The genus <Emphasis Type="Italic">Diaporthe</Emphasis>: a rich source of diverse and bioactive metabolites

The genus Diaporthe (asexual state: Phomopsis) comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions. Although species of Diaporthe have in the past chiefly been distinguished based on host association, studies have confirmed several taxa to have wide host ranges, suggesting that they move freely between hosts, frequently co-colonizing diseased or dead tissue, while some species are known to be host-specific. They are also very frequently isolated as endophytes of seed plants. Due to their importance as plant pathogens, the genus has been thoroughly investigated for secondary metabolites, including during screening programs aimed at the discovery of novel bioactive natural products, but the respective information has never been compiled. Therefore, we have examined the relevant literature to explore and highlight the major classes of metabolites of Diaporthe and their Phomopsis conidial states. These fungi predominantly produce a large number of polyketides, but cytochalasins and other types of commonly encountered fungal secondary metabolites are also predominant in some species. Interestingly, not a single metabolite which is also known from the host plant has ever been isolated as a major component from an endophytic Diaporthe strain, despite the fact that many of the recent studies were targeting endophytic fungi of medicinal plants.     

Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants

Main conclusion

Medicinal and aromatic plants are known to produce secondary metabolites that find uses as flavoring agents, fragrances, insecticides, dyes and drugs. Biotechnology offers several choices through which secondary metabolism in medicinal plants can be altered in innovative ways, to overproduce phytochemicals of interest, to reduce the content of toxic compounds or even to produce novel chemicals. Detailed investigation of chromatin organization and microRNAs affecting biosynthesis of secondary metabolites as well as exploring cryptic biosynthetic clusters and synthetic biology options, may provide additional ways to harness this resource. Plant secondary metabolites are a fascinating class of phytochemicals exhibiting immense chemical diversity. Considerable enigma regarding their natural biological functions and the vast array of pharmacological activities, amongst other uses, make secondary metabolites interesting and important candidates for research. Here, we present an update on changing trends in the biotechnological approaches that are used to understand and exploit the secondary metabolism in medicinal and aromatic plants. Bioprocessing in the form of suspension culture, organ culture or transformed hairy roots has been successful in scaling up secondary metabolite production in many cases. Pathway elucidation and metabolic engineering have been useful to get enhanced yield of the metabolite of interest; or, for producing novel metabolites. Heterologous expression of putative plant secondary metabolite biosynthesis genes in a microbe is useful to validate their functions, and in some cases, also, to produce plant metabolites in microbes. Endophytes, the microbes that normally colonize plant tissues, may also produce the phytochemicals produced by the host plant. The review also provides perspectives on future research in the field.

     

Spore abortion index (SAI) as a promising tool of evaluation of spore fitness in ferns: an insight into sexual and apomictic species

Ferns reproduce through small and usually haploid spores. The general paradigm states that whereas species produce good shaped spores, hybrids are sterile and form aborted spores. Apomictic fern species represent an unusual case, and it is believed that they produce an unbalanced spore spectrum. Until now, no comprehensive comparison of sexual and apomictic taxa using extensive spore fitness data has been published. Based on a representative data set of 109 plants from 23 fern taxa, we accomplished the first robust analysis of spore fitness using spore abortion index (SAI), the ratio of aborted to all examined spores. One thousand spores were analyzed for each plant. Focusing mainly on two major European fern taxa (Asplenium, Dryopteris), we compared this trait for different fern reproductive types (sexual/apomicts/hybrids) and ploidy levels (diploid versus polyploid). Our results confirmed the general assumption that shows higher SAI for apomictic taxa (18%) when compared to sexual taxa (3%). Furthermore, hybrids are characterized by having almost all spores aborted (99.8%) with the notable exception of pentaploid Dryopteris × critica (93.1%), the hybrid between sexual and apomictic taxa. We found no significant difference in SAI between sexual taxa of various ploidy levels or between sexual taxa of genera Dryopteris and Asplenium. Additionally, we carried out an optimization of the SAI method, outlying important guidelines for the use of this method in the future.     

Advances in phytochemistry,pharmacology and biotechnology of Bulgarian <Emphasis Type="Italic">Astragalus</Emphasis> species

The genus Astragalus L., Fabaceae (Leguminosae) is presented in Bulgarian flora by 31 species including local endemics, nine of them listed in the Red Data Book. The main biologically active compounds found in the Bulgarian Astragalus plants are saponins and flavonoids with a variety of effects: immunomodulatory, hepatoprotective, cytotoxic, antioxidant, antihypoxic and others. In the recent years volatiles with cytotoxic activity have also been determined. Saponins and flavonoids are still preferably produced by plants due to their complex structures. Variable quantities and qualities of the wild plant material, plants that need to grow several years before they are ready for harvesting and over-collecting of endangered Astragalus species are just a few of the problems connected with the production of these natural products. Therefore, cultured cells are possible alternative for production of high-value secondary metabolites. This review is focused on phytochemical, pharmacological and biotechnological studies of Bulgarian Astragalus plants and includes also results from our investigations.     

