A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a key rotational crop for sustainable farming systems, whose grain is high in protein content. It is a gluten-free, non-genetically modified, alternative protein source to soybean (Glycine max) and as such has gained interest as a human food ingredient. Here, we present a chromosome-length reference genome for the species and a pan-genome assembly comprising 55 NLL lines, including Australian and European cultivars, breeding lines and wild accessions. We present the core and variable genes for the species and report on the absence of essential mycorrhizal associated genes. The genome and pan-genomes of NLL and its close relative white lupin (Lupinus albus) are compared. Furthermore, we provide additional evidence supporting LaRAP2-7 as the key alkaloid regulatory gene for NLL and demonstrate the NLL genome is underrepresented in classical NLR disease resistance genes compared to other sequenced legume species. The NLL genomic resources generated here coupled with previously generated RNA sequencing datasets provide new opportunities to fast-track lupin crop improvement.

Characterization of the genetic factors affecting quinolizidine alkaloid biosynthesis and its response to abiotic stress in narrow-leafed lupin (Lupinus angustifolius L.).

Quinolizidine alkaloids (QAs) are toxic secondary metabolites that complicate the end use of narrow-leafed lupin (NLL; Lupinus angustifolius L.) grain, as levels sometimes exceed the industry limit for its use as a food and feed source. The genotypic and environmental influences on QA production in NLL are poorly understood. Here, the expression of QA biosynthetic genes was analysed in vegetative and reproductive tissues of bitter (high QA) and sweet (low QA) accessions. It was demonstrated that sweet accessions are characterized by lower QA biosynthetic gene expression exclusively in leaf and stem tissues than bitter NLL, consistent with the hypothesis that QAs are predominantly produced in aerial tissues and transported to seeds, rather than synthesized within the seed itself. This analysis informed our identification of additional candidate genes involved in QA biosynthesis. Drought and temperature stress are two major abiotic stresses that often occur during NLL pod set. Hence, we assessed the effect of drought, increased temperature, and their combination, on QA production in three sweet NLL cultivars. A cultivar-specific response to drought and temperature in grain QA levels was observed, including the identification of a cultivar where alkaloid levels did not change with these stress treatments.

A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant-microbe interactions and legume evolution.

Lupins are important grain legume crops that form a critical part of sustainable farming systems, reducing fertilizer use and providing disease breaks. It has a basal phylogenetic position relative to other crop and model legumes and a high speciation rate. Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is gaining popularity as a health food, which is high in protein and dietary fibre but low in starch and gluten-free. We report the draft genome assembly (609 Mb) of NLL cultivar Tanjil, which has captured >98% of the gene content, sequences of additional lines and a dense genetic map. Lupins are unique among legumes and differ from most other land plants in that they do not form mycorrhizal associations. Remarkably, we find that NLL has lost all mycorrhiza-specific genes, but has retained genes commonly required for mycorrhization and nodulation. In addition, the genome also provided candidate genes for key disease resistance and domestication traits. We also find evidence of a whole-genome triplication at around 25 million years ago in the genistoid lineage leading to Lupinus. Our results will support detailed studies of legume evolution and accelerate lupin breeding programmes.

Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches.

BACKGROUND: The major proteins in lupin seeds are conglutins that have primary roles in supplying carbon, sulphur and nitrogen and energy for the germinating seedling. They fall into four families; α, ß, γ and δ. Interest in these conglutins is growing as family members have been shown to have beneficial nutritional and pharmaceutical properties. RESULTS: An in-depth transcriptome and draft genome from the narrow-leafed lupin (NLL; Lupinus angustifolius) variety, Tanjil, were examined and 16 conglutin genes were identified. Using RNAseq data sets, the structure and expression of these 16 conglutin genes were analysed across eight lupin varieties from five lupin species. Phylogenic analysis suggest that the α and γ conglutins diverged prior to lupin speciation while ß and δ members diverged both prior and after speciation. A comparison of the expression of the 16 conglutin genes was performed, and in general the conglutin genes showed similar levels of RNA expression among varieties within species, but quite distinct expression patterns between lupin species. Antibodies were generated against the specific conglutin families and immunoblot analyses were used to compare the levels of conglutin proteins in various tissues and during different stages of seed development in NLL, Tanjil, confirming the expression in the seed. This analysis showed that the conglutins were expressed highly at the mature seed stage, in all lupin species, and a range of polypeptide sizes were observed for each conglutin family. CONCLUSIONS: This study has provided substantial information on the complexity of the four conglutin families in a range of lupin species in terms of their gene structure, phylogenetic relationships as well as their relative RNA and protein abundance during seed development. The results demonstrate that the majority of the heterogeneity of conglutin polypeptides is likely to arise from post-translational modification from a limited number of precursor polypeptides rather than a large number of different genes. Overall, the results demonstrate a high degree of plasticity for conglutin expression during seed development in different lupin species.

Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence on plant stress gene regulation and defense.

Mitochondria are both a source of ATP and a site of reactive oxygen species (ROS) production. However, there is little information on the sites of mitochondrial ROS (mROS) production or the biological role of such mROS in plants. We provide genetic proof that mitochondrial complex II (Complex II) of the electron transport chain contributes to localized mROS that regulates plant stress and defense responses. We identify an Arabidopsis mutant in the Complex II subunit, SDH1-1, through a screen for mutants lacking GSTF8 gene expression in response to salicylic acid (SA). GSTF8 is an early stress-responsive gene whose transcription is induced by biotic and abiotic stresses, and its expression is commonly used as a marker of early stress and defense responses. Transcriptional analysis of this mutant, disrupted in stress responses 1 (dsr1), showed that it had altered SA-mediated gene expression for specific downstream stress and defense genes, and it exhibited increased susceptibility to specific fungal and bacterial pathogens. The dsr1 mutant also showed significantly reduced succinate dehydrogenase activity. Using in vivo fluorescence assays, we demonstrated that root cell ROS production occurred primarily from mitochondria and was lower in the mutant in response to SA. In addition, leaf ROS production was lower in the mutant after avirulent bacterial infection. This mutation, in a conserved region of SDH1-1, is a unique plant mitochondrial mutant that exhibits phenotypes associated with lowered mROS production. It provides critical insights into Complex II function with implications for understanding Complex II's role in mitochondrial diseases across eukaryotes.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing.

BACKGROUND: Lupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species. RESULTS: A NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers. CONCLUSIONS: The NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species.

Identification and characterisation of seed storage protein transcripts from Lupinus angustifolius.

BACKGROUND: In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L.), also known as narrow-leaf lupin (NLL) is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch. RESULTS: Genes encoding the seed storage proteins of NLL were characterised by sequencing cDNA clones derived from developing seeds. Four families of seed storage proteins were identified and comprised three unique α, seven ß, two γ and four δ conglutins. This study added eleven new expressed storage protein genes for the species. A comparison of the deduced amino acid sequences of NLL conglutins with those available for the storage proteins of Lupinus albus (L.), Pisum sativum (L.), Medicago truncatula (L.), Arachis hypogaea (L.) and Glycine max (L.) permitted the analysis of a phylogenetic relationships between proteins and demonstrated, in general, that the strongest conservation occurred within species. In the case of 7S globulin (ß conglutins) and 2S sulphur-rich albumin (δ conglutins), the analysis suggests that gene duplication occurred after legume speciation. This contrasted with 11S globulin (α conglutin) and basic 7S (γ conglutin) sequences where some of these sequences appear to have diverged prior to speciation. The most abundant NLL conglutin family was ß (56%), followed by α (24%), δ (15%) and γ (6%) and the transcript levels of these genes increased 103 to 106 fold during seed development. We used the 16 NLL conglutin sequences identified here to determine that for individuals specifically allergic to lupin, all seven members of the ß conglutin family were potential allergens. CONCLUSION: This study has characterised 16 seed storage protein genes in NLL including 11 newly-identified members. It has helped lay the foundation for efforts to use molecular breeding approaches to improve lupins, for example by reducing allergens or increasing the expression of specific seed storage protein(s) with desirable nutritional properties.

Foliar resistance to Rhizoctonia solani in Arabidopsis is compromised by simultaneous loss of ethylene, jasmonate and PEN2 mediated defense pathways.

Rhizoctonia solani causes damaging yield losses on most major food crops. R. solani isolates belonging to anastomosis group 8 (AG8) are soil-borne, root-infecting pathogens with a broad host range. AG8 isolates can cause disease on wheat, canola and legumes, however Arabidopsis thaliana is heretofore thought to possess non-host resistance as A. thaliana ecotypes, including the reference strain Col-0, are resistant to AG8 infection. Using a mitochondria-targeted redox sensor (mt-roGFP2) and cell death staining, we demonstrate that both AG8 and a host isolate (AG2-1) of R. solani are able to infect A. thaliana roots. Above ground tissue of A. thaliana was found to be resistant to AG8 but not AG2. Genetic analysis revealed that ethylene, jasmonate and PENETRATION2-mediated defense pathways work together to provide resistance to AG8 in the leaves which subsequently enable tolerance of root infections. Overall, we demonstrate a significant difference in defense capabilities of above and below ground tissue in providing resistance to R. solani AG8 in Arabidopsis.

