James E. Dombrowski

Salt Stress Activation of Wound-Related Genes in Tomato Plants

Plants respond to various stresses by expressing distinct sets of genes. The effects of multiple stresses on plants and their interactions are not well understood. We have discovered that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato (Lycopersicon esculentum) plants. Salt stress was also found to enhance the plants response to wounding locally and systemically. The tomato mutant (def-1), which has an impairment in the octadecanoid pathway, displayed a severe reduction in the accumulation of proteinase inhibitors under salt stress, indicating that salt stress-induced accumulation of proteinase inhibitors was jasmonic acid dependent. The analysis of salt stress in another tomato mutant, spr-1, which carries a mutation in a systemin-specific signaling component, and transgenic tomato plants that express an antisense-prosystemin cDNA, showed that prosystemin activity was not required for the salt-induced accumulation of proteinase inhibitors, but was necessary to achieve maximal levels. These results suggest that a prosystemin independent- but jasmonic acid-dependent pathway is utilized for proteinase inhibitor accumulation in response to salt stress.

Identification and characterization of a salt stress-inducible zinc finger protein from Festuca arundinacea

BackgroundIncreased biotic and abiotic plant stresses due to climate change together with an expected global human population of over 9 billion by 2050 intensifies the demand for agricultural production on marginal lands. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. Forage grasses are a critical component of feed used in livestock production worldwide, with many of these same species of grasses being utilized for lawns, erosion prevention, and recreation. Consequently, it is important to develop a better understanding of salt tolerance in forage and related grass species.FindingsA gene encoding a ZnF protein was identified during the analysis of a salt-stress suppression subtractive hybridization (SSH) expression library from the forage grass species Festuca arundinacea. The expression pattern of FaZnF was compared to that of the well characterized gene for delta 1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in proline biosynthesis, which was also identified in the salt-stress SSH library. The FaZnF and P5CS genes were both up-regulated in response to salt and drought stresses suggesting a role in dehydration stress. FaZnF was also up-regulated in response to heat and wounding, suggesting that it might have a more general function in multiple abiotic stress responses. Additionally, potential downstream targets of FaZnF (a MAPK [Mitogen-Activated Protein Kinase], GST [Glutathione-S-Transferase] and lipoxygenase L2) were found to be up-regulated in calli overexpressing FaZnF when compared to control cell lines.ConclusionsThis work provides evidence that FaZnF is an AN1/A20 zinc finger protein that is involved in the regulation of at least two pathways initiated by the salt stress response, thus furthering our understanding of the mechanisms of cellular action during a stress that is applicable to commercial crops worldwide.

Methanol and ethanol modulate responses to danger- and microbe-associated molecular patterns.

Methanol is a byproduct of cell wall modification, released through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play not only a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. Molecular mechanisms that explain how methanol affects plant defenses are poorly understood. Here we show that exogenously supplied methanol alone has weak effects on defense signaling in three dicot species, however, it profoundly alters signaling responses to danger- and microbe-associated molecular patterns (DAMPs, MAMPs) such as the alarm hormone systemin, the bacterial flagellum-derived flg22 peptide, and the fungal cell wall-derived oligosaccharide chitosan. In the presence of methanol the kinetics and amplitudes of DAMP/MAMP-induced MAP kinase (MAPK) activity and oxidative burst are altered in tobacco and tomato suspension-cultured cells, in Arabidopsis seedlings and tomato leaf tissue. As a possible consequence of altered DAMP/MAMP signaling, methanol suppressed the expression of the defense genes PR-1 and PI-1 in tomato. In cell cultures of the grass tall fescue (Festuca arundinacea, Poaceae, Monocots), methanol alone activates MAPKs and increases chitosan-induced MAPK activity, and in the darnel grass Lolium temulentum (Poaceae), it alters wound-induced MAPK signaling. We propose that methanol can be recognized by plants as a sign of the damaged self. In dicots, methanol functions as a DAMP-like alarm signal with little elicitor activity on its own, whereas it appears to function as an elicitor-active DAMP in monocot grasses. Ethanol had been implicated in plant stress responses, although the source of ethanol in plants is not well established. We found that it has a similar effect as methanol on responses to MAMPs and DAMPs.

Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses

Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grass species Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly propagated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPK activity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to the wound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the wound response in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity of the 44 kDa MAPK over the activity in control plants.

Enhanced Oxidative Stress Resistance through Activation of a Zinc Deficiency Transcription Factor in Brachypodium distachyon

A zinc deficiency transcription factor moderates oxidative stress resistance. Identification of viable strategies to increase stress resistance of crops will become increasingly important for the goal of global food security as our population increases and our climate changes. Considering that resistance to oxidative stress is oftentimes an indicator of health and longevity in animal systems, characterizing conserved pathways known to increase oxidative stress resistance could prove fruitful for crop improvement strategies. This report argues for the usefulness and practicality of the model organism Brachypodium distachyon for identifying and validating stress resistance factors. Specifically, we focus on a zinc deficiency B. distachyon basic leucine zipper transcription factor, BdbZIP10, and its role in oxidative stress in the model organism B. distachyon. When overexpressed, BdbZIP10 protects plants and callus tissue from oxidative stress insults, most likely through distinct and direct activation of protective oxidative stress genes. Increased oxidative stress resistance and cell viability through the overexpression of BdbZIP10 highlight the utility of investigating conserved stress responses between plant and animal systems.