Complete chloroplast genome sequence of a tree fern Alsophila spinulosa : insights into evolutionary changes in fern chloroplast genomes

Background  

Ferns have generally been neglected in studies of chloroplast genomics. Before this study, only one polypod and two basal ferns had their complete chloroplast (cp) genome reported. Tree ferns represent an ancient fern lineage that first occurred in the Late Triassic. In recent phylogenetic analyses, tree ferns were shown to be the sister group of polypods, the most diverse group of living ferns. Availability of cp genome sequence from a tree fern will facilitate interpretation of the evolutionary changes of fern cp genomes. Here we have sequenced the complete cp genome of a scaly tree fern Alsophila spinulosa (Cyatheaceae).     

Siberian plants: untapped repertoire of bioactive endosymbionts

Background

Endosymbionts are microorganisms present in all plant species, and constitute the subject of interest among the scientific community. These symbionts have gained considerable attention in recent years, owing to their emerging biological roles. Global challenges, such as antimicrobial resistance, treatment of infectious diseases such as HIV and tuberculosis, cancer, and many genetic disorders, exist. Endosymbionts can help address these challenges by secreting valueadded bioactive compounds with various activities.

Objective

Herein, we describe the importance of plants inhabiting Siberian niches. These plants are considered to be among the least studied organisms in the plant kingdom worldwide. Barcoding these plants can be of interest for exploring bioactive endosymbionts possessing myriad biological properties.

Methods

A systematic survey of relevant scientific reports was conducted using the PubMed search engine. The reports were analyzed, and compiled to draft this review.

Results

The literature survey on Siberian plants regarding endosymbionts included a few reports, since extremely few exploratory studies have been conducted on the plants in these regions. Studies on the endosymbionts of these plants are highly valuable, as they report potent endosymbionts possessing numerous biological properties. Based on these considerations, this review aims to create awareness among the global scientific community working on related areas.

Conclusion

This review could provide the basis for barcoding novel endosymbionts of Siberian plants and their ecological importance, which can be exploited in various sectors. The main purpose of this review is to create awareness of Siberian plants, which are among the least studied organisms in the plant kingdom, with respect to endosymbionts, among the scientific community.

     

Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores

Summary Chemical protection plays a decisive role in the resistance of plants against pathogens and herbivores. The so-called secondary metabolites, which are a characteristic feature of plants, are especially important and can protect plants against a wide variety of microorganisms (viruses, bacteria, fungi) and herbivores (arthropods, vertebrates). As is the situation with all defense systems of plants and animals, a few specialized pathogens have evolved in plants and have overcome the chemical defense barrier. Furthermore, they are often attracted by a given plant toxin. During domestication of our crop and food plants secondary metabolites have sometimes been eliminated. Taking lupins as an example, it is illustrated that quinolizidine alkaloids are important as chemical defense compounds and that the alkaloid-free varieties (sweet lupins), which have been selected by plant breeders, are highly susceptible to a wide range of herbivores to which the alkaloid-rich wild types were resistant. The potential of secondary metabolites for plant breeding and agriculture is discussed.     

Carbon catabolite regulation in <Emphasis Type="Italic">Streptomyces</Emphasis>: new insights and lessons learned

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.     

The genus <Emphasis Type="Italic">Lindera</Emphasis>: a source of structurally diverse molecules having pharmacological significance

Lindera plants not only have good ornamental and economic uses but also have great medicinal and therapeutic values. The genus Lindera consists of approximately 100 species that are widely distributed in tropical and subtropical areas throughout the world. This extensive geographical distribution allows Lindera plants to produce diverse secondary metabolites having novel structures. Phytochemical investigations have shown that Lindera plants produce 341 constituents, including sesquiterpenoids, alkaloids, butanolides, lucidones, flavonoids, and phenylpropanoids. Moreover, some Lindera plants show significant chemotaxonomic reference under family Lauraceae and tribe Litseae. Although Lindera plants have various pharmacological and biological properties, their anticancer, antihypertensive, anti-inflammatory, and analgesic properties have been focused in many studies. Butanolides and lucidones have shown great potential in developing anticancer agents while aporphine alkaloids have shown great potential in developing antiarthritic and antinociceptive agents. However, these compounds need to be assessed further by performing in-depth and systematic research.     

