RNAi-mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing.

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O-methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down-regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3' direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down-regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.

Expression of a functional jasmonic acid carboxyl methyltransferase is negatively correlated with strawberry fruit development.

The volatile metabolite methyl jasmonate (MeJA) plays an important role in intra- and interplant communication and is involved in diverse biological processes. In this study, we report the cloning and functional characterization of a S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Fragaria vesca and Fragaria×ananassa. Biochemical assays and comprehensive transcript analyses showed that JMT has been erroneously annotated as gene fusion with a carboxyl methyltransferase (CMT) (gene15184) in the first published genome sequence of F. vesca. Recombinant FvJMT catalyzed the formation of MeJA with KM value of 22.3µM while FvCMT and the fusion protein were almost inactive. Activity of JMT with benzoic acid and salicylic acid as substrates was less than 1.5% of that with JA. Leucine at position 245, an amino acid missing in other JMT sequences is essential for activity of FvJMT. In accordance with MeJA levels, JMT transcript levels decreased steadily during strawberry fruit ripening, as did the expression levels of JA biosynthesis and regulatory genes. It appears that CMT has originated by a recent duplication of JMT and lost its enzymatic activity toward JA. In the newest version of the strawberry genome sequence (June 2014) CMT and JMT are annotated as separate genes in accordance with differential temporal and spatial expression patterns of both genes in Fragaria sp. In conclusion, MeJA, the inactive derivative of JA, is probably involved in early steps of fruit development by modulating the levels of the active plant hormone JA.

Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background.

Multifunctional flavonoid dioxygenases: flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are "multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.

Developmental, genetic and environmental factors affect the expression of flavonoid genes, enzymes and metabolites in strawberry fruits.

The influence of internal (genetic and developmental) and external (environmental) factors on levels of flavonoid gene transcripts, enzyme activity and metabolites was studied in fruit of six cultivated strawberry (Fragaria x ananassa Duch.) genotypes grown at two Italian locations. Gene expression and enzyme activity showed development- and genotype-associated patterns, revealing gene coordination. Analysis clarified the regulation mechanism of the hydroxylation status of the B-ring of the major flavonoid pools and pointed out examples of genotype-specific post-transcriptional regulation mechanisms and key steps of pathway regulation in strawberry fruits. Metabolite profiles were strongly affected by development and genotype. Flavan-3-ols, their proanthocyanidin (PA) derivatives and anthocyanins were the most abundant metabolites. Flavonol levels and PA-associated traits (epicatechin/catechin ratio and mean degree of polymerization) showed significant environmental effects. Multivariate and correlation analyses determined the relationships among genes, enzymes and metabolites. The combined molecular and biochemical information elucidated more in depth the role of genetic and environmental factors on flavonoid metabolism during strawberry fruit development, highlighting the major impact of developmental processes, and revealing genotype-dependent differences and environmental effects on PA-related traits.

Arabidopsis thaliana expresses a second functional flavonol synthase.

Arabidopsis thaliana L. produces flavonoid pigments, i.e. flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of flavonols.

Influence of type of luting cement used with all-ceramic crowns on load capability of post-restored endodontically treated maxillary central incisors.

Adhesive luting of the final restorations is supposed to support the overall strength of post-endodontically restored teeth. For evaluating adhesive vs conventional cementation, the load capability of adhesively restored endodontically treated teeth (ETT) with glass fibre-reinforced composite posts (GFRCP), resin composite cores and all-ceramic crowns was determined. Thirty-two caries-free human upper, central incisors were randomly assigned to four groups, endodontically treated and cut 2 mm above the cemento-enamel junction. All teeth were restored with GFRCP, composite core build-ups and all-ceramic crowns. All-ceramic crowns were cemented with zinc phosphate cement (I), glass ionomer cement (II), and a self-adhesive resin cement (III and IV). After thermo-mechanical loading, the specimens were loaded to fracture. Maximum load capability F(max) and fracture patterns were compared using non-parametric statistics. Median values for the maximum load capability F(max) (minimum/maximum) in [N] were: I = 503 (416/1,038), II = 442 (369/711), III = 502 (326/561) and IV = 518 (416/652). No statistically significant differences were found for F(max) (p = 0.761) and fracture patterns (p = 0.094) between the experimental groups. The type of cementation of all-ceramic crowns, whether self-adhesive or conventional, appears to have no impact on the load capability of ETT restored with GFRCP and composite core build-ups.

Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria xananassa).

The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria xananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products.

Reinforcement effect of adhesively luted fiber reinforced composite versus titanium posts.

OBJECTIVE: The aim of the present study was to investigate the influence of the rigidity of different post materials (titanium versus glass fiber reinforced composite [FRC]) on the fracture resistance of endodontically treated teeth (ETT). METHOD: Forty-eight caries-free maxillary central incisors were randomly assigned to six groups (n=8). After endodontic treatment, teeth of four groups were flattened 2mm above and two groups at the cemento-enamel junction (CEJ). As control, specimens of one group received neither composite build-up nor post, and teeth were left empty (E). In another group only a resin composite build-up was placed (C). In two groups, both titanium and FRC posts were inserted. One group with titanium (2/T) and FRC (2/FRC) post received a 2mm deep ferrule preparation. For one group with titanium (0/T) and FRC (0/FRC) post no ferrule design was provided. All-ceramic crowns were adhesively luted on all specimens. Specimens were exposed to thermomechanical loading and finally statically loaded until failure. RESULTS: The median fracture load values (min-max) were: E=317 (242-404); C=387 (335-475); 0/FRC=352 (0-440); 2/FRC=502 (326-561); 0/T=420 (0-548), 2/T=517 (416-653). Statistically significant differences were computed between E, C, 2/FRC, 0/T and 2/T; between C, 2/FRC and 2/T; between 0/FRC, 2/FRC and 2/T; between 0/T and 2/T regarding maximum fracture load. SIGNIFICANCE: Fracture resistance of ETT is not influenced by the rigidity of the post material. The combination of ferrule preparation and endodontic post results in higher load resistance after TML than any other build-up design.

