Rhonda C. Foley

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.

Interactions between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences.

Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.

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 IIs role in mitochondrial diseases across eukaryotes.

Isolation of a Vicia faba metallothionein-like gene: expression in foliar trichomes

Animal metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins that bind to heavy metals and are believed to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants although their function remains to be elucidated. We describe the isolation and characterization of a bean cDNA encoding an MT-like protein. The bean gene, called MT, was isolated as a result of a differential screen for genes that are expressed in leaves but not in the most common cell type, the mesophyll cell. MT contained two regions with abundant cysteines and sequence comparison found that MT had greatest homology to MT-like subtype 2 from other plant species. Northern blot analysis demonstrated that MT was expressed in the leaf, stem and flower, at very low levels in roots and was not detectable in mesophyll protoplasts. MT transcript levels were not significantly affected by treatment with Cu, Zn or Cd. In the leaf, in situ hybridization studies demonstrated striking cell specificity with MT expression confined predominantly to trichomes. Possible explanations for the pronounced expression of MT in leaf trichomes are discussed.

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.

Analysis of type 1 metallothionein cDNAs in Vicia faba

In animals and fungi, small cysteine-rich proteins called metallothioneins (MTs) play a role in heavy metal tolerance. MT genes have been isolated in plants, but their function remains to be elucidated. We have isolated two distinct Vicia faba MT genes that belong to the type 1 group of plant MT genes in contrast to a MT gene we previously isolated that belongs to type 2. We found similarities and differences between the V. faba MT genes. The RNA expression patterns differed and this was most pronounced in roots, which contained high MT1 but very low MT2 RNA levels. Like MT2, MT1 transcript levels were not significantly affected by treatment with Cd, Cu, Fe and Zn, at least under the experimental conditions. MT RNA levels varied in leaves and stem internodes of different developmental ages, with the highest expression in the older tissue. The levels of MT RNA correlated inversely with endogenous Cd, Cu and Fe levels within different internodes, but not with a number of other metals tested (including Zn). The three bean MTs were expressed in Escherichia coli and found to bind Cd, Cu and Zn but not to Fe. The MTs were tested to determine if they differed in their ability to bind a specific metal but no significant differences in binding were observed.

Identification and characterisation of seed storage protein transcripts from Lupinus angustifolius

BackgroundIn 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.ResultsGenes 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 β, 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 (β 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 β (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 β conglutin family were potential allergens.ConclusionThis 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.

Mutant Analysis in Arabidopsis Provides Insight into the Molecular Mode of Action of the Auxinic Herbicide Dicamba

Herbicides that mimic the natural auxin indole-3-acetic acid are widely used in weed control. One common auxin-like herbicide is dicamba, but despite its wide use, plant gene responses to dicamba have never been extensively studied. To further understand dicambas mode of action, we utilized Arabidopsis auxin-insensitive mutants and compared their sensitivity to dicamba and the widely-studied auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The mutant axr4-2, which has disrupted auxin transport into cells, was resistant to 2,4-D but susceptible to dicamba. By comparing dicamba resistance in auxin signalling F-box receptor mutants (tir1-1, afb1, afb2, afb3, and afb5), only tir1-1 and afb5 were resistant to dicamba, and this resistance was additive in the double tir1-1/afb5 mutant. Interestingly, tir1-1 but not afb5 was resistant to 2,4-D. Whole genome analysis of dicamba-induced gene expression showed that 10 hours after application, dicamba stimulated many stress-responsive and signalling genes, including those involved in biosynthesis or signalling of auxin, ethylene, and abscisic acid (ABA), with TIR1 and AFB5 required for the dicamba-responsiveness of some genes. Research into dicamba-regulated gene expression and the selectivity of auxin receptors has provided molecular insight into dicamba-regulated signalling and could help in the development of novel herbicide resistance in crop plants.

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.

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

BackgroundLupinus 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.ResultsA 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.ConclusionsThe 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.