Just in time: Circadian defense patterns and the optimal defense hypothesis

The optimal defense hypothesis (ODH) provides a functional explanation for the inhomogeneous distribution of defensive structures and defense metabolites throughout a plant’s body: tissues that are most valuable in terms of fitness and have the highest probability of attack are generally the best defended. In a previous review,¹ we argue that ontogenically-controlled accumulations of defense metabolites are likely regulated through an integration of developmental and defense signaling pathways. In this addendum, we extend the discussion of ODH patterns by including the recent discoveries of circadian clock-controlled defenses in plants.     

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.Ferns (Pteridophyta) are nonflowering vascular plants comprised of 250 genera, the second largest group of diversified species in the plant kingdom (Gifford and Foster, 1989). Many interesting traits are inherent in various fern species, such as arsenic hyperaccumulation (Pteris vittata; Ma et al., 2001), insecticide production and allelopathy (Pteridium aquilinum; Marrs and Watt, 2006), and antimicrobial compound production (Acrostichum aureum; Lai et al., 2009). Ferns occupy the evolutionary niche between nonvascular land plants, bryophytes, and higher vascular plants such as gymnosperms and angiosperms. Therefore, extant fern species still hold a living record of the initial adaptations required for plants to thrive on land. Of these adaptations, the most important is tracheids that comprise xylem tissues for water and mineral transport and structural support. The vascular system allowed pteridophytes to grow upright during the sporophyte generation, leading to greater resource acquisition capacity. Ultimately, sufficient resources allowed for greater spore production and upright growth, which facilitated spore spread. Recent endeavors have investigated lower plant genomics, including the sequencing of the bryophyte Physcomitrella patens (Rensing et al., 2008) and the lycophyte Selaginella moellendorffii (Banks et al., 2011). Both basic and applied biology of ferns lag far behind that for angiosperms and even bryophytes.Stable genetic transformation has been accomplished in a few species outside angiosperms and gymnosperms, especially among bryophytes (Schaefer et al., 1991; Chiyoda et al., 2008; Ishizaki et al., 2008), but never in the Pteridophyta. Transient transformation methods have been developed both in Ceratopteris richardii ‘C-fern’ and P. vittata, which were limited to heterologous expression in gametophytes (Rutherford et al., 2004; Indriolo et al., 2010). While transient expression of transgenic constructs does enable research, there is no substitute for stable transformation in functional genomics and plant biology. Researchers have resorted to studying fern gene function using heterologous expression in the angiosperm model plant Arabidopsis (Arabidopsis thaliana; Dhankher et al., 2006; Sundaram et al., 2009; Sundaram and Rathinasabapathi, 2010), which is far from optimal. Overexpression and knockdown analysis of individual genes in a wide variety of fern species would undoubtedly accelerate our ability to learn more about their biology and subsequently develop novel products from ferns. Furthermore, a facile transformation system would accelerate functional genomics and systems biology of ferns. For example, knockdown analysis of genes involved in interesting biosynthetic pathways can greatly facilitate gene and biochemical discovery.P. vittata has the unparalleled ability to accumulate more arsenic per gram biomass than any other plant species and is highly tolerant to arsenic (Gumaelius et al., 2004). It can thrive in soils containing up to 1,500 µg mL^–1 arsenic, whereas most plants cannot survive 50 µg mL^–1 arsenic (Ma et al., 2001). Therefore, P. vittata has been the subject of extensive basic and applied research for arsenic hyperaccumulation, translocation, and resistance (Gumaelius et al., 2004) and has been used for arsenic phytoremediation (Shelmerdine et al., 2009). For example, this fern might be of great utility for the production of a safer rice (Oryza sativa) crop; arsenic can be transported and stored in the grain, resulting in serious human health ramifications (Srivastava et al., 2012). In a recent greenhouse study, P. vittata has been used to remediate arsenic-contaminated soil. Following remediation, rice plants were subsequently grown, and it was found that arsenic uptake by rice grains was reduced by 52%, resulting in less than 1 µg mL^–1 arsenic after two rounds of remediation using P. vittata phytoremediation (Mandal et al., 2012). Furthermore, this treatment also resulted in increased rice grain yield by 14% (w/w) compared with control.Ceratopteris is a subtropical-to-tropical fern genus containing four to six species living in aquatic habitats. The C-fern cultivar was developed as a model fern for teaching (http://www.c-fern.org) and research owing to its small size, short life cycle (120 d), and its amenability for in vitro culture (Banks, 1999). The C-fern Express cultivar was developed by Leslie G. Hickok by crossing two Japanese varieties of Ceratopteris thalictroides (L. Hickok, personal communication). cv C-fern Express, a tetraploid, develops spores in 60 d of culture. Though cv C-fern spores have been shown to be a useful single cell model system and a rapid and efficient system for studying RNA interference in ferns (Stout et al., 2003), no stable transformation studies have been reported. In bryophytes, immature thalli are most often used as explants (Chiyoda et al., 2008; Ishizaki et al., 2008), whereas in fungi, spores are routinely used for stable transformation studies (Michielse et al., 2005; Utermark and Karlovsky, 2008).The objective of our research was to develop, for the first time, a facile stable transformation system for P. vittata and C. thalictroides using spores as the transformation targets. This method can be used as an additional tool to further substantiate and strengthen the molecular mechanism studies in pteridophytes.     

