Plant invaders and their novel natural enemies: who is naïve?

Introduced exotic species encounter a wide range of non‐coevolved enemies and competitors in their new range. Evolutionary novelty is a key aspect of these interactions, but who benefits from novelty: the exotic species or their new antagonists? Paradoxically, the novelty argument has been used to explain both the release from and the suppression by natural enemies. We argue that this paradox can be solved by considering underlying interaction mechanisms. Using plant defenses as a model, we argue that mismatches between plant and enemy interaction traits can enhance plant invasiveness in the case of toxin‐based defenses, whereas invasiveness is counteracted by mismatches in recognition‐based defenses and selective foraging of generalist herbivores on plants with rare toxins. We propose that a mechanistic understanding of ecological mismatches can help to explain and predict when evolutionary novelty will enhance or suppress exotic plant invasiveness. This knowledge may also enhance our understanding of plant abundance following range expansion, or during species replacements along successional stages.     

Ecophysiological response of Crambe maritima to airborne and soil-borne salinity

Background and Aims

There is a need to evaluate the salt tolerance of plant species that can be cultivated as crops under saline conditions. Crambe maritima is a coastal plant, usually occurring on the driftline, with potential use as a vegetable crop. The aim of this experiment was to determine the growth response of Crambe maritima to various levels of airborne and soil-borne salinity and the ecophysiological mechanisms underlying these responses.

Methods

In the greenhouse, plants were exposed to salt spray (400 mm NaCl) as well as to various levels of root-zone salinity (RZS) of 0, 50, 100, 200 and 300 mm NaCl during 40 d. The salt tolerance of Crambe maritima was assessed by the relative growth rate (RGR) and its components. To study possible salinity effects on the tissue and cellular level, the leaf succulence, tissue Na⁺ concentrations, Na⁺ : K⁺ ratio, net K⁺/Na⁺ selectivity, N, P, K⁺, Ca²⁺, Mg²⁺, proline, soluble sugar concentrations, osmotic potential, total phenolics and antioxidant capacity were measured.

Key Results

Salt spray did not affect the RGR of Crambe maritima. However, leaf thickness and leaf succulence increased with salt spray. Root zone salinities up to 100 mm NaCl did not affect growth. However, at 200 mm NaCl RZS the RGR was reduced by 41 % compared with the control and by 56 % at 300 mm NaCl RZS. The reduced RGR with increasing RZS was largely due to the reduced specific leaf area, which was caused by increased leaf succulence as well as by increased leaf dry matter content. No changes in unit leaf rate were observed but increased RZS resulted in increased Na⁺ and proline concentrations, reduced K⁺, Ca²⁺ and Mg²⁺ concentrations, lower osmotic potential and increased antioxidant capacity. Proline concentrations of the leaves correlated strongly (r = 0·95) with RZS concentrations and not with plant growth.

Conclusions

Based on its growth response, Crambe maritima can be classified as a salt spray tolerant plant that is sensitive to root zone salinities exceeding 100 mm NaCl.     

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [¹H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [¹H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.     

Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS

A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.     

Fitness costs of chemical defense in Plantago lanceolata L.: effects of nutrient and competition stress

Fitness costs of defense are often invoked to explain the maintenance of genetic variation in levels of chemical defense compounds in natural plant populations. We investigated fitness costs of iridoid glycosides (IGs), terpenoid compounds that strongly deter generalist insect herbivores, in ribwort plantain (Plantago lanceolata L.) using lines that had been artificially selected for high and low leaf IG concentrations for four generations. Twelve maternal half-sib families from each selection line were grown in four environments, consisting of two nutrient and two competition treatments. We tested whether: (1) in the absence of herbivores and pathogens, plants from lines selected for high IG levels have a lower fitness than plants selected for low IG levels; and (2) costs of chemical defense increase with environmental stress. Vegetative biomass did not differ between selection lines, but plants selected for high IG levels produced fewer inflorescences and had a significantly lower reproductive dry weight than plants selected for low IG levels, indicating a fitness cost of IG production. Line-by-nutrient and line-by-competition interactions were not significant for any of the fitness-related traits. Hence, there was no evidence that fitness costs increased with environmental stress. Two factors may have contributed to the absence of higher costs under environmental stress. First, IGs are carbon-based chemicals. Under nutrient limitation, the relative carbon excess may result in the production of IGs without imposing a further constraint on growth and reproduction. Second, correlated responses to selection on IG levels indicate the existence of a positive genetic association between IG level and cotyledon size. At low nutrient level, a path analysis based on family means revealed that in the presence of competitors, the negative direct effect of a high IG level on aboveground plant dry weight was partly offset by a positive direct effect of the associated larger cotyledon size. This indicates that fitness costs of defense may be modulated by environment-specific fitness effects of genetically associated traits.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Modification of flavonoid biosynthesis in crop plants

Flavonoids comprise the most common group of polyphenolic plant secondary metabolites. In plants, flavonoids play an important role in biological processes. Beside their function as pigments in flowers and fruits, to attract pollinators and seed dispersers, flavonoids are involved in UV-scavenging, fertility and disease resistance. Since they are present in a wide range of fruits and vegetables, flavonoids form an integral part of the human diet. Currently there is broad interest in the effects of dietary polyphenols on human health. In addition to the potent antioxidant activity of many of these compounds in vitro, an inverse correlation between the intake of certain polyphenols and the risk of cardiovascular disease, cancer and other age related diseases has been observed in epidemiological studies. The potential nutritional effects of these molecules make them an attractive target for genetic engineering strategies aimed at producing plants with increased nutritional value. This review describes the current knowledge of the molecular regulation of the flavonoid pathway and the state of the art with respect to metabolic engineering of this pathway in crop plants.     

Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specificity

Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a beta-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lücker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClgammaTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of beta-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClgammaTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClgammaTS and Cl(-)betaPINS.     

The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea

Ethylene, jasmonate, and salicylate play important roles in plant defense responses to pathogens. To investigate the contributions of these compounds in resistance of tomato (Lycopersicon esculentum) to the fungal pathogen Botrytis cinerea, three types of experiments were conducted: (a) quantitative disease assays with plants pretreated with ethylene, inhibitors of ethylene perception, or salicylate; (b) quantitative disease assays with mutants or transgenes affected in the production of or the response to either ethylene or jasmonate; and (c) expression analysis of defense-related genes before and after inoculation of plants with B. cinerea. Plants pretreated with ethylene showed a decreased susceptibility toward B. cinerea, whereas pretreatment with 1-methylcyclopropene, an inhibitor of ethylene perception, resulted in increased susceptibility. Ethylene pretreatment induced expression of several pathogenesis-related protein genes before B. cinerea infection. Proteinase inhibitor I expression was repressed by ethylene and induced by 1-methylcyclopropene. Ethylene also induced resistance in the mutant Never ripe. RNA analysis showed that Never ripe retained some ethylene sensitivity. The mutant Epinastic, constitutively activated in a subset of ethylene responses, and a transgenic line producing negligible ethylene were also tested. The results confirmed that ethylene responses are important for resistance of tomato to B. cinerea. The mutant Defenseless, impaired in jasmonate biosynthesis, showed increased susceptibility to B. cinerea. A transgenic line with reduced prosystemin expression showed similar susceptibility as Defenseless, whereas a prosystemin-overexpressing transgene was highly resistant. Ethylene and wound signaling acted independently on resistance. Salicylate and ethylene acted synergistically on defense gene expression, but antagonistically on resistance.