Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca²⁺/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.     

Somatic embryos cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> Rupr. in air-lift bioreactors for the production of biomass and resveratrol

Resveratrol are the most important bioactive compounds found in Vitis amurensis. In this study, a somatic embryo induction system for V. amurensis was established in air-lift bioreactors for the production of biomass and resveratrol. The somatic embryos biomass growth was low on solid medium (69.60 g L^?1) compared to in liquid medium in bioreactor (329.45 g L^?1). Bioreactor cultures were found to be superior compared with solid medium culture not only in terms of biomass but also resveratrol productivity. Various culture parameters, including culture method, inoculum density, carbon source, and organic compounds were optimized. An inoculum density of 20 g L^?1 embryogenic calli was found suitable for the accumulation of biomass and resveratrol production, whereas 10 g L^?1 embryogenic calli increased the amount of resveratrol per fresh weight in somatic embryos. For bioreactor culturing, sucrose was an optimum carbon source and 500 mg L^–1 casein hydrolysate acid was conducive to the biomass and resveratrol production. This result indicates that an efficient protocol for the large-scale production of resveratrol can be achieved by bioreactor culturing of V. amurensis somatic embryos and can be used as a source of medicinal raw materials.     

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga,a halophytic species

Key message

QTL mapping in F ₂ population [ V. luteola × V. marina subsp. oblonga ] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL.

Abstract

The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F₂ population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F_2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50 % of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.     

Introduction of chromosome segment carrying the seed storage protein genes from chromosome 1V of Dasypyrum villosum showed positive effect on bread-making quality of common wheat

Key message

Development of wheat- D. villosum 1V#4 translocation lines; physically mapping the Glu - V1 and Gli - V1 / Glu - V3 loci; and assess the effects of the introduced Glu - V1 and Gli - V1 / Glu - V3 on wheat bread-making quality.

Abstract

Glu-V1 and Gli-V1/Glu-V3 loci, located in the chromosome 1V of Dasypyrum villosum, were proved to have positive effects on grain quality. However, there are very few reports about the transfer of the D. villosum-derived seed storage protein genes into wheat background by chromosome manipulation. In the present study, a total of six CS-1V#4 introgression lines with different alien-fragment sizes were developed through ionizing radiation of the mature female gametes of CS––D. villosum 1V#4 disomic addition line and confirmed by cytogenetic analysis. Genomic in situ hybridization (GISH), chromosome C-banding, twelve 1V#4-specific EST–STS markers and seed storage protein analysis enabled the cytological physical mapping of Glu-V1 and Gli-V1/Glu-V3 loci to the region of FL 0.50–1.00 of 1V#4S of D. villosum. The Glu-V1 allele of D. villosum was Glu-V1a and its coded protein was V71 subunit. Quality analysis indicated that Glu-V1a together with Gli-V1/Glu-V3 loci showed a positive effect on protein content, Zeleny sedimentation value and the rheological characteristics of wheat flour dough. In addition, the positive effect could be maintained when specific Glu-V1 and Gli-V1/Glu-V3 loci were transferred to the wheat genetic background as in the case of T1V#4S-6BS·6BL, T1V#4S·1BL and T1V#4S·1DS translocation lines. These results showed that the chromosome segment carrying the Glu-V1 and Gli-V1/Glu-V3 loci in 1V#4S of D. villosum had positive effect on bread-making quality, and the T1V#4S-6BS·6BL and T1V#4S·1BL translocation lines could be useful germplasms for bread wheat improvement. The developed 1V#4S-specific molecular markers could be used to rapidly identify and trace the alien chromatin of 1V#4S in wheat background.     

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of calcium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02?% dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1–0.8?% DW). Treatment with the CI at a 1?μM concentration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10?μM significantly decreased resveratrol production by 2–4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1?μM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expression of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concentrations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.     

Characterization of bio-related vanadium and zinc complexes containing tetradentate dithiolate-disulfide, -diamine and -amine-amide ligands

