Proteomics analysis of UV‐irradiated Lonicera japonica Thunb. with bioactive metabolites enhancement

A previous study showed that the contents of caffeoylquinic acids and iridoids, the major bioactive components in the postharvest Lonicera japonica Thunb., were induced by enhanced ultraviolet (UV)‐A or UV‐B irradiation. To clarify the UV‐responsive key enzymes in the bioactive metabolites biosynthetic pathway and the related plant defense mechanism in L. japonica, 2DE in combination with MALDI‐TOF/TOF MS was employed. Seventy‐five out of 196 differential proteins were positively identified. Based on the functions, these proteins were grouped into nine categories, covering a wide range of molecular processes including the secondary metabolites (caffeoylquinic acids and iridoids) biosynthetic‐related proteins, photosynthesis, carbohydrate and energy metabolism, stress, DNA, transport‐related proteins, lipid metabolism, amino acid metabolism, cell wall. Of note is the increasing expression of 1‐deoxy‐d ‐xylulose 5‐phosphate reductoisomerase and 5‐enol‐pyruvylshikimate‐phosphate synthase, which was crucial to supply more precursor for the secondary metabolites including caffeoylquinic acids and iridoids. Thus, this study provides both the clues at the protein level for the increase of the two bioactive components upon UV irradiation and the profile of UV‐responsive proteins in L. japonica.     

Some aspects of overproduction of secondary metabolites

Different approaches used to increase production of secondary metabolites and construct overproducing strains of microorganisms are reviewed. Overproduction of secondary metabolites incuudes the physiological control,e.g. feed-back inhibition, carbon and energy source regulation, nitrogen source regulation, phosphate regulation and the effect of autoregulatory compounds. The genetic control of overproduction of secondary metabolites includes mechanisms similar to those controlling the expression of primary metabolism coding genes, although the genes specifying biosynthesis of secondary metabolites and their expression have some particular features. Possible future trends in the study of overproduction of secondary metabolites are discussed. Dedicated to the 70th birthday of Dr. Z. Vanêk     

beta-Glucosidases as detonators of plant chemical defense

Some plant secondary metabolites are classified as phytoanticipins. When plant tissue in which they are present is disrupted, the phytoanticipins are bio-activated by the action of beta-glucosidases. These binary systems--two sets of components that when separated are relatively inert--provide plants with an immediate chemical defense against protruding herbivores and pathogens. This review provides an update on our knowledge of the beta-glucosidases involved in activation of the four major classes of phytoanticipins: cyanogenic glucosides, benzoxazinoid glucosides, avenacosides and glucosinolates. New aspects of the role of specific proteins that either control oligomerization of the beta-glucosidases or modulate their product specificity are discussed in an evolutionary perspective.     

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi

A secondary metabolite is a chemical compound produced by a limited number of fungal species in a genus, an order, or even phylum. A profile of secondary metabolites consists of all the different compounds a fungus can produce on a given substratum and includes toxins, antibiotics and other outward-directed compounds. Chemotaxonomy is traditionally restricted to comprise fatty acids, proteins, carbohydrates, or secondary metabolites, but has sometimes been defined so broadly that it also includes DNA sequences. It is not yet possible to use secondary metabolites in phylogeny, because of the inconsistent distribution throughout the fungal kingdom. However, this is the very quality that makes secondary metabolites so useful in classification and identification. Four groups of organisms are particularly good producers of secondary metabolites: plants, fungi, lichen fungi, and actinomycetes, whereas yeasts, protozoa, and animals are less efficient producers. Therefore, secondary metabolites have mostly been used in plant and fungal taxonomy, whereas chemotaxonomy has been neglected in bacteriology. Lichen chemotaxonomy has been based on few biosynthetic families (chemosyndromes), whereas filamentous fungi have been analysed for a wide array of terpenes, polyketides, non-ribosomal peptides, and combinations of these. Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few basidiomycete genera, but not in Zygomycota and Chytridiomycota.     

