Erik Andreasson

Myrosinase: gene family evolution and herbivore defense in Brassicaceae

Glucosinolates are a category of secondary products present primarily in species of the order Capparales. When tissue is damaged, for example by herbivory, glucosinolates are degraded in a reaction catalyzed by thioglucosidases, denoted myrosinases, also present in these species. Thereby, toxic compounds such as nitriles, isothiocyanates, epithionitriles and thiocyanates are released. The glucosinolate-myrosinase system is generally believed to be part of the plants defense against insects, and possibly also against pathogens. In this review, the evolution of the system and its impact on the interaction between plants and insects are discussed. Further, data suggesting additional functions in the defense against pathogens and in sulfur metabolism are reviewed.

Convergence and specificity in the Arabidopsis MAPK nexus

Although mitogen-activated protein kinase (MAPK) signal transduction cascades are known regulators of various aspects of plant biology, our knowledge of these systems has been largely restricted to a small subset of the MAPKs. However, global analyses are now revealing that many more of these kinases are probably engaged in modulating developmental and fitness adaptation processes in the plant kingdom. In this review, we show how these new findings are beginning to define the overall architecture of plant MAPK signaling, with a particular focus on the interplay between the terminal MPKs and their activators, inactivators and cellular targets.

Update on glucosinolate metabolism and transport

Abstract Glucosinolates are secondary plant metabolites found mainly in the order Capparales. Tissue disruption allows rapid enzymatic degradation of glucosinolates by specific thioglucosidases, denoted myrosinases. Within the last few years, significant progresses in our understanding of glucosinolate biosynthesis and degradation have been achieved. In particular, the Arabidopsis thaliana genome-sequencing project has accelerated the identification and characterization of genes involved in the glucosinolate metabolism. More evidence has accumulated for the hypothesis that the glucosinolate-myrosinase system has evolved from the prevalent system of cyanogenic glucosides and corresponding O -β-glucosidases. Glucosinolates have been shown to be taken up by a specific carrier system and transported by phloem. The de novo biosynthesis, degradation and transport of glucosinolates may constitute a delicately regulated dynamic diagram, through which various physiological functions are fulfilled. There is a rising interest in controlling the level of glucosinolates in crops to improve pest resistance and nutritional value. Genes identified in Arabidopsis thaliana will provide important tools to initiate molecular strategies to modulate the quantity and quality of glucosinolates in a tissue-specific manner in closely related Brassica crops. This review summarizes current knowledge on glucosinolate biosynthesis, degradation and mobilization, and provides a comprehensive discussion and update on the regulation, physiological functions and genetic engineering of glucosinolate metabolism and transport.

Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses

pH is a highly variable environmental factor for the root, and plant cells can modify apoplastic pH for nutrient acquisition and in response to extracellular signals. Nevertheless, surprisingly few effects of external pH on plant gene expression have been reported. We have used microarrays to investigate whether external pH affects global gene expression. In Arabidopsis thaliana roots, 881 genes displayed at least twofold changes in transcript abundance 8 h after shifting medium pH from 6.0 to 4.5, identifying pH as a major affector of global gene expression. Several genes responded within 20 min, and gene responses were also observed in leaves of seedling cultures. The pH 4.5 treatment was not associated with abiotic stress, as evaluated from growth and transcriptional response. However, the observed patterns of global gene expression indicated redundancies and interactions between the responses to pH, auxin and pathogen elicitors. In addition, major shifts in gene expression were associated with cell wall modifications and Ca(2+) signalling. Correspondingly, a marked overrepresentation of Ca(2+)/calmodulin-associated motifs was observed in the promoters of pH-responsive genes. This strongly suggests that plant pH recognition involves intracellular Ca(2+). Overall, the results emphasize the previously underappreciated role of pH in plant responses to the environment.

An Arabidopsis Protein Phosphorylated in Response to Microbial Elicitation, AtPHOS32, Is a Substrate of MAP Kinases 3 and 6

Although mitogen-activated protein kinases (MAPKs) have been shown to be activated by a wide range of biotic and abiotic stimuli in diverse plant species, few in vivo substrates for these kinases have been identified. While studying proteins that are differentially phosphorylated upon treatment of Arabidopsis suspension cultures with the general bacterial elicitor peptide flagellin-22 (flg22), we identified two proteins with endogenous nickel binding properties that become phosphorylated after flg22 elicitation. These highly related proteins, AtPHOS32 and AtPHOS34, show similarity to bacterial universal stress protein A. We identified one of the phosphorylation sites on AtPHOS32 by nanoelectrospray ionization tandem mass spectrometry. Phosphorylation in a phosphoSer-Pro motif indicated that this protein may be a substrate of MAPKs. Using in vitro kinase assays, we confirmed that AtPHOS32 is a substrate of both AtMPK3 and AtMPK6. Specificity of phosphorylation was demonstrated by site-directed mutagenesis of the first phosphorylation site. In addition, immunosubtraction of both MAPKs from protein extracts removed detectable kinase activity toward AtPHOS32, indicating that the two MAPKs were the predominate kinases recognizing the motif in this protein. Finally, the target phosphorylation site in AtPHOS32 is conserved in AtPHOS34 and among apparent orthologues from many plant species, indicating that phosphorylation of these proteins by AtMPK3 and AtMPK6 orthologues has been conserved throughout evolution.