A Plant Stress-Responsive Bioreporter Coupled With Transcriptomic Analysis Allows Rapid Screening for Biocontrols of Necrotrophic Fungal Pathogens.

Streptomyces are soil-borne Actinobacteria known to produce a wide range of enzymes, phytohormones, and metabolites including antifungal compounds, making these microbes fitting for use as biocontrol agents in agriculture. In this study, a plant reporter gene construct comprising the biotic stress-responsive glutathione S-transferase promoter GSTF7 linked to a luciferase output (GSTF7:luc) was used to screen a collection of Actinobacteria candidates for manipulation of plant biotic stress responses and their potential as biocontrol agents. We identified a Streptomyces isolate (KB001) as a strong candidate and demonstrated successful protection against two necrotrophic fungal pathogens, Sclerotinia sclerotiorum and Rhizoctonia solani, but not against a bacterial pathogen (Pseudomonas syringe). Treatment of Arabidopsis plants with either KB001 microbial culture or its secreted compounds induced a range of stress and defense response-related genes like pathogenesis-related (PR) and hormone signaling pathways. Global transcriptomic analysis showed that both treatments shared highly induced expression of reactive oxygen species and auxin signaling pathways at 6 and 24 h posttreatment, while some other responses were treatment specific. This study demonstrates that GSTF7 is a suitable marker for the rapid and preliminary screening of beneficial bacteria and selection of candidates with potential for application as biocontrols in agriculture, including the Streptomyces KB001 that was characterized here, and could provide protection against necrotrophic fungal pathogens.

Plants versus pathogens: an evolutionary arms race.

The analysis of plant-pathogen interactions is a rapidly moving research field and one that is very important for productive agricultural systems. The focus of this review is on the evolution of plant defence responses and the coevolution of their pathogens, primarily from a molecular-genetic perspective. It explores the evolution of the major types of plant defence responses including pathogen associated molecular patterns and effector triggered immunity as well as the forces driving pathogen evolution, such as the mechanisms by which pathogen lineages and species evolve. Advances in our understanding of plant defence signalling, stomatal regulation, R gene-effector interactions and host specific toxins are used to highlight recent insights into the coevolutionary arms race between pathogens and plants. Finally, the review considers the intriguing question of how plants have evolved the ability to distinguish friends such as rhizobia and mycorrhiza from their many foes.

The role of jasmonate signalling in quinolizidine alkaloid biosynthesis, wounding and aphid predation response in narrow-leafed lupin.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites produced in lupin species that protect the plant against insects. They form in vegetative tissues and accumulate to a different extent in the grains: high levels in 'bitter' narrow-leafed lupin (NLL) and low levels in 'sweet' NLL. Grain QA levels vary considerably, and sometimes exceed the industry limit for food and feed purposes. We hypothesised that jasmonates regulate QA biosynthesis in response to environmental stresses such as wounding and aphid predation, which may explain non-genetic variability in grain QA levels. Methyl jasmonate (MeJA)-inducible genes were identified and verified in NLL. Exogenous MeJA application-induced expression of QA biosynthetic genes and QA levels for bitter, but not sweet NLL. Although MeJA-inducible genes responded to wounding, the expression of QA biosynthetic genes was not induced for bitter and sweet NLL. We assessed the effect of aphid predation on QA production for two cultivars - one moderately resistant and one susceptible to aphid predation. Although MeJA-inducible genes responded to aphid predation, no change in QA levels was found for either cultivar. These findings offer insights into the regulation of QA biosynthesis in bitter and sweet NLL and concludes that aphids are not a concern for increasing grain QAs in NLL cultivars.

The Arabidopsis RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) is a biotic stress susceptibility gene.

