Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank accession no. EU677841) was successfully isolated from young leaves of C. acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp long and contained a 1413-bp open reading frame encoding a polypeptide of 471 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Southern hybridization analysis showed that there were at least two HMGS gene members in the C. acuminata genome. CPT content was found to be much higher in cotyledons and hypocotyls as compared to roots. RT-PCR analysis revealed strong expression in hypocotyls and cotyledons, but no expression in roots, indicating good correlation between CaHMGS expression and CPT content in the tested tissues. The expression of CaHMGS could be regulated by exogenous elicitors, including salicylic acid and methyl jasmonate, suggesting that CaHMGS was elicitor-responsive. This work is a first step to acquire a better understanding on the role of HMGS in CPT biosynthesis.     

Isolation and characterization of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> LY214, a camptothecin-producing endophytic bacterium from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptothecin (CPT) is mainly produced and extracted from Camptotheca acuminata and Nothapodytes foetida for pharmaceutical use, i.e., the starting material for chemical conversion to the clinical CPT-type drugs. As the third largest plant anticancer drug, the heavy demand on CPT from global market leads to many research efforts to identify new sources for CPT production. Herein we report the isolation and characterization of a CPT-producing endophytic bacterium Paenibacillus polymyxa LY214 from Camptotheca acuminata. A 10.7 μg l^?1 of CPT was presented in the fermentation broth of P. polymyxa LY214. Its CPT production decreased sharply when the strain of the 2nd generation of P. polymyxa LY214 was cultured and fermented. However, the CPT production remained relatively constant from 2.8 μg l^?1 of the 2nd generation to 0.8 μg l^?1 of the 8th generation of P. polymyxa LY214 under optimized fermentation conditions. A 15- to 30-fold increase of CPT yield was observed when the optimized fermentation conditions, together with the addition of putative biosynthetic precursors of CPT and adsorbent resin XAD16, were applied to ferment the strains of the 7th and 8th generation of P. polymyxa LY214. Bioinformatics analysis of the relative species of P. polymyxa LY214 indicates its potential to produce CPT, which will be helpful to decipher the mysteries of CPT biosynthesis.     

Metatrancriptomic analysis from the Hepatopancreas of adult white leg shrimp (<Emphasis Type="Italic">Litopenaeus vannamei)</Emphasis>

The amoeba, Mayorella viridis contains several hundred symbiotic green algae in its cytoplasm. Transmission electron microscopy revealed strong resemblance between symbiotic algae from M. viridis the symbiotic Chlorella sp. in the perialgal vacuoles of Paramecium bursaria and other ciliates. Although it is thought that the M. viridis and symbiotic algae could be model organisms for studying endosymbiosis between protists and green algae, few cell biological observations of the endosymbiosis between M. viridis and their symbiotic algae have been published. In this study, we characterized the specificity of endosymbiotic relationships between green algae and their hosts. Initially, we established stable cultures of M. viridis in KCM medium by feeding with Chlorogonium capillatum. Microscopic analyses showed that chloroplasts of symbiotic algae in M. viridis occupy approximately half of the algal cells, whereas those in P. bursaria occupy entire algal cells. The symbiotic algae in P. bursaria contain several small spherical vacuoles. The labeling of actin filaments using Acti-stain? 488 Fluorescent Phalloidin revealed no relationship between host actin filaments and symbiotic algal localization, although the host mitochondria were localized around symbiotic algae. Symbiotic algae from M. viridis could infect algae-free P. bursaria but could not support P. bursaria growth without feeding, whereas the original symbiotic algae of P. bursaria supported its growth without feeding. These data indicated the specificity of endosymbiotic algae relationships in M. viridis and P. bursaria.     

Biosynthesis of <Emphasis Type="Italic">Veratrum californicum</Emphasis> specialty chemicals in <Emphasis Type="Italic">Camelina sativa</Emphasis> seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.     

Population demographics and trade-offs to reproduction of an invasive and noninvasive species of <Emphasis Type="Italic">Rubus</Emphasis>