Load capability of excessively flared teeth restored with fiber-reinforced composite posts and all-ceramic crowns.

This investigation evaluated the stabilizing effect of glass fiber reinforced posts (FRP) luted with self-adhesive universal cement on the fracture resistance of excessively flared endodontically treated teeth (ETT). Values were compared to teeth with no ferrule, 2 mm ferrule and resin cement for luting with 2 mm ferrule. Thirty-two caries-free maxillary central incisors were randomly assigned to 4 groups (n=8) and endodontically treated. Two groups were flattened 2 mm above and 2 groups at the cemen-to-enamel junction (CEJ). The teeth received FRPs as follows: 1) post was cemented with self-adhesive cement (RelyX Unicem, 3M ESPE) (U), no ferrule (F) was prepared, root canal entrance was excessively flared with a remaining wall thickness of 0.5 - 0.75 mm (UNF/flared); 2) post was cemented with U, no F was prepared; 3) post was luted with U, F was prepared; 4) post was cemented with a resin cement (Panavia F, Kuraray, Japan), F was prepared. All specimens were built-up using a resin composite (Clearfil Core, Kuraray). All-ceramic crowns were adhesively luted (U). Specimens were exposed to thermo-mechanical loading and statically loaded until failure. The mean fracture load values [N](SD) were: UNF/flared=68 (126); UNF=315 (136); UF=488 (72); PF=860 (190). All groups exhibited statistically significant differences regarding maximum fracture load (p<0.05).

The effect of alveolar bone loss on the load capability of restored endodontically treated teeth: a comparative in vitro study.

OBJECTIVES: The aim of the present investigation was to study the influence of a reduced bone support on the fracture resistance of endodontically treated teeth restored with glass fibre-reinforced posts (FRC). METHODS: 30 caries-free maxillary central incisors were divided into 3 groups (n=10). Endodontic treatment was performed. Teeth were flattened 2 mm above the cemento-enamel junction. Group I (control) simulated a clinical situation without horizontal bone loss. In group II a horizontal bone loss of 25% and in group III of 50% was simulated. All specimens received FRC posts and composite core restorations. All-ceramic crowns were adhesively cemented. Specimens were exposed to thermal cycling and mechanical loading (TCML) and finally statically loaded until failure in a universal testing machine (v=1 mm/min). Non-parametric tests were used to compare median fracture loads between groups. Fracture modes were compared using Fisher's exact test. RESULTS: The median fracture load values (min/max) in [N] were: group I=501 (326/561), group II=422 (323/495); group III=352 (266/406), p=0.004. Two specimens in each group II and III failed during TCML. Statistical analysis revealed statistically significant differences between all test groups regarding maximum fracture load and mode of fracture. CONCLUSIONS: The fracture resistance of endodontically treated teeth restored adhesively with a FRC post, composite core, and all-ceramic crown is dependent on the level of surrounding supporting periodontal bone. Loss of alveolar bone loss due to periodontal disease may lead to an increased risk of failure.

Effect of incomplete crown ferrules on load capacity of endodontically treated maxillary incisors restored with fiber posts, composite build-ups, and all-ceramic crowns: an in vitro evaluation after chewing simulation.

OBJECTIVE: The aim of this study was to compare the fracture resistance of endodontically treated maxillary central incisors with incomplete crown ferrules after chewing simulation. MATERIAL AND METHODS: Forty caries-free maxillary central incisors were divided into 4 groups (n = 10). Endodontic treatment was performed. Teeth were decoronated 2 mm above the cemento-enamel junction (CEJ). Group I (control) provided a 360 degrees circumferential 2-mm ferrule. In group II, a 2-mm ferrule was present on the palatal aspect (status after occlusal overload), and in group III on the facial aspect (status after traumatic injury). In group IV, the ferrule was interrupted by bi-proximal cavitations (simulating caries treatment). The teeth received glass fiber reinforced posts and composite core restorations. All-ceramic crowns were adhesively cemented. Specimens were simultaneously exposed to thermal cycling and mechanical loading (1.2 million cycles; 6,000 cycles 5 degrees/55 degrees C) and finally statically loaded until failure in a universal testing machine (crosshead speed = 1 mm/min). For statistical analysis, the Kruskal-Wallis test was applied followed by the Mann-Whitney U-test as post hoc testing. RESULTS: The median fracture load values (min./max.) were: group I = 502 (326/561), group II = 658 (280/827), group III = 899 (396/1176), and group IV = 360 (279/646). Analysis revealed statistically significant differences between test groups, except between groups I and IV and groups II and IV. CONCLUSIONS: The fracture resistance of endodontically and post/core restored teeth is dependent on the degree of tooth conservation. An incomplete crown ferrule is associated with greater variation in load capacity and, despite high fracture values, inclines to fracture.