The reaction of [VCl₃(PMe₂Ph)₃] _{with HSS′S′SH (where the HS are thiophenolate and the S′ thioether functions, respectively), H2–1, yields [VCl(μ-SS′S′S)]2 (3) with one of the thiolate groups of each of the two ligands in the bridging mode. Reaction of Na2–1 with [VOCl2(thf)2] leads to a polymeric product of composition [VO(SS′S′S)]x (4). The products obtained from the reaction between [VOCl2(thf)2] and NaSNNSNa, Na2–2, (S is thiophenolate, N the amine function) depend on subtle changes in the diamine backbone of this ligand: If the amine functions are linked by -CH2CH2– (2a), the tetranuclear V^IV complex [V(SNNS)μ-O]4 (5) is formed alongside the V^III complex [VCl(SNNS)]. If the backbone is -CH(Me)CH(Me)- (2b), [VO(SNNS)] (7) and the dinuclear, asymmetrically oxo-bridged V^IV complex [{(SNN ^– S)(thf)V}μ-O{V(SNN ^– S)}] (8) are obtained. In 8, one amine of each of the two ligands is deprotonated to the amide group. In either case, the complexation is accompanied by oxidation of the thiolates to disulfides, leading to the generation of teraazatetrathio-cycloeicosanes (6a/b). Compounds 5 and 8·2THF have been structurally characterized by X-ray analyses. The connectivities have further been established for 3·2CH2Cl2 and for 6b, which exhibits the same conformation as formally characterized 6a. The cluster compound 5 is stabilized by an extended intramolecular N-H...O and N-H...S) hydrogen-bonding network. In 7·2THF, one of the THFs of crystallization is hydrogen-bonded to the NH of the penta-coordinated {VO(SNN ^– S)} moiety; further, there is an intramolecular hydrogen bond between one of the thiolates of this tetragonal-pyramidal half of the molecule and the NH of the octahedral {VO(SNN ^– S)thf} half. The generation of the ligand 2b from its precursor compound, the zinc complex [Zn(SNNS)] (9) leads to the structural characterization of 9·CH3OH with a large SZnS bite angle and a strong hydrogen bond between the methanolic OH and one of the thiolate sulfurs. The relevance of these compounds in biological systems is discussed.}     

Biotransformation of Methylphenylacetonitriles by Brazilian Marine Fungal Strain Aspergillus sydowii CBMAI 934: Eco-friendly Reactions

This study reports the biotransformation of methylphenylacetonitriles by Brazilian marine filamentous fungus Aspergillus sydowii CBMAI 934 under eco-friendly reaction conditions. The phenylacetonitrile 1, 2-methylphenylacetonitrile 2, 3-methylphenylacetonitrile 3, and 4-methylphenylacetonitrile 4 were quantitatively biotransformed into 2-hydroxyphenylacetic 1a, 2-methylphenylacetic acid 2a, 3-methylphenylacetic acid 3a, and 4-methylphenylacetic acid 4a by enzymatic processes using whole cell as biocatalyst. The marine fungus A. sydowii CBMAI 934 is thus a promising biocatalyst for the preparation of important carboxylic acids under mild conditions (pH 7.5 and 32 °C) from nitrile compounds.     

Multiple loss-of-function 5-O-glucosyltransferase alleles revealed in Vitis vinifera,but not in other Vitis species

Key message

Wild and loss-of-function alleles of the 5 - O - glucosyltransferase gene responsible for synthesis of diglucoside anthocyanins in Vitis were characterized. The information aids marker development for tracking this gene in grape breeding.

Abstract

Anthocyanins in red grapes are present in two glycosylation states: monoglucoside (3-O-glucoside) and diglucoside (3, 5-di-O-glucoside). While monoglucoside anthocyanins are present in all pigmented grapes, diglucoside anthocyanins are rarely found in the cultivated grape species Vitis vinifera. Biochemically 3-O-glucoside anthocyanins can be converted into 3,5-di-O-glucoside anthocyanins by a 5-O-glucosyltransferase. In this study, we surveyed allelic variation of the 5-O-glucosyltransferase gene (5GT) in 70 V. vinifera ssp. vinifera cultivars, 52 V. vinifera ssp. sylvestris accessions, 23 Vitis hybrid grapes, and 22 accessions of seven other Vitis species. Eighteen 5GT alleles with apparent loss-of-function mutations, including seven premature stop codon mutations and six frameshift indel mutations, were discovered in V. vinifera, but not in the other Vitis species. A total of 36 5GT alleles without apparent loss-of-function mutations (W-type) were identified. These W-type alleles were predominantly present in wild Vitis species, although a few of them were also found in some V. vinifera accessions. We further evaluated some of these 5GT alleles in producing diglucoside anthocyanins by analyzing the content of diglucoside anthocyanins in a set of representative V. vinifera cultivars. Through haplotype network analysis we revealed that V. vinifera ssp. vinifera and its wild progenitor V. vinifera ssp. sylvestris shared many loss-of-function 5GT alleles and extensive divergence of the 5GT alleles was evident within V. vinifera. This work advances our understanding of the genetic diversity of 5GT and provides a molecular basis for future marker-assisted selection for improving this important wine quality trait.     

Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine VvMybA1 gene

Key message

This study established an efficient method of regenerating plants of Ficus lyrata and producing purple-leaved F. lyrata plants through genetic transformation using a VvMybA1 gene of grapevine.

Abstract

Ficus lyrata, a species with unique violin- or guitar-shaped leaves, was regenerated from leaf-derived calli cultured on Murashige and Skoog (MS) basal medium supplemented with 4.5 μM N-phenyl-N’-1, 2, 3-thiadiazol-5-yl urea (TDZ) and 0.5 μM α-naphthalene acetic acid (NAA). Leaf discs were inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector DEAT that contains the VvMybA1 gene and neomycin phosphotransferase (npt II) gene and subsequently cultured on the established regeneration medium supplemented with 100 mg l^?1 kanamycin. Results showed that 87.5 % of the leaf discs produced kanamycin-resistant callus, and 68.8 % of them produced adventitious shoots. Transgenic plants with three leaf colors including green, green-purple, and purple were produced. Regular and quantitative real-time PCR analyses confirmed the integration of transgenes into the host genome. Semi-quantitative RT-PCR analysis indicated that the VvMybA1 gene was responsible for the purple-colored phenotype. Purple-leaved plants with strong color stability grew vigorously in a greenhouse. This study illustrated the feasibility of using a genetically engineered VvMybA1 gene for drastic modification of leaf color of an important woody ornamental plant.     