Do trade-offs between growth and reproduction differ between an invasive and noninvasive plant species and how do such trade-offs relate to population demographics? To help address these questions, we compared demographics for an invasive plant species, Rubus discolor, with a noninvasive congener, R. ursinus, in several populations of varying density. Removal of floral buds from reproductive canes increased the size of juvenile canes that arose from clonal sprouting in R. ursinus, suggesting a trade-off between current reproduction and growth. Removal of floral buds had no effect on growth of R. discolor. R. ursinus displayed trade-offs between reproduction (sexual and vegetative) and future growth based on negative correlations between leaf area production and both clonal sprouting and seedling production during the previous year. R. discolor did not exhibit these trade-offs. Both species had high population growth rates in low-density populations, but exhibited little or no growth in high-density populations. A life table response experiment was used to determine the underlying cause for the effect of density on population growth. For R. ursinus, lack of population growth in high-density populations was due primarily to increased mortality of clonally sprouting canes, while for R. discolor, it was due to decreased clonal cane production. Elasticity analysis revealed that clonal growth was more important than sexual reproduction for population growth of both species. However, elasticity values for sexual reproduction in R. discolor were greater in high- than low-density populations. This suggests an increased reliance on sexual reproduction in populations that had reached stable sizes, which could increase the capacity of R. discolor to disperse to new sites. Elasticity analyses were also used to simulate the efficacy of various control strategies for R. discolor. Control of this species could be attained by reducing clonal production within existing populations while reducing seed production to limit establishment of new populations.     

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.     

Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.     

Effects of light on production of camptothecin and expression of key enzyme genes in seedlings of <Emphasis Type="Italic">Camptotheca acuminate</Emphasis> Decne

Camptotheca acuminata (C. acuminata) is utilized in preparation of drugs and as constituent in functional foods of China due to high camptothecin (CPT) content in different plant parts. Light intensity is one of the most critical factors which affect plant growth and secondary metabolites. Pot experiment was conducted to study the effect of light intensity (i.e., 100 % irradiance (control), 75 % irradiance, 50 % irradiance and 25 % irradiance) on contents of CPT, activity of enzymes and genes expression related to CPT biosynthesis of C. acuminata seedlings. The study examined total leaf biomass, CPT content, activities of tryptophan synthase (TSB) and tryptophan decarboxylase (TDC), and relative expression of TSB, TDC1, and TDC2 genes. Plants grown in 75 % irradiance possessed the greatest leaf biomass compared with 100 % light irradiance. Highest values of CPT contents were found after 60 days in plants grown in 50 % irradiance, followed by 25, 75 % and full sunlight. Furthermore, activities of TSB, TDC and relative expression of genes of TSB, TDC1, and TDC2, were significantly increased after 60 days of 50 % irradiance compared with full sunlight. Irradiance of 50 % up-regulated the expression of CPT biosynthesis-related genes and induced CPT biosynthesis. In addition to that lower or higher irradiance inhibited the expression of CPT biosynthesis-related genes and CPT biosynthesis. It is concluded that manipulating light intensity can be an effective means to achieve highest CPT yield in medicinal plants.     

Rhizobial diversity associated with the spontaneous legume <Emphasis Type="Italic">Genista saharae</Emphasis> in the northeastern Algerian Sahara

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N₂-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4 % (w/v) NaCl and grew at 45 °C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Structural and functional organization of the plasmid regulons of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> symbiotic genes

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym^2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P ₀ < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.     

Enhanced production of camptothecin and biological preparation of <Emphasis Type="Italic">N</Emphasis><Superscript>1</Superscript>-acetylkynuramine in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> cell suspension cultures

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ～500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N ¹-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.     

Characterization of surface motility in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>: regulation and role in symbiosis

Sinorhizobium meliloti can exhibit diverse modes of surface translocation whose manifestation depends on the strain. The mechanisms involved and the role played by the different modes of surface motility in the establishment of symbiosis are largely unknown. In this work, we have characterized the surface motility shown by two S. meliloti reference strains (Rm1021 and GR4) under more permissive conditions for surface spreading and analyzed the symbiotic properties of two flagella-less S. meliloti mutants with different behavior on surfaces. The use of Noble agar in semisolid minimal medium induces surface motility in GR4, a strain described so far as non-motile on surfaces. The motility exhibited by GR4 is swarming as revealed by the non-motile phenotype of the flagella-less flaAB mutant. Intriguingly, a flgK mutation which also abolishes flagella production, triggers surface translocation in GR4 through an as yet unknown mechanism. In contrast to GR4, Rm1021 moves over surfaces using mostly a flagella-independent motility which is highly reliant on siderophore rhizobactin 1021 production. Surprisingly, this motility is absent in a flagella-less flgE mutant. In addition, we found that fadD loss-of-function, known to promote surface motility in S. meliloti, exerts different effects on the two reference strains: while fadD inactivation promotes a flagella-independent type of motility in GR4, the same mutation interferes with the surface translocation exhibited by the Rm1021 flaAB mutant. The symbiotic phenotypes shown by GR4flaAB and GR4flgK, non-flagellated mutants with opposite surface motility behavior, demonstrate that flagella-dependent motility positively influences competitiveness for nodule occupation, but is not crucial for optimal infectivity.