Efficient transformation of <Emphasis Type="Italic">Actinidia arguta</Emphasis> by reducing the strength of basal salts in the medium to alleviate callus browning

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of Murashige and Skoog (MS) basal salt media was used to overcome the problem of browning and/or necrosis of explants and calli. Callus browning was significantly reduced, resulting in regenerated adventitious shoots when the MS basal salt concentration in the culture medium was reduced to half-strength at low light intensity (3.4 μmol m^?2 s^?1) conditions. Inoculated leaf strips produced putative transformed shoots of Actinidia arguta on half-MS basal salt medium supplemented with 3.0 mg l^?1 zeatin, 0.5 mg l^?1 6-benzyladenine, 0.05 mg l^?1 naphthalene acetic acid, 150 mg l^?1 kanamycin and 300 mg l^?1 Timentin^®. All regenerated plantlets were deemed putative transgenic by histochemical GUS assay and polymerase chain-reaction analysis.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

The mitochondrial genome of the moss <Emphasis Type="Italic">Brachythecium rivulare</Emphasis> (Hypnales,Brachytheciaceae)

The mitochondrial genome of the pleurocarpous moss Brachythecium rivulare has been sequenced and annotated. The genome consists of 104,460 base pairs and has approximately the same gene set and organization as other bryophyte mitogenomes. Whole mitochondrial genome comparison between B. rivulare and Physcomitrella patens, Tetraphis pellucida, Anomodon rugelii, and Anomodon attenuatus was performed. The primary cause of bryophyte mitochondrial gene length variation was found to be numerous indels in the introns. Bryophyte mitochondrial gene conservation level was estimated, and it was in a good congruence with the overall phylogeny of bryophytes with the percentage of mitogenome similarity being proportional to the age estimated by phylochronologic analysis. Annotation discrepancies in the analyzed mitogenome sequences were identified. The simple sequence repeat (SSR) content was evaluated, and candidate sites of RNA editing were predicted in the B. rivulare mitochondrial genome.     

Bioengineering of the Plant Culture of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> with Vanillin Synthase Gene for the Production of Vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.     

The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Callus and cell suspension culture of <Emphasis Type="Italic">Viola odorata</Emphasis> as in vitro production platforms of known and novel cyclotides

Callus from Opuntia streptacantha (cv. Tuna loca), Opuntia megacantha (cv. Rubí reina), and Opuntia ficus-indica (cv. Rojo vigor) were exposed to jasmonic acid (JA) and abiotic stress (drought and UV light) to improve the metabolite production. The callus growth curves, phenolic acids and flavonoids content, antioxidant activity and phenylalanine ammonia lyase (PAL) activity were analyzed under normal and stress conditions. In O. streptacantha callus, the phenolics concentration increased 1.6 to 3 times times in presence of 5% PEG or after irradiation with UV light for 240 min, respectively, while flavonoids triplicate with UV light. A significant increase in antioxidant activity was observed in calli from the three Opuntia species in media with 50 µM JA. The relationships between metabolites/PAL activity, and metabolites/antioxidant activity were analyzed using a surface response methodology. Results showed that PAL activity, induced with PEG and UV, correlated with flavonoids content in O. megacantha and O. ficus-indica calli; PAL activity was related to both flavonoids and phenolics compounds in O. ficus-indica and O. megacantha calli exposed to JA, but only to flavonoids in O. streptacantha callus. In general, the JA stimulated simultaneously the metabolic pathways for phenolics and flavonoids synthesis, while abiotic stress induced mainly flavonoids route. As the stressed Opuntia calli exhibited as high antioxidant activity as cladodes, they are a promising system for research on antioxidant biosynthesis and/or to identify new compounds with antioxidant properties.     

Interactive effects of wood decomposer fungal activities and bryophytes on spruce seedling regeneration on coarse woody debris

Decaying logs are important seedbeds in boreal and subalpine forests. However, biotic and abiotic factors and their interactions related to seedling colonization patterns on logs remain unclear. In the present study, we evaluated the influence of bryophyte communities, wood decay type (white-, brown-, and soft-rot) owing to decomposer fungal activities, and environmental abiotic factors on seedling establishment in an old-growth subalpine coniferous forest in Japan. Among the tree species recorded on the conifer logs, Picea jezoensis var. hondoensis was the most dominant. Log surfaces were covered with distinctive patches of liverwort Scapania bolanderi and moss Pleurozium schreberi (approximately 33% cover for each). Redundancy analysis showed that brown-rot in sapwood significantly affects the bryophyte and seedling community on the logs. Generalized linear models suggested that the total bryophyte cover, Scapania cover, and white-rot in heartwood positively associate with Picea seedling density, whereas Pleurozium cover and basal area of adjacent Picea adults negatively associate with Picea seedling density. Results of structural equation modeling suggested that the brown-rot of sapwood positively associates with Scapania cover that has a positive effect on Picea seedling density. Furthermore, brown-rot of sapwood inhibited the Pleurozium cover, thus contributing to the Scapania dominance on the logs. These results suggest that fungal wood decomposer activities affect colonization of Picea seedlings in an indirect way via structuring bryophyte community on the logs.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

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.     

Titanium Dioxide Nanoparticles Affect the Percentage of Free Radical Scavenging,Protein Content and DNA Mismatch Repair Genes in <Emphasis Type="Italic">Zea mays</Emphasis> L. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

This paper identifies the potential molecular markers predicting the impact of nTiO₂ on plants and explores the new statistical correlations between the biomarkers and growth parameters. The quantitative mRNA expression of the three genes involved in DNA mismatch repair (MLH1) and cell division (PCNA1 and PCNA2) in Zea mays and Triticum aestivum seedlings were related to the growth parameters measured in response to five nTiO2 treatments. The results indicated that the higher concentrations were harmless to Z. mays but not to T. aestivum. nTiO₂ treatments increased the total protein levels in both species and significantly inhibited the percentage of DPPH radical scavenging in Z. mays compared with T. aestivum seedlings. The exposure to both 50 μg/ml and 30 μg/ml concentrations of nTiO₂ significantly induced the expression of MLH1 and PCNA1 genes in both species; however, the exposure to 30 μg/ml of nTiO₂ also significantly induced the expression of PCNA2 genes in T. aestivum. The exposure to 50, 70 and 140 μg/ml significantly inhibited the expression of PCNA2 in both species, while 70 and 140 μg/ml repressed the expression of MLH1 and PCNA1 in the seedlings of Z. mays. The induction and repression of the expression of the three genes were correlated with some growth parameters and biological indices in both species. This key finding suggests that the above genes may play a vital role in mediating plant stress response to nTiO₂ and could be used as sensitive molecular biomarkers indicative of the oxidative stress of nTiO₂ exposure.