Fertile introgression products generated via somatic hybridization between wheat and Thinopyrum intermedium

Key message

Fertile hybrids were produced with genetic material transferred from Th. intermedium into a wheat background and supply a source of genetic variation to wheat improvement.

Abstract

Both symmetric and asymmetric somatic hybrids have been obtained from the combination of wheatgrass (Thinopyrum intermedium) and bread wheat (Triticum aestivum). Two wheat protoplast populations, one derived from embryogenic calli and the other from a non-regenerable, rapidly dividing cell line, were fused with Th. intermedium protoplasts which had been (or not been) pre-irradiated with UV. Among the 124 regenerated calli, 64 could be categorized as being of hybrid origin on the basis of plant morphology, peroxidase isozyme, RAPD DNA profiling and karyological analysis. Numerous green plantlets were regenerated from 13 calli recovered from either the symmetric hybrid (no UV pre-treatment) or the asymmetric one (30 s UV irradiation). One of these hybrid plants proved to be vigorous and self-fertile. The regenerants were all closer in phenotype to wheat than to Th. intermedium. Genomic in situ hybridization analysis showed that the chromosomes in the hybrids were largely intact wheat ones, although a few Th. intermedium chromosome fragments had been incorporated within them.     

Effect of N-Methyl Substituents in 2-Methoxycarbonylphenylsulfamides on Kinetics and Mechanism of Formation of (1H)-2,1,3-Benzothiadiazin-4(3H)-One-2,2-Dioxides

The cyclization reactions of N-methyl-N’-(2-methoxycarbonylphenyl)sulfamide (1a), N-methyl-N-(2-methoxycarbonylphenyl)-sulfamide (2a), and 2-methoxycarbonylphenylsulfamide (3a) were studied in aqueous amine buffers (butylamine, ethanolamine, morpholine, glycinamide). The dependences observed between the rate constants and buffer concentrations show that the reactions are subject to base catalysis in all the three cases, the decomposition of the tetrahedral intermediate being rate limiting. The ratio of the relative rate constants of the base catalyzed cyclizations reactions of the three derivatives is 1a: 2a: 3a = 1: 20000: 100. The logarithm of rate constants of the base catalyzed cyclization reactions was plotted against the pK_a values of conjugated acids of the individual amines used as the buffers in the cyclization of compound 1a, and the value of the Brönsted coefficient obtained was about 0.1, which means that the proton transfer from the intermediate to the basic buffer component is thermodynamically favorable. The intermediate is a much weaker base, and the reaction is controlled by diffusion. The slope of an analogous dependence for compound 2a gradually decreases from values near to 0.5 to values near to zero, which means that the intermediate formed from compound 2a (pK_a ≈ 9.3) has a pK_a value comparable with that of the acid buffer component.     

Synthesis of Pyrrolo[2′,3′:4,5]Furo[3,2-c]Pyridine-2-Carboxylic Acids

1H-Pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylic acid (6a) and its 1-methyl (6b) and 1-benzyl (6c) derivatives were synthesized. 3-(5-Methoxycarbonyl-4H-furo[3,2-b]-pyrrole-2-yl)propenoic acid (1) was converted to the corresponding azide 2, which in turn was cyclized to give 3 by heating in diphenylether. The pyridone 3 obtained was aromatized with phosphorus oxychloride, then reduced with zinc in acetic acid to give methyl 1H-pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylate (5), which by hydrolysis gave the corresponding carboxylic acid 6a.     

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Key message

Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance.

Abstract

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. ‘Richter 110’ rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.     

New genera and species of ericaceae (Vaccinieae) from Costa Rica and Panama

Two new monotypic genera,Didonica andUtleya, are described, withD. pendula from Panama andU. costaricensis from Costa Rica.Disterigma trimera (Panama),D. utleyorum (Costa Rica, Panama, Colombia, and Ecuador),Lateropora santafeensis (Panama),Lysiclesia panamensis (Panama),Macleania talamancensis (Costa Rica),Themistoclesia costaricensis (Costa Rica) andT. horquetensis (Panama),Vaccinium costaricense andV. orosiense (both from Costa Rica) and V.jefense (Panama) are all described as new. New combinations are provided for the PanamanianVaccinium floccosum (=Symphysia floccosa) and the West IndianVaccinium racemosum (=Symphysia racemosa). Keys are provided for the Central American species ofDisterigma andThemistoclesia, the species ofLateropora andLysiclesia, and the Costa Rican and Panamanian species ofVaccinium. Six new species are illustrated.