Abiotic stresses activate a MAPkinase in the model grass species Lolium temulentum.

Forage and turf grasses are utilized in diverse environments that expose them to a variety of abiotic stresses, however very little is known concerning the perception or molecular responses to these various stresses. In the model grass species Lolium temulentum, a 46kDa mitogen-activated protein kinase (MAPK) was activated in the leaf within 10min of exposing the roots to salt stress. When plants were subjected cold stress, no significant activation of the MAPK was observed. However, the 46kDa MAPK was rapidly activated in the leaves of plants within 3min of exposure to heat stress. Previously, mechanical wounding has been shown to rapidly activate a 46kDa and a 44kDa MAPK in L. temulentum. The wound activation of the MAPKs was delayed and diminished in plants undergoing cold treatment. In plants subjected simultaneously to 40°C and wounding, the activation of the 46kDa MAPK was enhanced. However if plants were subjected to heat and cold stress for more than 2h or exposed to 300mM NaCl for 24h prior to wounding, the wound activation of the 46kDa and a 44kDa MAPKs were significantly inhibited. These results suggest that the 46kDa MAPK plays a role in the response to various environmental stimuli.

Transformation of Epichloë typhina by electroporation of conidia

BackgroundChoke, caused by the endophytic fungus Epichloë typhina, is an important disease affecting orchardgrass (Dactylis glomerata L.) seed production in the Willamette Valley. Little is known concerning the conditions necessary for successful infection of orchardgrass by E. typhina. Detection of E. typhina in plants early in the disease cycle can be difficult due to the sparse distribution of hyphae in the plant. Therefore, a sensitive method to detect fungal infection in plants would provide an invaluable tool for elucidating the conditions for establishment of infection in orchardgrass. Utilization of a marker gene, such as the green fluorescent protein (GFP), transformed into Epichloë will facilitate characterization of the initial stages of infection and establishment of the fungus in plants.FindingsWe have developed a rapid, efficient, and reproducible transformation method using electroporation of germinating Epichloë conidia isolated from infected plants.ConclusionsThe GFP labelled E. typhina provides a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke disease in orchardgrass.

Virus induced gene silencing in Lolium temulentum

Lolium temulentum L. is valuable as a model species for studying abiotic stress in closely related forage and turf grasses, many of which are polyploid outcrossing species. As with most monocot species, Agrobacterium-mediated transformation of L. temulentum is still challenging, time consuming and inefficient. The aim of this study was to use the Barley stripe mosaic virus (BSMV) vector to develop a virus induced gene silencing (VIGS) system in L. temulentum to facilitate functional gene analysis. Plants infected with the BSMV vector containing a small region of the L. temulentumphytoene desaturase (PDS) gene, which is commonly used as a phenotypic marker in VIGS studies, displayed the characteristic albino phenotype observed in PDS silenced plants. Decreased PDS gene expression in these albino leaves was also confirmed by quantitative reverse transcriptase polymerase chain reaction. To evaluate the utility of using the VIGS vector for gene interactions in L. temulentum, a region of the translation factor eIF4A gene was incorporated into the VIGS vector. Inoculation of young seedlings with this modified vector resulted in a partial reduction in the expression of eIF4A suggesting that this vector will be a useful tool to examine gene interactions in L. temulentum.

Genome-wide (ChIP-seq) identification of target genes regulated by BdbZIP10 during paraquat-induced oxidative stress

BackgroundbZIP transcription factors play a significant role in many aspects of plant growth and development and also play critical regulatory roles during plant responses to various stresses. Overexpression of the Brachypodium bZIP10 (Bradi1g30140) transcription factor conferred enhanced oxidative stress tolerance and increased viability when plants or cells were exposed to the herbicide paraquat. To gain a better understanding of genes involved in bZIP10 conferred oxidative stress tolerance, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) was performed on BdbZIP10 overexpressing plants in the presence of oxidative stress.ResultsWe identified a transcription factor binding motif, TGDCGACA, different from most known bZIP TF motifs but with strong homology to the Arabidopsis zinc deficiency response element. Analysis of the immunoprecipitated sequences revealed an enrichment of gene ontology groups with metal ion transmembrane transporter, transferase, catalytic and binding activities. Functional categories including kinases and phosphotransferases, cation/ion transmembrane transporters, transferases (phosphorus-containing and glycosyl groups), and some nucleoside/nucleotide binding activities were also enriched.ConclusionsBrachypodium bZIP10 is involved in zinc homeostasis, as it relates to oxidative stress.