Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon

Proanthocyanidins(PAs) are specialized metabolites that infuence persimmon fruit quality.Normal astringent(A)-type and non-astringent(NA)-type mutants show significant variation in PA accumulation, but the infuencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by Dk DTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pa...     

Identification of c-type cytochromes involved in anaerobic, bacterial U(IV) oxidation

Anaerobic, bacterial reduction of water-soluble U(VI) complexes to the poorly soluble U(IV) mineral uraninite has been intensively studied as a strategy for in situ remediation of uranium-contaminated groundwater. A novel and potentially counteracting metabolic process, anaerobic, nitrate-dependent U(IV) oxidation, has recently been described in two bacterial species (Geobacter metallireducens and Thiobacillus denitrificans), but the underlying biochemistry and genetics are completely unknown. We report here that two diheme, c-type cytochromes (putatively c ₄ and c ₅ cytochromes) play a major role in nitrate-dependent U(IV) oxidation by T. denitrificans. Insertion mutations in each of the two genes encoding these cytochromes resulted in a greater than 50% decrease in U(IV) oxidation activity, and complementation in trans restored activity to wild-type levels. Sucrose-density-gradient ultracentrifugation confirmed that both cytochromes are membrane-associated. Insertion mutations in genes encoding other membrane-associated, c-type cytochromes did not diminish U(IV) oxidation. This is the first report of proteins involved in anaerobic U(IV) oxidation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pyrrolizidine alkaloid profiles in Crotalaria species from Brazil: Chemotaxonomic significance

Gas chromatography–mass spectrometry analysis of seeds from 28 species of Crotalaria from Brazil (sections Calycinae, Crotalaria, Chrysocalycinae and Hedriocarpae) showed that pyrrolizidine alkaloids (PAs) are important as chemotaxonomic markers at the infrageneric level. The sections Calycinae and Crotalaria were characterized by 11-membered macrocyclic monocrotaline-type PAs. In the section Chrysocalycinae, a single species in the subsection Incanae, C. incana, showed integerrimine, a 12-membered macrocyclic senecionine-type, as main PA. The group close to the subsection Stipulosae (C. micans and C. maypurensis) showed distinctive PA patterns: C. micans presented mainly the 12-membered macrocyclic integerrimine, and C. maypurensis the unusual 7-hydroxy-1-methylene-8-pyrrolizidine. In the group close to the subsection Glaucae, the PAs with otonecine as the necine base were the main alkaloids, except in C. rufipila which showed an assamicadine-like PA (monocrotaline-type). The section Hedriocarpae showed main 12-membered macrocyclic senecionine-type PAs.     

Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker

A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 μg/g fresh wt in different marker-free transgenic tomato lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3′5′ hydroxylase (F3′5′H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3′ hydroxylation (F3′H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3′5′H to F3′H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3′5′H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.     

Root damage and aboveground herbivory change concentration and composition of pyrrolizidine alkaloids of Senecio jacobaea

Thus far not many studies focussed on how herbivory in one plant part affects plant defence in the other. The effects of root damage and a leaf-feeding herbivore (Mamestra brassicae) on pyrrolizidine alkaloid (PA) levels of Senecio jacobaea were investigated in a controlled environment. Three cloned S. jacobaea genotypes, which differed in PA concentrations, received four treatments: (1) no damage, (2) root damage (removing half of the root system), (3) shoot herbivory by M. brassicae larvae, (4) root damage and shoot herbivory.Shoot herbivory did not significantly affect shoot biomass, while root damage decreased both root and shoot biomass. Shoot herbivory decreased PA concentrations in the roots. Conversely, root damage increased PA concentrations in the roots. Alkaloid concentrations in the shoot showed a weak response to root damage, shoot herbivory had no effect on PA levels in the shoot. The effect of damage on the allocation of PAs to shoot and roots depended on genotype. One genotype allocated more PAs to the damaged site, another genotype did not change allocation and the third genotype allocated more PAs to the shoot if the roots were damaged. Changes in PA composition were observed in one genotype. Shoot herbivory increased erucifoline concentrations in the shoot and decreased concentrations of senecionine in the roots. In conclusion, we have shown that even in an alleged constitutively defended plant, damage of one compartment affects secondary metabolite level in the other.     

Effects of seasonal temperature changes on <Emphasis Type="Italic">DkMyb4</Emphasis> expression involved in proanthocyanidin regulation in two genotypes of persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA). Fruits of the mutant non-astringent (NA) type lose their ability to accumulate PA at an early stage of fruit development, whereas fruits of the normal astringent (A) type sustain PA accumulation until ripening. This allelotype is determined by the genotype of a single ASTRINGENCY (AST) locus. It is possible that the reduction in PA accumulation in NA-type fruits is due to phenological down-regulation of DkMyb4 (a PA regulator) and the resultant down-regulation of structural genes in the PA pathway. In this study, attempts were made to identify the regulatory mechanisms of phenological PA accumulation in A- and NA-type fruits, focusing particularly on the effects of ambient temperature. Continuous cool temperature conditions caused sustained expression of DkMyb4 in NA-type fruits, as well as in A-type fruits, resulting in increased expression of PA pathway genes and PA accumulation. However, the expression of some A/NA phenotypic marker genes was not significantly affected by the cool temperature conditions. In addition, PA composition in NA-type fruits exposed to cool temperatures differed from that in A-type fruits. These results indicate that a cool ambient temperature may have induced DkMyb4 expression and resultant PA accumulation, but did not directly affect the expression of the AST gene.     

On the mechanism of dormancy release in grapevine buds: a comparative study between hydrogen cyanamide and sodium azide

The effect of low doses (LD)—0.1 kJ m⁻² d⁻¹ and high doses (HD)—0.3 kJ m⁻² d⁻¹ of UV-C irradiation on free, conjugated and bound spermine, spermidine and putrescine in leaves of young pea plants after 7 and 14 days of consecutive treatment was studied. Free polyamine (PA) fractions increased mainly in LD treated plants, while conjugated fractions decreased. Bound fractions accumulated mainly at the end of the experiment (after 14 days of UV-C irradiation). The results are interpreted in relation to the possible role of endogenous bound PAs in the prevention of membrane damage induced by UV-C irradiation. Stress markers (malondialdehyde and electrolyte leakage) increased after 7 days of UV-C treatment, and reached control values by the end of the experiment (mainly after HD treatment). Malondialdehyde concentration correlated negatively with UV-C—induced bound fraction and total PAs. The results support the conclusion that endogenous PAs lessen membrane damage in young pea plants provoked by UV-C irradiation.