Amanda J. Able

Role of reactive oxygen species in the response of barley to necrotrophic pathogens.

Summary. The interactions between Hordeum vulgare (barley) and two fungal necrotrophs, Rhynchosporium secalis and Pyrenophora teres (causal agents of barley leaf scald and net blotch), were investigated in a detached-leaf system. An early oxidative burst specific to epidermal cells was observed in both the susceptible and resistant responses to R. secalis, and later on, a second susceptible-specific burst was observed. Time points of the first and the second burst correlated closely with pathogen contact to the plasma membrane and subsequent cell death, respectively. HO2•/O2− levels in resistant and susceptible responses to P. teres were limited in comparison. During later stages, HO2•/O2− was only detected in 2 to 3 epidermal cells immediately adjacent to phenolic browning and cell death observed during the susceptible response. However, H2O2 was detected in the majority of mesophyll cells adjacent to the observed lesion caused by P. teres. In contrast to observations during challenge with R. secalis, no direct contact between P. teres and the plasma membrane at sites of reactive oxygen species production was evident. Preinfiltration of leaves with antioxidants prior to challenge with either pathogen had no effect on resistance responses but did limit the growth of the pathogens and inhibit the extent of cell death during susceptible responses. These results suggest a possible role for reactive oxygen species in the induction of cell death during the challenge of a susceptible plant cell with a necrotrophic fungal leaf pathogen.

Induction of a grapevine germin-like protein (VvGLP3) gene is closely linked to the site of Erysiphe necator infection: A possible role in defense?

Germin-like proteins (GLP) have various proposed roles in plant development and defense. Seven novel GLP cDNA clones were isolated from grapevine (Vitis vinifera cv. Chardonnay). Reverse transcriptase-polymerase chain reaction expression analysis revealed that the VvGLP genes exhibit diverse and highly specific patterns of expression in response to a variety of abiotic and biotic treatments, including challenge by Erysiphe necator, Plasmopara viticola, and Botrytis cinerea, suggesting a diversity of roles for each of the GLP family members. Significantly, one of the grapevine GLP genes, VvGLP3, is induced specifically by E. necator infection and expression is closely linked to the site of infection. Subcellular localization of VvGLP3 determined by transient expression of a VvGLP3:GFP fusion construct in onion cells indicated that the recombinant protein was targeted to the cell wall. Recombinant VvGLP3 was successfully expressed in Arabidopsis thaliana and the partially purified recombinant protein was demonstrated to have superoxide dismutase activity. This data has provided an insight into the diverse nature of the GLP family in grapevine and suggests that VvGLP3 may be involved in the defense response against E. necator.

The effects of 1-methylcyclopropene on the shelf life of minimally processed leafy asian vegetables

The effect of 1-methylcyclopropene (1-MCP) on shelf life and the ethylene sensitivity of detached leaves from six leafy asian vegetables (Chinese mustard, choy sum, garland chrysanthemum, mibuna, mizuna and tatsoi) commonly used in minimal processing was examined. All vegetables studied were significantly affected by the presence of 1 μl/l ethylene. This reduction was primarily due to yellowing except in mizuna and mibuna. In the absence of ethylene, 1-MCP treatment only increased shelf life for mizuna and tatsoi (21 and 67% increase, respectively). In contrast, 1-MCP treatment in the presence of ethylene significantly protected Chinese mustard, choy sum, garland chrysanthemum and tatsoi. 1-MCP may, therefore, be ideal for use on minimally processed leafy asian vegetables in situations of co-storage or co-retail with ethylene-producing commodities.

Production of reactive oxygen species during non-specific elicitation, non-host resistance and field resistance expression in cultured tobacco cells

We examined production of reactive oxygen species (ROS) and induction of cell death in tissue-cultured tobacco cells undergoing different disease resistance responses. A superoxide-dependent hypersensitive response occurs during both the race-specific resistance response of tobacco cells challenged with incompatible zoospores of Phytophthora nicotianae and during non-specific elicitation of tobacco cells challenged with Phytophthora glucan elicitors extracted from the fungal cell wall. Inhibition studies are consistent with dependence upon endogenous Ca2+ levels, and with involvement of NAD(P)H oxidase and peroxidases in production of ROS during both specific and non-specific elicitation. The patterns of resistance expression during non-host resistance or field resistance responses appear to be similar to race-specific resistance expression with regard to the timing and order of events. However, the intensity of the response is very much reduced. In contrast, during non-specific elicitation, these temporal patterns are significantly altered. The differences in timing, intensity and extent of responses during different modes of disease resistance expression indicate that stimulation of cultured plant cells with non-specific soluble fractions in order to model in planta events during plant / Oomycete and, by implication, plant / fungal interactions, has significant limitations.

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.

Natural variation for Fe-efficiency is associated with upregulation of Strategy I mechanisms and enhanced citrate and ethylene synthesis in Pisum sativum L.