Secretion systems for secondary metabolites: how producer cells send out messages of intercellular communication

Many secondary metabolites (e.g. antibiotics and mycotoxins) are toxic to the microorganisms that produce them. The clusters of genes that are responsible for the biosynthesis of secondary metabolites frequently contain genes for resistance to these toxic metabolites, such as different types of multiple drug resistance systems, to avoid suicide of the producer strains. Recently there has been research into the efflux systems of secondary metabolites in bacteria and in filamentous fungi, such as the large number of ATP-binding cassette transporters found in antibiotic-producing Streptomyces species and that are involved in penicillin secretion in Penicillium chrysogenum. A different group of efflux systems, the major facilitator superfamily exporters, occur very frequently in a variety of bacteria that produce pigments or antibiotics (e.g. the cephamycin and thienamycin producers) and in filamentous fungi that produce mycotoxins. Such efflux systems include the CefT exporters that mediate cephalosporin secretion in Acremonium chrysogenum. The evolutionary origin of these efflux systems and their relationship with current resistance determinants in pathogenic bacteria has been analyzed. Genetic improvement of the secretion systems of secondary metabolites in the producer strain has important industrial applications.     

Root Secreted Metabolites and Proteins Are Involved in the Early Events of Plant-Plant Recognition Prior to Competition

The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually (Col-0, Ler and Cap grown alone) and the plants co-cultured with two homozygous individuals (Col-Col, Ler-Ler and Cap-Cap) or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress- related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions.     

Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses

Arabidopsis thaliana is a successful model plant for studying wide‐ranging topics including plant development, genetics and pathogen resistance. In addition, significant research has been conducted in the area of secondary metabolite biochemical genetics. The secondary metabolites in Arabidopsis include glucosinolates, terpenoids, phenylpropanoids, the alkaloid‐like camalexin, and other uncharacterized compounds. The genetic tools developed in studying secondary metabolite biochemistry are now being used to study how secondary metabolites control various biological processes. This includes compounds involved in plant/insect and plant/pathogen interactions, compounds preventing UV‐B damage, and compounds involved in hormone homeostasis. This review will describe what light Arabidopsis is shedding on the biological and ecological importance of specific secondary metabolites.     

The impact of Pseudomonas syringae type III effectors on transient protein expression in tobacco

The production of recombinant proteins in plants is often achieved by transient expression, e.g. following the injection or vacuum infiltration of Agrobacterium tumefaciens into tobacco leaves. We investigated the associated plant defence responses, revealing that callose deposition is triggered by T–DNA transfer and that subsets of secondary metabolites accumulate in response to mechanical wounding or the presence of bacteria. We also tested the ability of five co‐expressed type III effector proteins from Pseudomonas syringae to modulate these defence responses and increase the yield of two model proteins, the fluorescent marker DsRed and monoclonal antibody 2G12. HopF2 and AvrRpt2 induced necrotic lesions 5 days post‐injection (dpi) even at low doses (OD_600 nm = 0.0078), and increased the concentration of certain secondary metabolites. HopAO1 significantly reduced the number of callose deposits at 2 dpi compared to cells expressing DsRed and 2G12 alone, whereas HopI1 reduced the concentration of several secondary metabolites at 5 dpi compared to cells expressing DsRed and 2G12 alone. Co‐expression with HopAO1, AvrPtoB or HopI1 increased the concentrations of DsRed and 2G12 increased by ~6% but this was not a significant change. In contrast, HopF2 and AvrRpt2 significantly reduced the concentrations of DsRed and 2G12 by 34% and 22%, respectively. Our results show that type III effector proteins can modulate plant defence responses and secondary metabolite profiles but that transient co‐expression is not sufficient to increase the yields of target recombinant proteins in tobacco.     

Activation of chemical defenses in the tropical green algae Halimeda spp.

Halimeda spp. are among the most common seaweeds on tropical reefs where herbivory is intense. These calcified seaweeds produce diterpenoid feeding deterrents; the major metabolites are halimedatetraacetate and halimedatrial. We found that most species of Halimeda on Guam immediately convert the less-deterrent secondary metabolite halimedatetraacetate to the more potent feeding deterrent halimedatrial when plants are injured by grinding or crushing. This conversion would therefore occur when fishes bite or chew Halimeda plants. We term this process of rapid conversion “activation”. Extracts from injured plants contained higher amounts of halimedatrial and were more deterrent toward herbivorous fishes than extracts from control plants. Herbivore-activated defenses are common in many families of terrestrial plants: however, this is the first example of an activated defense in a marine plant.     

Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation

Whole genome duplication leads to autopolyploidy and brings about an increase in cell size, concentration of secondary metabolites and enhanced cytosine methylation. The increased cell size offers a positive advantage to polyploids for cell-surface-related activities, but there is a differential response to change in body size across species and taxonomic groups. Although polyploidy has been very extensively studied, having genetic, ecological and evolutionary implications, there is no report that underscores the significance of native secondary metabolites vis-à-vis body size with ploidy change. To address this problem we targeted unique diploid-autotetraploid paired sets of eight diverse clones of six species of Cymbopogon- a species complex of aromatic grasses that accumulate qualitatively different monoterpene essential oils (secondary metabolite) in their vegetative biomass. Based on the qualitative composition of essential oils and the plant body size relationship between the diploid versus autotetraploid paired sets, we show that polyploidy brings about enhanced accumulation of secondary metabolites in all cases, but exerts differential effects on body size in various species. It is observed that the accumulation of alcohol-type metabolites (e.g. geraniol) does not inhibit increase in body size with ploidy change from 2× to 4× (r = 0.854, P < 0.01), but aldehyde-type metabolites (e.g. citral) appear to drastically impede body development (r = -0.895). Such a differential response may be correlated to the metabolic steps involved in the synthesis of essential oil components. When changed to tetraploidy, the progenitor diploids requiring longer metabolic steps in production of their secondary metabolites are stressed, and those having shorter metabolite routes better utilize their resources for growth and vigour. In situ immunodetection of 5-methylcytosine sites reveals enhanced DNA methylation in autopolyploids. It is underpinned that the qualitative composition of secondary metabolites found in the vegetative biomass of the progenitor diploid has a decisive bearing on the body size of the derived autotetraploids and brings about an enhancement in genome-wide cytosine methylation.     

The emerging role of ICP-MS in proteomic analysis

Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics.In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The “heteroatom (isotope)-tagged proteomics” concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a “hard” ion source, the ICP, to complement well-established “soft” ion sources for mass spectrometry to tackle present proteomic analysis.     

Pathogens pulling the strings: Effectors manipulating salicylic acid and phenylpropanoid biosynthesis in plants

During evolution, plants have developed sophisticated ways to cope with different biotic and abiotic stresses. Phytohormones and secondary metabolites are known to play pivotal roles in defence responses against invading pathogens. One of the key hormones involved in plant immunity is salicylic acid (SA), of which the role in plant defence is well established and documented. Plants produce an array of secondary metabolites categorized in different classes, with the phenylpropanoids as major players in plant immunity. Both SA and phenylpropanoids are needed for an effective immune response by the plant. To successfully infect the host, pathogens secrete proteins, called effectors, into the plant tissue to lower defence. Secreted effectors can interfere with several metabolic or signalling pathways in the host to facilitate infection. In this review, we will focus on the different strategies pathogens have developed to affect the levels of SA and phenylpropanoids to increase plant susceptibility.     

Transporters of secondary metabolites

The membrane transport of plant secondary metabolites is a newly developing research area. Recent progress in genome and expressed sequence tag (EST) databases has revealed that many transporters and channels exist in plant genome. Studies of the genetic sequences that encode these proteins, and of phenotypes caused by the mutation of these sequences, have been used to characterize the membrane transport of plant secondary metabolites. Such studies have clarified that membrane transport is fairly specific and highly regulated for each secondary metabolite. Not only genes that are involved in the biosynthesis of secondary metabolites but also genes that are involved in their transport will be important for systematic metabolic engineering aimed at increasing the productivity of valuable secondary metabolites in planta.     

Host plant secondary metabolite profiling shows a complex, strain-dependent response of maize to plant growth-promoting rhizobacteria of the genus Azospirillum

Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and/or phytohormone production, but little is known about their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars. After 10d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain×plant cultivar combination. Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.     

Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.     

Stress and aberrant phenotypes in vitro culture

Stress responses are largely conserved in eukaryotic cells, but with plants having certain distinctive reactions to specific stresses, e.g. the induction of pathogenesis-related proteins. General responses to stress involve signaling stress detection via the redox system, checkpoints arresting the cell cycle and DNA repair processes stimulated in response to DNA damage. Specific responses to stress include the induction of protective metabolites, such as betaines, and protective proteins, for example, heat shock proteins. Chemical signals, e.g. reactive oxygen species, Ca²⁺ and plant hormones, acting through signal transduction cascades activate genomic re-programming. Genome plasticity in plants allows adaptation to environmental conditions and includes genomic or epigenetic changes (histone acetylation, methylation, chromatin remodeling etc.) and possibly directed mutation. In plants, recent research has indicated that intricate stress response mechanisms and `cross talk' between stress responses exist. Here, changes in the plant genome and in genomic expression in development and as a response to environmental stress are reviewed as background to a discussion of the basis of aberrant genomic expression in vitro. Markers are discussed which may be used to characterize the stress exposure of in vitro tissues.