Crop breeding for improved disease resistance may be achieved through the manipulation of host susceptibility genes. Previously we identified multiple Arabidopsis mutants known as enhanced stress response1 (esr1) that have defects in a KH-domain RNA-binding protein and conferred increased resistance to the root fungal pathogen Fusarium oxysporum. Here, screening the same mutagenized population we discovered two further enhanced stress response mutants that also conferred enhanced resistance to F. oxysporum. These mutants also have enhanced resistance to a leaf fungal pathogen (Alternaria brassicicola) and an aphid pest (Myzus persicae), but not to the bacterial leaf pathogen Pseudomonas syringae. The causal alleles in these mutants were found to have defects in the ESR1 interacting protein partner RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) and subsequently given the allele symbols cpl1-7 and cpl1-8. These results define a new role for CPL1 as a pathogen and pest susceptibility gene. Global transcriptome analysis and oxidative stress assays showed these cpl1 mutants have increased tolerance to oxidative stress. In particular, components of biotic stress responsive pathways were enriched in cpl1 over wild-type up-regulated gene expression datasets including genes related to defence, heat shock proteins and oxidative stress/redox state processes.

Characterization of narrow-leaf lupin (Lupinus angustifolius L.) recombinant major allergen IgE-binding proteins and the natural ß-conglutin counterparts in sweet lupin seed species.

ß-conglutin has been identified as a major allergen for Lupinus angustifolius seeds. The aim of this study was to evaluate the binding of IgE to five recombinant ß-conglutin isoforms (rß) that we overexpressed and purified and to their natural counterparts in different lupin species and cultivars. Western blotting suggested ß-conglutins were the main proteins responsible for the IgE reactivity of the lupin species and cultivars. Newly identified polypeptides from "sweet lupin" may constitute a potential new source of primary or cross-reactive sensitization to lupin, particularly to L. albus and L. angustifolius seed proteins. Several of them exhibited qualitative and quantitative differences in IgE-binding among these species and cultivars, mainly in sera from atopic patients that react to lupin rather than peanut. IgE-binding was more consistent to recombinant ß2 than to any of the other isoforms, making this protein a potential candidate for diagnosis and immunotherapy.

Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites found within the genus Lupinus, some species of which are commercially important grain legume crops including Lupinus angustifolius (narrow-leafed lupin, NLL), L. luteus (yellow lupin), L. albus (white lupin), and L. mutabilis (pearl lupin), with NLL grain being the most largely produced of the four species in Australia and worldwide. While QAs offer the plants protection against insect pests, the accumulation of QAs in lupin grain complicates its use for food purposes as QA levels must remain below the industry threshold (0.02%), which is often exceeded. It is not well understood what factors cause grain QA levels to exceed this threshold. Much of the early work on QA biosynthesis began in the 1970-1980s, with many QA chemical structures well-characterized and lupin cell cultures and enzyme assays employed to identify some biosynthetic enzymes and pathway intermediates. More recently, two genes associated with these enzymes have been characterized, however, the QA biosynthetic pathway remains only partially elucidated. Here, we review the research accomplished thus far concerning QAs in lupin and consider some possibilities for further elucidation and manipulation of the QA pathway in lupin crops, drawing on examples from model alkaloid species. One breeding strategy for lupin is to produce plants with high QAs in vegetative tissues while low in the grain in order to confer insect resistance to plants while keeping grain QA levels within industry regulations. With the knowledge achieved on alkaloid biosynthesis in other plant species in recent years, and the recent development of genomic and transcriptomic resources for NLL, there is considerable scope to facilitate advances in our knowledge of QAs, leading to the production of improved lupin crops.

Narrow-leafed lupin (Lupinus angustifolius L.) ß-conglutin proteins modulate the insulin signaling pathway as potential type 2 diabetes treatment and inflammatory-related disease amelioration.

SCOPE: We have investigated the potential use of ß-conglutin protein isoforms from narrow-leafed lupin (Lupinus angustifolius L.) as a diabetes treatment. METHODS AND RESULTS: We produced purified recombinant ß1-, ß2-, ß3-, ß4-, and ß6-conglutin proteins and showed that ß1, ß3, and ß6 could bind to insulin. To assess ß-conglutin proteins modulatory effect on insulin activation meditated kinases, whole blood and peripheral blood mononuclear cell cultures from type 2 diabetes (T2D) and healthy control subjects (C) were incubated with conglutin proteins. The treatment of peripheral blood mononuclear cells from T2D patients with ß1, ß3, and ß6 proteins increased up to threefold mRNA and protein levels of genes important in insulin signaling pathways, namely insulin receptor substrate 1/p85/AKT/glucose transporter type 4. This was accompanied by a comparable fold-change decrease in the mRNA expression level of pro-inflammatory genes (iNOS and IL-1ß) and proteins compared to healthy controls. The ß2 and ß4 isoforms had no effect on the insulin signaling pathway. However, these ß-conglutin proteins elicited pro-inflammatory effects since levels of mRNA and proteins of inducible nitric oxide synthase and IL 1 beta were increased. CONCLUSION: Our results raise the possibility of using these particular ß-conglutin proteins in the prevention and treatment of diabetes, as well as their potential as anti-inflammatory molecules.