Iron (Fe)-deficiency is a common abiotic stress in Pisum sativum L. grown in many parts of the world. The aim of the study was to investigate variation in tolerance to Fe deficiency in two pea genotypes, Santi (Fe-efficient) and Parafield (Fe-inefficient). Fe deficiency caused greater declines in chlorophyll score, leaf Fe concentration and root–shoot development in Parafield compared to Santi, suggesting greater Fe-efficiency in Santi. Fe chelate reductase activity and ethylene production were increased in the roots of Santi and to a lesser extent in Parafield under Fe deficiency, while proton extrusion was only occurred in Santi. Moreover, expression of the Fe chelate reductase gene, FRO1, and Fe transporter, RIT1 were upregulated in Fe-deficient roots of Santi. Expression of HA1 (proton extrusion) was also significantly higher in Santi when compared to Parafield grown in Fe-deficient conditions. Furthermore, the application of the ethylene biosynthesis inhibitor, 1-aminoisobutyric acid reduced the Fe chelate reductase activity, supporting a direct role for ethylene in its induction. A significant increase in root citrate was only observed in Santi under Fe deficiency indicating a role for citrate in the Fe-efficiency mechanism. Taken together, our physiological and molecular data indicate that genotypic variation in tolerance to Fe deficiency in Santi and Parafield plants is a result of variation in a number of Strategy I mechanisms and also suggest a direct role for ethylene in Fe reductase activity. The pea cultivar, Santi provides a new source of Fe-efficiency that can be exploited to breed more Fe-efficient peas.

Genome-Wide Identification of MicroRNAs in Leaves and the Developing Head of Four Durum Genotypes during Water Deficit Stress.

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in plant development and abiotic stress responses. The miRNA transcriptome (miRNAome) under water deficit stress has been investigated in many plant species, but is poorly characterised in durum wheat (Triticum turgidum L. ssp. durum). Water stress during early reproductive stages can result in significant yield loss in durum wheat and this study describes genotypic differences in the miRNAome between water deficit tolerant and sensitive durum genotypes. Small RNA libraries (96 in total) were constructed from flag leaf and developing head tissues of four durum genotypes, with or without water stress to identify differentially abundant miRNAs. Illumina sequencing detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins with 66 conserved miRNAs and five novel miRNA hairpins differentially abundant under water deficit stress. Ten miRNAs (seven conserved, three novel) were validated through qPCR. Several conserved and novel miRNAs showed unambiguous inverted regulatory profiles between the durum genotypes. Several miRNAs also showed differential abundance between two tissue types regardless of treatment. Predicted mRNA targets (130) of four novel durum miRNAs were characterised using Gene Ontology (GO) which revealed functions common to stress responses and plant development. Negative correlation was observed between several target genes and the corresponding miRNA under water stress. For the first time, we present a comprehensive study of the durum miRNAome under water deficit stress. The identification of differentially abundant miRNAs provides molecular evidence that miRNAs are potential determinants of water stress tolerance in durum wheat. GO analysis of predicted targets contributes to the understanding of genotypic physiological responses leading to stress tolerance capacity. Further functional analysis of specific stress responsive miRNAs and their interaction with targets is ongoing and will assist in developing future durum wheat varieties with enhanced water deficit stress tolerance.

Identification and characterisation of barley (Hordeum vulgare) respiratory burst oxidase homologue family members

Respiratory burst oxidase homologues (RBOHs) of the human phagocyte gp91phox gene have been isolated from several plant species and the proteins that they encode have been shown to play important roles in the cellular response to biotic stress via the production of superoxide. In this study we have identified and preliminarily characterised six RBOHs from barley (Hordeum vulgare L.). Conservation of the genomic structure and conceptual protein sequence was observed between all six barley RBOH genes when compared with Arabidopsis and rice RBOH gene family members. Four of the six barley RBOH transcripts had wide-spread constitutive spatial expression patterns. The inducible expression profiles of HvRBOHF1 and HvRBOHF2 in response to infection by the necrotrophic fungal pathogens Pyrenophora teres f. teres Drechsler and Rhynchosporium secalis (Oudem) J. Davis were further characterised by quantitative real-time PCR (qPCR). Increased expression of both transcripts was observed in leaf epidermal tissue in response to infection, which is in keeping with a suggested role for both transcripts in the early oxidative burst during the plant response to pathogen invasion. This research provides a basis for further analysis and establishment of the roles of this RBOH family in various reactive oxygen species dependent processes in barley.

Antioxidant capacity and vitamin E in barley: Effect of genotype and storage

Antioxidants, including vitamin E, may have a positive effect on human health and prolong storage of food items. Vitamin E content and antioxidant capacity were measured in 25 barley genotypes before and after 4 months storage at 10 °C using high performance liquid chromatography (HPLC) and ability to scavenge DPPH radicals, respectively. As expected, α-tocotrienol (α-T3) and α-tocopherol (α-T) were the predominant tocol isomers. Vitamin E content and antioxidant capacity varied significantly among genotypes. Vitamin E ranged from 8.5 to 31.5 μg/g dry weight (DW) while ascorbic acid equivalent antioxidant capacity (AEAC) varied from 57.2 to 158.1 mg AEAC/100 g fresh weight (FW). Generally, lower vitamin E content or antioxidant capacity was observed in hulless or coloured genotypes. These results suggest that some genotypes are potential candidates for breeding of barley cultivars with high vitamin E content or antioxidant capacity at harvest, even after storage.

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5′ RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.