Cell culture system versus adventitious root culture system in Asian and American ginseng: a collation

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g^?1 DW, 12.88 mg·L^?1, respectively) than that of CS cell line (9.50 mg·g^?1 DW, 2.39 mg·L^?1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

A Comparative Analysis of Genetic Variability and Differentiation in <Emphasis Type="Italic">Panax vietnamensis</Emphasis> Ha et Grushv. and <Emphasis Type="Italic">P. ginseng</Emphasis> C.A. Meyer Using ISSR Markers

A comparative analysis of the genetic variability and differentiation of rare medicinal ginseng species, Panax vietnamensis Ha et Grushv. and P. ginseng C.A. Meyer, was carried out using inter-simple sequence repeat markers. It was demonstrated that all the genetic diversity parameters of Vietnamese ginseng were high and considerably exceeded those of P. ginseng. On the contrary, the level of genetic differentiation was higher in true ginseng. It is suggested that the differences in the levels of genetic variability and differentiation of the two ginseng species were influenced by the demographic history, peculiarities of the reproductive system, and human activity.     

<Emphasis Type="Italic">Leifiporia rhizomorpha</Emphasis> gen. et sp. nov. and <Emphasis Type="Italic">L. eucalypti</Emphasis> comb. nov. in Polyporaceae (Basidiomycota)

A new poroid wood-inhabiting fungal genus, Leifiporia, is proposed, based on morphological and molecular evidence, which is typified by L. rhizomorpha sp. nov. The genus is characterized by an annual growth habit, resupinate basidiocarps with white to cream pore surface, a dimitic hyphal system with generative hyphae bearing clamp connections and branching mostly at right angles, skeletal hyphae present in the subiculum only and distinctly thinner than generative hyphae, IKI–, CB–, and ellipsoid, hyaline, thin-walled, smooth, IKI–, CB– basidiospores. Sequences of ITS and LSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analysis based on molecular data of ITS + nLSU sequences showed that Leifiporia belonged to the core polyporoid clade and was closely related to Diplomitoporus overholtsii and Lopharia cinerascens, and then grouped with Pycnoporus and Trametes. Further investigation was obtained for more representative taxa in the Polyporaceae based on nLSU sequences, in which the results demonstrated that the genus Leifiporia formed a monophyletic lineage with a strong support (100 % BS, 100 % BP, 1.00 BPP). Both morphological and molecular evidence confirmed the placement of the new genus in the core polyporoid clade. In addition, a new combination, Leifiporia eucalypti, is proposed based on examination of its type material and phylogeny.     

Elemental composition and flavonol content of <Emphasis Type="Italic">Pentaphylloides fruticosa</Emphasis> (L.) O. Schwarz in connection with plant age

The elemental composition and flavonol content of Pentaphylloides fruticosa (L.) O. Schwartz under the conditions of the Altai mountains are investigated. Some differences in the concentrations of elements and flavonols were detected in the leaves of P. fruticosa plants of different age classes corresponding to two periods of development: pregenerative (immature and virginal plants) and generative (young, middle-aged and old generative individuals). The maximum content of flavonols, in particular quercetin, in the leaves of P. fruticosa corresponds to the young generative age. A very high correlation between the flavonol content and the concentrations of Mn, Zn, Mo, and Se was revealed.     

Flower palate structure of the aquatic bladderworts <Emphasis Type="Italic">Utricularia bremii</Emphasis> Heer and <Emphasis Type="Italic">U. minor</Emphasis> L. from section <Emphasis Type="Italic">Utricularia</Emphasis> (Lentibulariaceae)

There is an enormous diversity in the structure of the flower palate of the carnivorous rootless genus Utricularia. This study aims to examine the structure of the palates in Utricularia bremii Heer and U. minor L of the Utricularia sect. Utricularia, which have a glandular palate type. In both species, the palate has only one type of glandular trichomes. Because of the occurrence of cell wall ingrowths in its glandular cells, any exudation may be transported via eccrinous secretion. It was proposed that the palate trichomes of the examined species act as scent glands and that the palate may play a role as an unguentarium. Both U. bremii and U. minor are of an open flower type. Thus, U. bremii and U. minor flowers can be penetrated by small, weak insects, which then easily have access to their generative structure. Small Hymenoptera (member of families Mymaridae and Braconidae) were observed as flower visitors of the male-sterile species Utricularia bremii.     

Molecular characterization of 5-chlorophyll a/b-binding protein genes from Panax ginseng Meyer and their expression analysis during abiotic stresses

The chlorophyll _a/b-binding protein (CAB) serves in both photosystems (PS), I and II, as a coordinator of antenna pigments in the light-harvesting complex (LHC). The CABs constitute abundant and important proteins in the thylakoid membrane of higher plants. In our study, five CAB genes, which contained full-length cDNA sequences from the 4-year-old ginseng leaves (Panax ginseng Meyer), were isolated and named PgCAB. Phylogenetic comparison of the members of the subfamily between ginseng and higher plants, including Arabidopsis, revealed that the putative functions of these ginseng CAB proteins were clustered into the different family of Arabidopsis CABs; two PgCABs in LHCII family and three PgCABs in LHCI family. The expression analysis of PgCABs consistently showed dark-dependent inhibition in leaves. Expression analysis during abiotic stress identified that PgCAB genes responded to heavy metal, salinity, chilling, and UV stresses differently, suggesting their specific function during photosynthesis. This is the first comprehensive study of the CAB gene family in P. ginseng.     