Plant secretome proteomics

The plant secretome refers to the set of proteins secreted out of the plant cell into the surrounding extracellular space commonly referred to as the apoplast. Secreted proteins maintain cell structure and acts in signaling and are crucial for stress responses where they can interact with pathogen effectors and control the extracellular environment. Typically, secreted proteins contain an N-terminal signal peptide and are directed through the endoplasmic reticulum/Golgi pathway. However, in plants many proteins found in the secretome lack such a signature and might follow alternative ways of secretion. This review covers techniques to isolate plant secretomes and how to identify and quantify their constituent proteins. Furthermore, bioinformatical tools to predict secretion signals and define the putative secretome are presented. Findings from proteomic studies and important protein families of plant secretomes, such as proteases and hydrolases, are highlighted.

Induced resistance in potato to Phytphthora infestans —effects of BABA in greenhouse and field tests with different potato varieties

We have investigated to what degree induced resistance with β-aminobutyric acid (BABA) can protect potato from late blight infection under Swedish field conditions and if synergistic interactions occur if BABA is applied in combination with a commonly used fungicide, Shirlan. In greenhouse experiments we also investigated the durability of BABA induced resistance, the dose-response relationships in susceptible (Bintje) and partially resistant (Ovatio, Suberb) cultivars and effects of combined applications of BABA and fungicides. We found a clear effect of BABA on P. infestans infection of greenhouse grown potato plants. The lesion sizes were reduced by on average 40–50% compared to untreated control. However, this effect lasted for only 4–5 days after BABA treatment and then the efficacy was lower. When BABA was given in combination with the fungicides it appeared to have an additive effect both in greenhouse and field experiments. Higher concentrations of BABA gave a stronger protective effect. The partially resistant cultivars Ovatio and Superb reacted to lower concentrations of BABA where no effect was found in susceptible Bintje. According to our field data, 20–25% reduction of the fungicide dose in combination with BABA gave on average the same result on late blight development as full dose Shirlan alone; while reduced dose of Shirlan alone sometimes resulted in less effective protection. Our results indicate that induced resistance could be used in practice in combinations with fungicides in order to reduce the amount of toxic compounds under north European conditions.

Characterization of transgenic Arabidopsis thaliana with metabolically engineered high levels of p-hydroxybenzylglucosinolate.

Abstract. The cytochrome P450 CYP79A1 catalyzes the conversion of l-tyrosine to p-hydroxyphenylacetaldoxime, the first step in the biosynthetic pathway of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. We have demonstrated that introduction of CYP79A1 into Arabidopsis thaliana (L.) Heynh. results in the production of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate (p-OHBG), not found in wild-type A. thaliana (Bak et al., 1999, Plant J. 20: 663–671). In the present study, glucosinolate profiles and contents in various tissues (roots, leaves, stems, closed flower buds and green siliques) of A. thaliana plants expressing CYP79A1 were analyzed by high-performance liquid chromatography. The total glucosinolate content in these tissues was increased 3.5- to 4.5-fold in comparison with the level of the control plants. The increase was due solely to the production of p-OHBG, as the composition of the major endogenous aliphatic and indole glucosinolates was not affected. Conversely, in mature seeds the total glucosinolate content of CYP79A1 and control plants was similar, with p-OHBG accounting for ca. 30%. The transcript level of the post-oxime enzyme UDP-glucose:thiohydroximate glucosyltransferase in leaves of CYP79A1 plants was increased ca. 50% compared with control plants, indicating that the post-oxime enzymes in the biosynthetic pathway are up-regulated. Western blot analysis and activity measurements showed similar amounts and activities of myrosinase in CYP79A1 and control plants. Thus, the increase in glucosinolate content in CYP79A1 plants was not accompanied by an increase in content or activity of degradation enzyme. The present data demonstrate that the high biosynthetic capacity of the post-oxime enzymes combined with a low substrate-specificity of the post-oxime enzymes in A. thaliana provide a highly flexible system for metabolic engineering of glucosinolate profiles, including new (non-endogenous) glucosinolates derived from oximes introduced into the plant, e.g. by transformation with CYP79 homologues.

Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts.

Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production.

Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans

BackgroundPotato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling.ResultsPhosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with β-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid.ConclusionsOur study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.