, NaHS decreased Al accumulation, but elevated concentrations of S, P, Ca, Mg and Fe in plants. These data suggest that H2S-induced alleviation in Al toxicity is attributed to reduced Al uptake and MDA accumulation, improved uptake of P, Ca, Mg and Fe, and elevated ATPase and photosynthetic performance.

ChlorophyllaFluorescence—A Useful Tool for the Early Detection of Temperature Stress in Spring Barley (Hordeum vulgareL.)

The photosynthetic activity of two Syrian barley (Hordeum vulgare L.) landraces, Arabi Abiad (A. Abiad) and Arabi Aswad (A. Aswad), grown under low- and high- temperature stresses, were studied by analyzing the measured chlorophyll fluorescence signals. Both the applied stresses influenced photosystem II (PSII) activity. However, the effects depend on the stress type and the duration of its application. Phenomenological parameters were shifted shortly after the application of both stresses, whereas fluorescence ratios and yield values were altered most significantly after 7 days of stress imposition. The earliest changes in PSII activity of both cultivars were observed in the case of high temperature treatment. The maximal quantum efficiency of the photosynthetic apparatus (F(v)/F(M)) did not alter after stress application. Therefore, we could not recommend this parameter for early detection of such stress. In contrast, the results from the present investigation strongly indicate that the most significantly changed chlorophyll a fluorescence parameters could be used as an efficient tool for the early diagnosis of temperature stress in barley.

Importance of ABA homeostasis under terminal drought stress in regulating grain filling events

Recent studies suggest that abscisic acid (ABA) at its basal level plays an important role during seed set and grain filling events. Under drought stress ABA levels were found to be significantly enhanced in the developing seed. Until now we lack an understanding of (A) ABA homeostasis in developing seeds under terminal drought and (B) the interactive role of ABA in regulating the starch biosynthesis pathway in developing grains under terminal drought. We have recently reported the possible regulation of ABA homeostasis in source (flag leaf) and sink (developing grains) tissues under post-anthesis drought stress in barley and concluded that significantly enhanced ABA levels in developing grains are due to strong activation of the ABA deconjugation pathway and fine regulation of the ABA biosynthesis-degradation pathway.1 Additionally, we provided evidence for the role of ABA in differential regulation of starch biosynthesis genes and a significant upregulation of starch degradation beta amylase genes under drought, i.e. ABA not only influences the rate of starch accumulation but also starch quality.