Regulating the regulators: responses of four plant growth regulators during clonal propagation of <Emphasis Type="Italic">Lachenalia montana</Emphasis>

Geraniol, the precursor of terpenoid indole alkaloids can be converted to the 10-hydroxy geraniol by the function of geraniol 10-hydroxylase. In our study, for the first time, a full-length cytochrome P450 monooxygenase (P450) geraniol 10-hydroxylase (PgCYP76C9) cDNA was isolated and characterized from Panax ginseng Meyer. The gene has an open reading frame (ORF) of 1503 base pairs and encodes a precursor protein of 501 amino acids residues. The calculated molecular mass of the protein is approximately 56.3 kDa with a predicated isoelectric point of 8.45. Amino acid identities between PgG10H and other P450s of the CYP76 family in the database had revealed that the deduced amino acid of PgG10H sharing a higher sequence homology with geraniol 10-hydroxylase-like proteins encoded by Cinchona calisaya and Lonicera japonica. We implemented a molecular modeling method to evaluate the possible interaction of geraniol with PgG10H active site. Our finding showed that the geraniol was the potential ligand for PgG10H in P. ginseng. Expression of PgG10H gene was tissue-regulated and showed high expression in 3-year-old ginseng flowers and roots. Expression of PgG10H was differentially induced in ginseng, not only during Pseudomonas syringae infection and wounding but also after exposure to methyl jasmonate and salt stress. Furthermore, overexpression of the newly identified ginseng geraniol 10-hydroxylase P450 gene in Arabidopsis caused terpenoid indole alkaloid dihydrositsirikine production and also conferred enhanced resistance to P. syringae.     

New systematic position of <Emphasis Type="Italic">Aurantiporus alborubescens</Emphasis> (Meruliaceae,Basidiomycota), a threatened old-growth forest polypore

Odoria (Meruliaceae, Basidiomycota) is described as a new genus established for the threatened old-growth forest polypore Phaeolus alborubescens that was previously discussed in Aurantiporus or Tyromyces. It is characterized by the annual pinkish white spongy basidiocarp with strong sweetish smell, turning pale brown when drying, and has a positive reaction with KOH solution, the lack of cystidia, and the monomitic hyphal system with clamped generative hyphae often covered with fine orange crystals. Multigene phylogenetic analyses based on a combined (5.8S–nucLSU–rpb1–rpb2) and a single locus (nrITS) dataset place Odoria in Meruliaceae (Phlebioid clade), close to Crustodontia, Sarcodontia, Luteoporia, and Phlebiporia. Molecular as well as anatomical study of the old type specimen of P. alborubescens is also provided.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

<Emphasis Type="Italic">Purpureocorticium microsporum</Emphasis> (Basidiomycota) gen. et sp. nov. from East Asia

A corticioid fungal species found in mountainous Taiwan, and Yunnan Province of China, is described as a new genus and new species: Purpureocorticium microsporum (Agaricomycetes, Basidiomycota). Morphological study and phylogenetic analyses based on sequence data respectively derived from the large subunit nuclear ribosomal RNA gene (LSU nrDNA) alone and the combined dataset of LSU nrDNA and translation elongation factor 1-α gene (tef1-α) indicated that Purpureocorticium does not belong to any clade of the Agaricomycetes. Purpureocorticium and P. microsporum are characterized by having a resupinate basidiocarp with smooth hymenial surface which turns purple in KOH, and becomes purplish after storage, microscopically having compact texture of subiculum, nodose-septate thin-walled generative hyphae, absence of cystidia, basidia subclavate with a median constriction, or utriform, bearing ovate-ellipsoid small-sized basidiospores, which are smooth and thin-walled, inamyloid, and nondextrinoid. The new combination Rhizochaete rubescens is proposed, based on morphological and phylogenetic evidences.     

Shade tolerance of <Emphasis Type="Italic">Ailanthus altissima</Emphasis> revisited: novel insights from southern Switzerland

The tree of heaven (Ailanthus altissima (Mill.) Swingle) is considered to be an early-successional, gap-obligate pioneer species with vigorous height growth, low shade tolerance, early fecundity and large seed production. It is a highly invasive species in many temperate and Mediterranean ecosystems outside its natural range, especially after disturbance. Due to its low shade tolerance, the potential of A. altissima to colonise undisturbed forests is thought to be low. In this study we analysed the potential of juvenile A. altissima to grow and survive in sweet chestnut (Castanea sativa Mill.) forests in southern Switzerland. We used hemispherical photography to assess the light conditions of 204 individuals of A. altissima (31 % generative, 69 % vegetative) aged between 1 and 7 years (median: 3 years) in six sites. Generative (seed-borne) and vegetative (clonal ramet) offspring of A. altissima are able to grow in light conditions well below the requirements of shade-intolerant tree species such as European larch (Larix decidua Mill.) and Scots pine (Pinus sylvestris L.). The relatively low light conditions found to be sufficient for the growth and survival of generative regeneration of A. altissima suggest a higher shade tolerance for this species than previously stated, at least for early regeneration. Consequently, the colonisation frontier of A. altissima should be intensively monitored in both forest openings but also in closed canopy forests in the vicinity of seed-bearing A. altissima.