Transcription factors in plant defense and stress responses.

Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic and biotic stresses. Research carried out in the past few years has been productive in identifying transcription factors that are important for regulating plant responses to these stresses. These studies have also revealed some of the complexity and overlap in the responses to different stresses, and are likely to lead to new ways to enhance crop tolerance to disease and environmental stress.

Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi, Rhizoctonia solani in Wheat.

Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS) production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT), 3,3'-diaminobenzidine (DAB) and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction.

Narrow-Leafed Lupin (Lupinus angustifolius) ß1- and ß6-Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae.

Vicilins (7S globulins) are seed storage proteins and constitute the main protein family in legume seeds, particularly in narrow-leafed lupin (Lupinus angustifolius L.; NLL), where seven vicilin genes, called ß1- to ß7-conglutin have been identified. Vicilins are involved in germination processes supplying amino acids for seedling growth and plant development, as well as in some cases roles in plant defense and protection against pathogens. The roles of NLL ß-conglutins in plant defense are unknown. Here the potential role of five NLL ß-conglutin family members in protection against necrotrophic fungal pathogens was investigated and it was demonstrated that recombinant purified 6xHis-tagged ß1- and ß6-conglutin proteins exhibited the strongest in vitro growth inhibitory activity against a range of necrotrophic fungal pathogens compared to ß2, ß3, and ß4 conglutins. To examine activity in vivo, two representative necrotrophic pathogens, the fungus Sclerotinia sclerotiorum and oomycete Phytophthora nicotianae were used. Transient expression of ß1- and ß6-conglutin proteins in Nicotiana benthamiana leaves demonstrated in vivo growth suppression of both of these pathogens, resulting in low percentages of hyphal growth and elongation in comparison to control treated leaves. Cellular studies using ß1- and ß6-GFP fusion proteins showed these conglutins localized to the cell surface including plasmodesmata. Analysis of cellular death following S. sclerotiorum or P. nicotianae revealed both ß1- and ß6-conglutins suppressed pathogen induced cell death in planta and prevented pathogen induced suppression of the plant oxidative burst as determined by protein oxidation in infected compared to mock-inoculated leaves.

Early induction of the Arabidopsis GSTF8 promoter by specific strains of the fungal pathogen Rhizoctonia solani.

The Arabidopsis glutathione S-transferase GSTF8 promoter directs root-specific responses to stress. In this study, the response of this promoter to plant infection with Rhizoctonia solani was investigated using a luciferase reporter system. Arabidopsis seedlings harboring the GSTF8:luciferase construct were monitored in vivo for bioluminescence following infection with R. solani. Although the reporter gene was induced in infected roots, the response differed markedly between R. solani strains and was not observed with aggressive strains that caused death of the seedlings. The three strains tested in detail progressed through typical stages of infection, but ZG1-1 induced the GSTF8 promoter in most seedlings, ZG3 induced it in approximately 25% of seedlings, and ZG5 caused little response. Induction of specific root segments occurred early in the infection process in root regions with very limited mycelium visible. In root segments with substantial mycelium, GSTF8 promoter activity no longer was observed. Induction by ZG1-1 also was observed in plants harboring a tetramer of the ocs element from the GSTF8 promoter, suggesting that this element helps mediate the response. Crossing GSTF8:luciferase plants with plants harboring an Nah-G construct that degrades salicylic acid did not abolish the response, indicating that the GSTF8 promoter response to R. solani may be mediated by signals other than salicylic acid.

TGA5 acts as a positive and TGA4 acts as a negative regulator of ocs element activity in Arabidopsis roots in response to defence signals.

TGA/OBF family members are bZIP transcription factors that bind to the octopine synthase (ocs) element, a plant promoter sequence that has been strongly linked to defence/stress responses. Intron-containing hairpin (ihp) constructs were used to generate Arabidopsis lines with reduced expression of TGA4 and TGA5. No visible phenotypic differences were observed between ihpTGA and wild-type (WT) plants. However, the ihpTGA4 and ihpTGA5 plants had opposite affects on ocs element activity, with the ihpTGA4 lines enhancing, and the ihpTGA5 lines reducing, the response of an ocs element construct to the key defence signals, salicylic acid (SA) and H(2)O(2), in roots.