Recent advances in the discovery of bioactive metabolites from <Emphasis Type="Italic">Pestalotiopsis</Emphasis>

Fungal endophytes have marked a significant impact on drug discovery reducing the burden and dependency on plants. The vast diversity of Pestalotiopsis sp. has emerged as promising source of wide range of bioactive natural compounds. Recently a series of numerous novel secondary metabolites have been discovered of which taxol has drawn attention of scientific community towards its medicinal potential. A wide variety of compounds like alkaloids, polyketides, terpenoids, flavonoids, coumarins, xanthones, quinones, semiquinones, peptides, phenols, phenolic acids, and lactones have been identified which have usage as antimicrobial, antifungal, antiviral antoneoplastic, and antioxidant activities. This review aims to highlight recent discoveries of different strains of Pestalotiopsis identified for producing natural bioactive compounds along with insights of their source of origin and potential in biotechnological applications.     

Biological activities associated to the chemodiversity of the brown algae belonging to genus <Emphasis Type="Italic">Lobophora</Emphasis> (Dictyotales,Phaeophyceae)

Although Lobophora belongs to a marine algal family (Dictyotaceae) that produces a large array of secondary metabolites, it has received little attention compared to other genera, such as Dictyota, in terms of natural compounds isolation and characterization. However, metabolites produced by Lobophora species have been found to exhibit a wide array of bioactivities including pharmacological (e.g. antibacterial, antiviral, antioxidant, antitumoral), pesticidal, and ecological. This review aims to report the state-of-the-art of the natural products isolated from Lobophora species (Dictyotales, Phaeophyceae) and their associated bioactivities. All bioactivities documented in the literature are reported, therefore including studies for which pure active substances were described, as well as studies limited to extracts or fractions. From the early 1980s until today, 49 scientific works have been published on Lobophora chemistry and bioactivity, among which 40 have reported bioactivities. Only six studies, however, have identified, characterized and tested no less than 23 bioactive pure compounds (three C₂₁ polyunsaturated alcohols, three fatty-acids, a macrolactone, 11 polyketides, a few sulfated polysaccharides, three sulfolipids, a tocopherol derivative). The present review intends to raise awareness of chemists and biologists given the recent significant taxonomic progress of this brown algal genus, which holds a promising plethora of natural products yet to be discovered with ecological and pharmacological properties.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Genome-wide dissection of the chalcone synthase gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis>

Enzymes of the chalcone synthase (CHS) family catalyze the generation of multiple secondary metabolites in fungi, plants, and bacteria. These metabolites have played key roles in antimicrobial activity, UV protection, flower pigmentation, and pollen fertility during the evolutionary process of land plants. We performed a genome-wide investigation about CHS genes in rice (Oryza sativa). The phylogenetic relationships, gene structures, chromosomal locations, and functional predictions of the family members were examined. Twenty-seven CHS family genes (OsCHS01–27) were identified in the rice genome and were found to cluster into six classes according to their phylogenetic relationships. The 27 OsCHS genes were unevenly distributed on six chromosomes, and 17 genes were found in the genome duplication zones with two segmental duplication and five tandem duplication events that may have played key roles in the expansion of the rice CHS gene family. In addition, the OsCHS genes exhibited diverse expression patterns under salicylic acid treatment. Our results revealed that the OsCHS genes exhibit both diversity and conservation in many aspects, which will contribute to further studies of the function of the rice CHS gene family and provide a reference for investigating this family in other plants.     

Micropropagation and genetic transformation of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merr.: a review

Key message

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties.

Abstract

Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.